Expression Of Peroxisome Proliferator-activated Receptor Gamma Research Articles

Peroxisome proliferator-activated receptor-gamma expression in monocytes/macrophages from rheumatoid arthritis patients: relation to disease activity and therapy efficacy--a pilot study

Peroxisome proliferator-activated receptor-gamma (PPARγ) is expressed by different cell types in the joints and plays a relevant anti-inflammatory role in various diseases. This pilot study aimed to evaluate PPARγ expression in monocytes/macrophages isolated from RA patients as compared with healthy subjects, the relationships between PPARγ expression, MMP-9 activity and disease, and the influence of therapy with anti-rheumatic drugs on these parameters. Thirty RA patients of both sexes (treated with CSs and MTX, mainly) and 15 healthy volunteers were enrolled in this study. Disease severity was evaluated by the 28-joint disease activity score (DAS-28). Monocytes and monocyte-derived macrophages (MDMs) were isolated by standard procedures. PPARγ protein and mRNA expression were assessed by immunoblotting and real-time PCR, respectively; MMP-9 activity was determined by gelatin zymography. Moreover, we checked the ability of 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ, a PPARγ agonist), MTX and methylprednisolone (MP) to affect PPARγ expression and lipopolysaccharide (LPS)-induced MMP-9 activity. Monocytes/MDMs from RA patients have significantly enhanced PPARγ expression (both protein and mRNA) and MMP-9 activity as compared with healthy donors. Interestingly, cells from patients with less active disease (DAS-28 <3.2) present higher PPARγ protein expression and lower MMP-9 activity than RA patients with DAS-28 >3.2. At therapeutic concentrations, MTX and MP increase in vitro PPARγ protein expression and inhibit LPS-induced MMP-9 activity. PPARγ expression in human monocytes/MDMs could represent an indicator of disease activity and therapy efficacy in RA because patients with a DAS-28 score <3.2 show the highest expression.

Role of PPARγ in COX-2 Activation in Mycobacterial Pulmonary Inflammation

Preliminary studies show that intranasal (i.n.) administration of BCG in mice induces M1 activation of alveolar macrophages (M∅) that increase TNF-α production and cyclooxygenase-2 (COX-2) expression but reduce constitutive peroxisome proliferator-activated receptor gamma (PPARγ) expression. However, COX-2 is catalytically inactive for prostaglandin E(2) release, unlike COX-2 that is active in M1 activation in vitro by BCG. In this study, we determined the role of PPARγ for BCG-induced M1 activation in vivo and in vitro. We found that treatment of mice with GW9662, a PPARγ antagonist, prior to i.n. BCG, partially restored PPARγ expression, and decreased TNF-α production and COX-2 expression. But COX-2 was still inactive. The decreased effects on TNF-α and COX-2 were also observed when alveolar M∅ were treated in vitro with GW9662/BCG, but COX-2 was still active. Our results indicate that PPARγ upregulates M1 activation of alveolar M∅, but inactive COX-2 formation is independent of PPARγ in mycobacterial pulmonary inflammation.

Vanin-1 Pantetheinase Drives Smooth Muscle Cell Activation in Post-Arterial Injury Neointimal Hyperplasia

The pantetheinase vanin-1 generates cysteamine, which inhibits reduced glutathione (GSH) synthesis. Vanin-1 promotes inflammation and tissue injury partly by inducing oxidative stress, and partly by peroxisome proliferator-activated receptor gamma (PPARγ) expression. Vascular smooth muscle cells (SMCs) contribute to neointimal hyperplasia in response to injury, by multiple mechanisms including modulation of oxidative stress and PPARγ. Therefore, we tested the hypothesis that vanin-1 drives SMC activation and neointimal hyperplasia. We studied reactive oxygen species (ROS) generation and functional responses to platelet-derived growth factor (PDGF) and the pro-oxidant diamide in cultured mouse aortic SMCs, and also assessed neointima formation after carotid artery ligation in vanin-1 deficiency. Vnn1 −/− SMCs demonstrated decreased oxidative stress, proliferation, migration, and matrix metalloproteinase 9 (MMP-9) activity in response to PDGF and/or diamide, with the effects on proliferation linked, in these studies, to both increased GSH levels and PPARγ expression. Vnn1−/− mice displayed markedly decreased neointima formation in response to carotid artery ligation, including decreased intima:media ratio and cross-sectional area of the neointima. We conclude that vanin-1, via dual modulation of GSH and PPARγ, critically regulates the activation of cultured SMCs and development of neointimal hyperplasia in response to carotid artery ligation. Vanin-1 is a novel potential therapeutic target for neointimal hyperplasia following revascularization.

