s2fieldsofstudy: Biology Medicine Engineering

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Human Endothelial-Like Differentiated Precursor Cells Maintain Their Endothelial Characteristics When Cocultured with Mesenchymal Stem Cell and Seeded onto Human Cancellous Bone

Introduction. Cancellous bone is frequently used for filling bone defects in a clinical setting. It provides favourable conditions for regenerative cells such as MSC and early EPC. The combination of MSC and EPC results in superior bone healing in experimental bone healing models. Materials and Methods. We investigated the influence of osteogenic culture conditions on the endothelial properties of early EPC and the osteogenic properties of MSC when cocultured on cancellous bone. Additionally, cell adhesion, metabolic activity, and differentiation were assessed 2, 6, and 10 days after seeding. Results. The number of adhering EPC and MSC decreased over time; however the cells remained metabolically active over the 10-day measurement period. In spite of a decline of lineage specific markers, cells maintained their differentiation to a reduced level. Osteogenic stimulation of EPC caused a decline but not abolishment of endothelial characteristics and did not induce osteogenic gene expression. Osteogenic stimulation of MSC significantly increased their metabolic activity whereas collagen-1α and alkaline phosphatase gene expressions declined. When cocultured with EPC, MSC's collagen-1α gene expression increased significantly. Conclusion. EPC and MSC can be cocultured in vitro on cancellous bone under osteogenic conditions, and coculturing EPC with MSC stabilizes the latter's collagen-1α gene expression.

Introduction
Autologous cell transplantation is a promising treatment option for large bone defects as it eliminates problems such as limited autologous bone availability, allogenic bone immunogenicity, and donor site morbidity and could be used for stabilizing loose alloplastic implant [1,2]. Marrowderived stromal cells (MSCs) alone and in combination with endothelial progenitor cells (EPCs) have been shown to promote bone healing in a variety of different settings [3][4][5].
The bone formation was shown to be improved when MSCs were preconditioned with osteogenic substances such as ascorbic acid and dexamethasone [6]. Another study demonstrated an enhanced callus formation after transplantation of osteogenic predifferentiated MSC during distraction osteogenesis in 3 patients [7]. In an animal study dexamethasone-pretreated MSC seeded on a collagen sponge result in a significant higher mineralization of the collagenous matrix. The mineralization could be clearly ascertained to the transplanted cells [8].
Improved bone healing has also been described in the presence of EPC. This effect has been attributed to EPC stimulation of early vascularization, a prerequisite for in vivo bone regeneration [9]. At least two major types of endothelial cell lines can be obtained by in vitro culture of mononuclear cells: first, the so-called "endothelial-like cells" or "early EPC" and, second, the so-called "outgrowth EPC" or "late EPC". Early EPCs are derived presumably from monocytic/dendritic precursors, and some authors therefore designate them as endothelial-like differentiated PBMC 2 Mediators of Inflammation [10,11]. Those cells can be generated in a sufficient amount within 3 to 5 days from a reasonable volume of blood [12]. Early EPCs are potent producers of vascular endothelial growth factor (VEGF) [13].
When transplanting MSC and/or EPC into bone defects, a scaffold is needed. Synthetic or processed bone-graft substitutes should be osteoinductive, enabling the osteogenic differentiation of cells, should provide appropriate mechanical stability, should permit the ingrowth of cells and vessels [14] thereby improving bone regeneration [15], and should be resorbable. Various porous ceramics are currently available [16]. Hydroxyapatite (HA) sintered ceramics are widely used due to their osteoconductivity but their bioresorbability are comparatively low. In contrast, tricalcium phosphate (TCP) ceramics were porous, resorbable, and biocompatible materials. They do not provoke an inflammatory response and permit the ingrowth of cells and vessels [17] during bone regeneration. Furthermore, TCP can be completely substituted for the bone tissue after stimulation of bone formation. Also the surface chemistry of the scaffold influences the behaviour, through either the influence of its charge density or atomic array on adherent or passing cell populations [18]. Moreover, the surface charge of the biomaterials influences the binding of matrix proteins or growth factors, which might also influence the cell behaviour locally [19].
Bone allografts consist of a collagen fibre network with attached hydroxyapatite crystals providing elasticity and perhaps osteoconductive properties to these scaffolds [20]. Those materials do not evoke any appreciable foreign-body immunogenic reaction. Although antigenic structures were destroyed during processing components of the extracellular matrix, various growth factors such as bone morphogenetic protein-2 (BMP-2) remain functionally active [21,22].
Bone graft substitutes are well described physically and chemically [23] as an osteoconductive scaffold, but even more of interest are biological properties like cell adhesion and function of processed and synthetic biomaterials as delivery system for bone tissue engineering in critical size defects. However, depending on the method used to process the bone allografts the active (osteoinductive) and passive (osteoconductive) biologic interaction between the scaffold and the transplanted cells may vary and greatly influence the proliferation of the latter [24]. This was proved in a comparative study in which a markedly increased survival and metabolic activity of MSC on human cancellous bone (Tutoplast) in comparison to five different synthetic materials were observed [20].
Recently it was shown that EPC and MSC can be successfully grafted onto a -TCP-matrix [25]; however, little data exists regarding the survival, proliferation, and differentiation of these cells when seeded on human cancellous bone (Tutoplast).
A beneficial effect on bone healing mediated by osteogenic preconditioned MSC has been repeatedly described [6][7][8]. But the effect of the osteogenic conditions on early EPC has not been addressed. Moreover, the influence of osteogenic culture conditions on the number, metabolic activity, and differentiation of early EPC alone or in coculture with MSC has not been addressed yet.
In the present study we investigated the influence of osteogenic culture conditions on the endothelial properties of human early EPC seeded alone on human cancellous bone chips and together with human MSC.