Abstract 466: Role of Janus Kinases and Signal Transducers and Activators of Transcription in Interferon-gamma--Induced Insulin Resistance in Human Adipocytes

Obesity induces chronic low-grade inflammation of the adipose tissue, accompanied by an infiltration of cytokine-secreting immune cells, driving adipose and systemic insulin resistance. Circulating levels of interferon-gamma (IFNγ) in obesity correlate with increased adiposity and insulin resistance. Furthermore, IFNγ inhibits insulin signaling in adipocytes. IFNγ receptors signal via the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway, but the specific molecules involved with IFNγ signaling in adipocytes have not been determined. Based on pharmacological data, we hypothesized that JAK1 and STAT1, but not JAK2 and STAT3 would mediate IFNγ signaling in human adipocytes. To test this hypothesis we developed lentivirus-delivered shRNA against JAK1, JAK2, STAT1, and STAT3. Knockdown of JAK1, JAK2, and STAT1 in adipocytes with shRNA significantly inhibited IFNγ-induced suppressor of cytokine signaling (SOCS)1 and SOCS3 expression. IFNγ downregulated insulin-signaling related gene (insulin receptor substrate-1, insulin receptor, and glucose transporter-4), and adipogenic gene (adiponectin, fatty acid synthase, lipoprotein lipase, perilipin, and peroxisome proliferator-activated receptor gamma) expression. JAK1, JAK2, and STAT1 shRNA attenuated IFNγ-induced downregulation of insulin signaling related and adipogenic gene expression, whereas STAT3 shRNA had no significant effect. These findings suggest that, in adipocytes, JAK1 and JAK2 as well as STAT1, but not STAT3, contribute to IFNγ signaling and the subsequent decrease in insulin sensitivity. Understanding the molecular mechanisms by which inflammation induces adipose insulin resistance may identify pathways for therapeutic treatment or prevention of obesity induced insulin resistance and diabetes.

PPARγ contributes to PKM2 and HK2 expression in fatty liver

Rapidly proliferating cells promote glycolysis in aerobic conditions, to increase growth rate. Expression of specific glycolytic enzymes, namely pyruvate kinase M2 and hexokinase 2, concurs to this metabolic adaptation, as their kinetics and intracellular localization favour biosynthetic processes required for cell proliferation. Intracellular factors regulating their selective expression remain largely unknown. Here we show that the peroxisome proliferator-activated receptor gamma transcription factor and nuclear hormone receptor contributes to selective pyruvate kinase M2 and hexokinase 2 gene expression in PTEN-null fatty liver. Peroxisome proliferator-activated receptor gamma expression, liver steatosis, shift to aerobic glycolysis and tumorigenesis are under the control of the Akt2 kinase in PTEN-null mouse livers. Peroxisome proliferator-activated receptor gamma binds to hexokinase 2 and pyruvate kinase M promoters to activate transcription. In vivo rescue of peroxisome proliferator-activated receptor gamma activity causes liver steatosis, hypertrophy and hyperplasia. Our data suggest that therapies with the insulin-sensitizing agents and peroxisome proliferator-activated receptor gamma agonists, thiazolidinediones, may have opposite outcomes depending on the nutritional or genetic origins of liver steatosis.

Semi-quantitative RT-PCR analysis of fat metabolism genes in mammary tissue of lactating and non-lactating water buffalo (Bubalus bubalis)

Understanding the mechanism of milk fat synthesis and secretion is important for dairy industry, as the nature of the cream fraction influences the manufacturing properties and organoleptic qualities of milk and dairy products. So, there is a need to understand the mechanism of milk fat synthesis and to elucidate the key genes regulating milk fat synthesis by studying the expression of genes involved in milk fat synthesis. Present manuscript reports the expression of genes involved in milk fat synthesis and metabolism in buffalo mammary tissue. The expression of lipogenic genes was studied in lactating and non-lactating mammary tissue of water buffalo by semi-quantitative reverse transcription PCR expression analysis. The genes studied were acetyl-CoA carboxylase (ACACA), stearoyl-CoA desaturase (SCD), 3 hydroxybutyrate dehydrogenase (BDH), LIPIN, lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma (PPARG), and sterol regulatory element binding protein (SREBF). The expression of ACACA, BDH, LIPIN, PPARG, LPL, and SREBF was higher in lactating as compared to non-lactating buffalo whereas no difference was found in the expression of SCD between both the stages.

070 Macrophages treated with interferon gamma and tumor necrosis factor alpha exhibit decreased cholesterol accumulation and are associated with decreased CD36 and peroxisome proliferator-activated receptor gamma expression

070 Macrophages treated with interferon gamma and tumor necrosis factor alpha exhibit decreased cholesterol accumulation and are associated with decreased CD36 and peroxisome proliferator-activated receptor gamma expression

Exercise training increases mitochondrial biogenesis in the brain

Increased muscle mitochondria are largely responsible for the increased resistance to fatigue and health benefits ascribed to exercise training. However, very little attention has been given to the likely benefits of increased brain mitochondria in this regard. We examined the effects of exercise training on markers of both brain and muscle mitochondrial biogenesis in relation to endurance capacity assessed by a treadmill run to fatigue (RTF) in mice. Male ICR mice were assigned to exercise (EX) or sedentary (SED) conditions (n = 16-19/group). EX mice performed 8 wk of treadmill running for 1 h/day, 6 days/wk at 25 m/min and a 5% incline. Twenty-four hours after the last training bout a subgroup of mice (n = 9-11/group) were euthanized, and brain (brain stem, cerebellum, cortex, frontal lobe, hippocampus, hypothalamus, and midbrain) and muscle (soleus) tissues were isolated for analysis of mRNA expression of peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC-1α), Silent Information Regulator T1 (SIRT1), citrate synthase (CS), and mitochondrial DNA (mtDNA) using RT-PCR. A different subgroup of EX and SED mice (n = 7-8/group) performed a treadmill RTF test. Exercise training increased PGC-1α, SIRT1, and CS mRNA and mtDNA in most brain regions in addition to the soleus (P < 0.05). Mean treadmill RTF increased from 74.0 ± 9.6 min to 126.5 ± 16.1 min following training (P < 0.05). These findings suggest that exercise training increases brain mitochondrial biogenesis, which may have important implications, not only with regard to fatigue, but also with respect to various central nervous system diseases and age-related dementia that are often characterized by mitochondrial dysfunction.

Human cholesteryl ester transfer protein enhances insulin-mediated glucose uptake in adipocytes

Aims Adipose tissue plays an important role in the pathogenesis of insulin resistance, obesity, and Type-2 diabetes. Human adipocytes express abundant cholesteryl ester transfer protein (CETP). However, the function and role of CETP in regulating lipoprotein metabolism are mostly unknown. In this study, we examined whether CETP affected the insulin-mediated responses in adipocytes. Main methods Because mouse 3T3-L1 preadipocytes do not express CETP, we established a stable cell line expressing human CETP by transfecting the cells with pcDNA3.1/human CETP. We used a standard approach to differentiate the cells into mature adipocytes, and we examined the cholesterol balance and insulin responses. Key findings The human CETP stable cell line expressed stable levels of CETP without affecting the expression of either peroxisome proliferator-activated receptor-gamma (PPARγ) or glucose transporter-4 (GLUT4) throughout cell differentiation. CETP expression significantly increased the level of both total and free cholesterol in the mature adipocytes. Upon insulin stimulation, CETP expressing cells had significantly higher protein kinase B (Akt) phosphorylation and 2- 3H-deoxyglucose uptake, as compared with 3T3-L1 cells and cells transfected with control vector. Significance Human CETP expression increased cellular cholesterol levels and enhanced insulin-stimulated Akt phosphorylation and glucose uptake in adipocytes. Thus, CETP may modulate glucose metabolism and insulin action in addition to its effects on lipoprotein metabolism.

Expression of COX-2 and PPARg in the livers of patients with acute on chronic HBV-related liver failure and their relationship with clinic parameters

To study the expressions of cyclooxygenase-2 (COX-2) and Peroxisome proliferator-activated receptor gamma (PPARg) in liver of patients with hepatitis B virus (HBV) related acute-on-chronic liver failure (ACLF) and their correlation with clinical parameters. 35 patients with ACLF, 35 patients with HBV related chronic liver failure (CLF), 27 patients with chronic hepatitis B(CHB) and 15 normal control were enrolled to study the expressions of COX-2 and PPARg in the liver tissues by immunohistochemical staining, and to analyze the correlation of the COX-2 and PPARg levels in liver tissues with clinical parameters. COX-2 was distinctly expressed in the cytoplasm of the hepatocytes, but PPARg was mostly expressed in the nuclei of the hepatocytes and also could be seen in the cytoplasm. The expressions of COX-2 in the liver of ACLF, CLF and CHB groups increased significantly as compared with NC group (z = -5.18, -4.50, -5.32, P is less than 0.01). The levels of COX-2 in ACLF livers also increased evidently as compared with CLF groups (z = -1.98, P is less than 0.05). The expression levels of PPARg in ACLF liver tissues were much higher than the other three groups, and statistical significances existed between ACLF group and the other two groups (CLF, NC groups) (z = -2.62, -4.28, P is less than 0.01). In ACLF group, the expression of COX-2 correlated with MELD score (r = 0.337, P is less than 0.05) and the expression of PPARg correlated with HBV DNA load (r = 0.348, P is less than 0.05). Clinical data showed that the levels of AST, TBil, CHOL, PT, INR, FIB and MELD score in ACLF group were significantly different from that in CLF, CHB and NC groups. COX-2 expressed in liver may be a marker to reflect the degree of inflammation and injury of liver tissue. The PPARg expression of liver could be increased during chronic HBV infection and may be a protective mechanism against liver injury.

The citrus flavonoid hesperidin induces p53 and inhibits NF-κB activation in order to trigger apoptosis in NALM-6 cells: involvement of PPARγ-dependent mechanism

Hesperidin, a flavanone present in citrus fruits, has been identified as a potent anticancer agent because of its proapoptotic and antiproliferative characteristics in some tumor cells. However, the precise mechanisms of action are not entirely understood. The main purpose of this study is to investigate the involvement of peroxisome proliferator-activated receptor-gamma (PPARγ) in hesperidin's anticancer actions in human pre-B NALM-6 cells, which expresses wild-type p53. The effects of hesperidin on cell-cycle distribution, proliferation, and caspase-mediated apoptosis were examined in NALM-6 cells in the presence or absence of GW9662. The expression of peroxisome proliferator-activated receptor-gamma (PPARγ), p53, phospho-IκB, Bcl-2, Bax, and XIAP proteins were focused on using the immunoblotting assay. The transcriptional activities of PPARγ and nuclear factor-kappaB (NF-κB) were analyzed by the transcription factor assay kits. The expression of PPARγ and p53 was analyzed using the RT-PCR method. Hesperidin induced the expression and transcriptional activity of PPARγ and promoted p53 accumulation and downregulated constitutive NF-κB activity in a PPARγ-dependent and PPARγ-independent manner. The growth-inhibitory effect of hesperidin was partially reduced when the cells preincubated with PPARγ antagonist prior to the exposure to hesperidin. The findings of this study clearly demonstrate that hesperidin-mediated proapoptotic and antiproliferative actions are regulated via both PPARγ-dependent and PPARγ-independent pathways in NALM-6 cells. These data provide the first evidence that hesperidin could be developed as an agent against hematopoietic malignancies.

Peroxisome proliferator-activated receptor-gamma (PPAR-γ) expression is downregulated in patients with active ulcerative colitis

Peroxisome proliferator-activated receptor-gamma (PPAR-γ) expression is downregulated in patients with active ulcerative colitis

Erratum to "Peroxisome Proliferator-Activated Receptor-Gamma Expression in the Lung Tissue of Obese Rats" by Hwang SJ, et al. (Yonsei Med J 2011;52:495-501.)

Erratum to "Peroxisome Proliferator-Activated Receptor-Gamma Expression in the Lung Tissue of Obese Rats" by Hwang SJ, et al. (Yonsei Med J 2011;52:495-501.)

Pigment Epithelium-Derived Factor Is an Intrinsic Antifibrosis Factor Targeting Hepatic Stellate Cells

The liver is the major site of pigment epithelium-derived factor (PEDF) synthesis. Recent evidence suggests a protective role of PEDF in liver cirrhosis. In the present study, immunohistochemical analyses revealed lower PEDF levels in liver tissues of patients with cirrhosis and in animals with chemically induced liver fibrosis. Delivery of the PEDF gene into liver cells produced local PEDF synthesis and ameliorated liver fibrosis in animals treated with either carbon tetrachloride or thioacetamide. In addition, suppression of peroxisome proliferator-activated receptor gamma expression, as well as nuclear translocation of nuclear factor-kappa B was found in hepatic stellate cells (HSCs) from fibrotic livers, and both changes were reversed by PEDF gene delivery. In culture-activated HSCs, PEDF, through the induction of peroxisome proliferator-activated receptor gamma, reduced the activity of nuclear factor-kappa B and prevented the nuclear localization of JunD. In conclusion, our observations that PEDF levels are reduced during liver cirrhosis and that PEDF gene delivery ameliorates cirrhosis suggest that PEDF is an intrinsic protector against liver cirrhosis. Direct inactivation of HSCs and the induction of apoptosis of activated HSCs may be two of the mechanisms by which PEDF suppresses liver cirrhosis.

Oxidized low-density lipoprotein activates adipophilin through ERK1/2 signal pathway in RAW264.7 cells

It has been reported that oxidized low-density lipoprotein (Ox-LDL) can increase the expression of adipophilin. However, the detailed mechanisms are not fully understood. The aim of this study was to investigate the mechanism of Ox-LDL on adipophilin expression and the intracellular lipid droplet accumulation. A mouse macrophage-like cell line, RAW264.7, was used throughout, and it was found that Ox-LDL induced adipophilin expression in a dose-dependent manner. Moreover, Ox-LDL induced peroxisome proliferator-activated receptor-gamma (PPARgamma) expression and PPARgamma-specific inhibitor T0070907 abrogated Ox-LDL-induced adipophilin expression, but specific agonist GW1929 not. Furthermore, Ox-LDL induced phosphorylation of ERK1/2, and ERK1/2-specific inhibition by PD98059 suppressed the Ox-LDL-induced PPARgamma and adipophilin expression. The results showed that ERK1/2 or PPARgamma-specific inhibition decreased the amounts of intracellular lipid droplets. Meanwhile, the PPARgamma-specific agonist increased intracellular lipid droplets. These results suggested that Ox-LDL-induced increase in adipophilin level via ERK1/2 activation is one of the mechanisms of inducing greater amounts of intracellular lipid droplets in RAW264.7 cells, which indicated that adipophilin is involved in atherosclerotic progression.

The small molecule phenamil is a modulator of adipocyte differentiation and PPARγ expression

We previously described the use of a cell-based screening approach to identify small molecules that regulate adipocyte differentiation. Here we identify the amiloride derivative phenamil as an adipogenic compound. Phenamil acutely induces expression of the key transcription factor of adipogenesis, peroxisome proliferator-activated receptor γ (PPARγ) and, consequently, promotes the differentiation of multiple preadipocyte cell lines, including 3T3-L1 and F442A. Interestingly, the adipogenic action of phenamil is distinct from and additive with both PPARγ ligands and the previously identified adipogenic small molecule harmine. To identify signaling pathways mediating phenamil's effects, we performed transcriptional profiling of 3T3-F442A preadipocytes. ETS variant 4 (ETV4) was identified as a gene rapidly induced by phenamil but not by other adipogenic small molecules or PPARγ agonists. Transient expression of ETV4 in preadipocytes enhances the expression of PPARγ. Stable overexpression of ETV4 promotes expression of PPARγ and its downstream target genes and enhances morphological differentiation. Finally, knockdown of PPARγ expression by shRNA blocks the effects of phenamil on adipocyte differentiation and gene expression, but it does not block phenamil induction of ETV4, which suggests that ETV4 acts upstream of PPARγ in differentiation processes. These results identify a phenamil as new small molecule tool for the probing of adipocyte differentiation that acts, at least in part, through induction of ETV4 expression.

Melatonin inhibits adipogenesis and enhances osteogenesis of human mesenchymal stem cells by suppressing PPARγ expression and enhancing Runx2 expression

Adipogenesis and osteogenesis, a reciprocal relationship in bone marrow, are complex processes including proliferation of precursor cells, commitment to the specific lineage, and terminal differentiation. Accumulating evidence from in vitro and in vivo studies suggests that melatonin affects terminal differentiation of osteoblasts and adipocytes, but little is known about the effect of melatonin on the process of adipogenesis and osteogenesis, especially adipogenesis. This study was performed to determine the effect of melatonin on adipogenesis and osteogenesis in human mesenchymal stem cells (hMSCs). Cell proliferation assays demonstrated that melatonin had no apparent effect on the proliferation of hMSCs. When melatonin was added to the adipogenic/osteogenic medium, it directly inhibited adipogenesis and simultaneously promoted osteogenesis of hMSCs in a dose-dependent manner. Furthermore, quantitative RT-PCR demonstrated that melatonin significantly suppressed peroxisome proliferator-activated receptor gamma (PPARγ) expression (day 3, 25% decrease; day 6, 47% decrease), but promoted Runx2 expression (day 3, 87% increase; day 6, 56% increase) in the early stages of adipogenesis and osteogenesis of hMSCs. Moreover, melatonin down-regulated several markers of terminal adipocyte differentiation, including leptin (30%), lipoprotein lipase (LPL, 41%), adiponectin (51%), and adipocyte protein 2 (αP2, 45%). Meanwhile, melatonin up-regulated several markers of osteoblast differentiation, including alkaline phosphatase (110%), osteopontin (218%), and osteocalcin (310%). These results suggest that melatonin directly inhibits hMSCs adipogenic differentiation and significantly enhances hMSCs osteogenic differentiation by suppressing PPARγ expression and enhancing Runx2 expression; this provides further evidence for melatonin as an anti-osteoporosis drug.

Tongxinluo Inhibits Oxidized Low-density Lipoprotein–induced Maturation of Human Dendritic Cells via Activating Peroxisome Proliferator-Activated Receptor Gamma Pathway

TongXinLuo (TXL) is a traditional Chinese herbal medicine with multiple vasoprotective activities. Dendritic cells (DCs) play an active role in the immunological processes related to atherosclerosis. The purpose of this study was to determine the effect and possible mechanisms of TXL on oxidized low-density lipoprotein (OX-LDL)-induced maturation and immune function of DCs. Human monocyte-derived DCs were incubated with TXL or ciglitazone and were subsequently stimulated with OX-LDL to induce maturation. Similar to ciglitazone, a peroxisome proliferator-activated receptor (PPAR) gamma agonist, TXL could significantly reduce the maturation-associated markers induced by OX-LDL, such as CD40, CD86, CD1a, and human leukocyte antigen-DR; improved the endocytotic function; and decreased secretions of cytokine interleukin-12 and tumor necrosis factor alpha. These inhibitory effects of TXL could be partly reversed by silencing the expression of PPAR gamma in DCs. In conclusion, TXL could inhibit OX-LDL-induced maturation of DCs through activating PPAR gamma pathway.

Platycodin D inhibits adipogenesis of 3T3‐L1 cells by modulating kruppel‐like factor 2 and peroxisome proliferator‐activated receptor γ

In this study, platycodin D was found to inhibit intracellular triglyceride accumulation in 3T3-L1 cells with an IC(50) of 7.1 microM. The expression levels of genes involved in lipid metabolism such as fatty-acid-binding protein 4 and lipoprotein lipase were significantly downregulated following treatment with platycodin D. Treatment with platycodin D also resulted in a reduction of Peroxisome proliferator-activated receptor(PPAR)gamma expression and its binding to target DNA sequence. Among the various upstream regulators of PPARgamma, the expression of Kruppel-like factor(KLF)2, an anti-adipogenic factor, was significantly upregulated following platycodin D treatment. When the upregulation of KLF2 was inhibited by KLF2 siRNA, the expression and binding of PPARgamma to its target sequence were significantly recovered under these conditions. The results of this study suggested that anti-adipogenic effect of platycodin D involves the upregulation of KLF2 and subsequent downregulation of PPARgamma.

Functional investigations on human mesenchymal stem cells exposed to magnetic fields and labeled with clinically approved iron nanoparticles

BackgroundFor clinical applications of mesenchymal stem cells (MSCs), labeling and tracking is crucial to evaluate cell distribution and homing. Magnetic resonance imaging (MRI) has been successfully established detecting MSCs labeled with superparamagnetic particles of iron oxide (SPIO). Despite initial reports that labeling of MSCs with SPIO is safe without affecting the MSC's biology, recent studies report on influences of SPIO-labeling on metabolism and function of MSCs. Exposition of cells and tissues to high magnetic fields is the functional principle of MRI. In this study we established innovative labeling protocols for human MSCs using clinically established SPIO in combination with magnetic fields and investigated on functional effects (migration assays, quantification of colony forming units, analyses of gene and protein expression and analyses on the proliferation capacity, the viability and the differentiation potential) of magnetic fields on unlabeled and labeled human MSCs. To evaluate the imaging properties, quantification of the total iron load per cell (TIL), electron microscopy, and MRI at 3.0 T were performed.ResultsHuman MSCs labeled with SPIO permanently exposed to magnetic fields arranged and grew according to the magnetic flux lines. Exposure of MSCs to magnetic fields after labeling with SPIO significantly enhanced the TIL compared to SPIO labeled MSCs without exposure to magnetic fields resulting in optimized imaging properties (detection limit: 1,000 MSCs). Concerning the TIL and the imaging properties, immediate exposition to magnetic fields after labeling was superior to exposition after 24 h. On functional level, exposition to magnetic fields inhibited the ability of colony formation of labeled MSCs and led to an enhanced expression of lipoprotein lipase and peroxisome proliferator-activated receptor-γ in labeled MSCs under adipogenic differentiation, and to a reduced expression of alkaline phosphatase in unlabeled MSCs under osteogenic differentiation as detected by qRT-PCR. Moreover, microarray analyses revealed that exposition of labeled MSCs to magnetic fields led to an up regulation of CD93 mRNA and cadherin 7 mRNA and to a down regulation of Zinc finger FYVE domain mRNA. Exposition of unlabeled MSCs to magnetic fields led to an up regulation of CD93 mRNA, lipocalin 6 mRNA, sialic acid acetylesterase mRNA, and olfactory receptor mRNA and to a down regulation of ubiquilin 1 mRNA. No influence of the exposition to magnetic fields could be observed on the migration capacity, the viability, the proliferation rate and the chondrogenic differentiation capacity of labeled or unlabeled MSCs.ConclusionsIn our study an innovative labeling protocol for tracking MSCs by MRI using SPIO in combination with magnetic fields was established. Both, SPIO and the static magnetic field were identified as independent factors which affect the functional biology of human MSCs. Further in vivo investigations are needed to elucidate the molecular mechanisms of the interaction of magnetic fields with stem cell biology.