Isolation, Cultivation, and Characterization of MSC.
Bone marrow cells were obtained from iliac crest aspirates of volunteer trauma patients ( = 5) undergoing pelvic surgery [26]. This was performed in accordance with and with the approval of our hospital's ethics committee. All patients signed informed consent. Briefly, MSCs were isolated from fresh bone marrow aspirate using Ficoll density gradient centrifugation (30 min, 1100 g, = 1, 077 g/mL, Biochrom, Berlin, Germany). Cells in the interphase were collected, washed twice with PBS containing 2% fetal bovine serum (FBS) (10 min, 900 g), resuspended in 3 mL MesenCult + Supplements (Cell-Systems, St. Katharinen, Germany), and were counted using a Neubauer chamber. 4 × 10 6 cells were seeded in a 25 cm 2 culture flask and then expanded over three to five passages prior to being used in the present experiments. Cells were detached by 10 min incubation with Accutase, then washed (10 min, 300 g), re-suspended in MesenCult + Supplements, and divided in 2 parts. One part was adjusted to a density of 2.5 × 10 5 cells in 100 L and used for the present experiments, and the other was used for confirmation of MSC, surface characteristics using flow cytometry (FACSCalibur, BD-Biosciences, Heidelberg, Germany). MSCs were negative for CD45 and CD34 and express CD73, CD90, and CD105 as described previously; all antibodies were purchased from BD-Biosciences [26].

Isolation, Identification, and Characterization of Early
EPC. EPCs were isolated according to procedures described elsewhere [25,27]. Briefly, PBMCs were isolated from buffy coat ( = 5) by density gradient centrifugation (20 min, 600 g) with Ficoll (1.077 g/mL, Biochrom, Berlin, Germany). PBMCs were washed twice with cold PBS without Ca 2+ and Mg 2+ (PBS w/o , 10 min, 350 g), and 4 * 10 6 cells were cultivated on a fibronectin-coated (10 g/mL, Sigma, Deisenhofen, Germany) 24-well culture dish in 1 mL of endothelial basal medium (EBM2, Cambrex, Verviers, Belgium) supplemented with EGM2 SingleQuots at 37 ∘ C, 5% CO 2 . After 48 hrs, non-and weakly adherent cells were removed, the medium was exchanged, and the cells were cultivated for an additional 72 hrs. A parallel preparation was performed to evaluate the percentage of endothelial cell-like differentiated cells. Cells were incubated for 1 h with 2.4 g/mL 1,1 -dioctadecyl-3,3,3 ,3 -tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (DiLDL, CellSystems, St. Katharinen, Germany) in EBM supplemented with 20% FCS. Cells were fixed with 2% paraformaldehyde for 10 min, and after washing with PBS +/+ FITC-labelled Ulex europaeus agglutinin-1 [10 g/mL] (lectin, Sigma, Deisenhofen, Germany) was incubated for 1 h. Cells presenting double-positive fluorescence were considered to be EPC. Only preparations with a percentage of endothelial like differentiated cells of greater than 80% were used. For the determination of the expression of the endothelial markers CD31 and von Willebrandt factor (vWF) a volume of 100 L DiLDL prestained cells was incubated for 20 min with either monoclonal antibody against vWF (Dako, Hamburg, Germany) followed by 15 min incubation with a FITC conjugated secondary antibody (Dako) or with a FITC-conjugated monoclonal antibody against CD31 (Chemikon, Hofheim, Germany). Antibodies of identical isotypes served as control. After two steps of washing, the cells were subjected to flow cytometry using a FACSCalibur (BD Biosciences).

Human-Processed Cancellous Bone (Tutoplast).
A processed human cancellous bone allograft material (Tutoplast, Tutogen Medical) was used. The solvent preserved, dried, and gamma-irradiated material has a particle size of 0.5-2 mm with a pore size ranging from 100 to 500 m. The material has a high mechanical stability and a rapid biodegradability (information provided by the manufacturer).

Seeding Cells on Scaffold.
Tutoplast granules were placed as a dense single layer in a 24-well plate (Nunc, Wiesbaden, Germany) using sterile forceps and prewetted with 200 L PBS with Ca 2+ and Mg 2+ (PBS +/+ ). 5 × 10 5 MSCs or EPCs in a volume of 200 L were dripped onto the Tutoplast layer and were incubated for 10 min at 37 ∘ C. When MSC together with EPCs were tested, 2.5 × 10 5 cells of each were mixed immediately prior to seeding.
After incubation, a medium containing the nonadhering cells was removed and dripped once again over the Tutoplast layer, followed by incubation as indicated above. This procedure was repeated three times. The granules were then gently transferred to another well containing 500 L of a mixture consisting of 2 parts Mesencult + supplements and 1 part EBM2+EGM2 Singlequots. Below this mixture is referred to as the "medium" and is described in [25]. All experiments were performed using the said medium. The remaining cells in the supernatant and the bottom of the initial seeding well were isolated, counted, and the percentage of adhering cells was calculated ((initial cell number − remaining cell number)/initial cell number) * 100.

Identification of MSC and Early EPC on Tutoplast.
MSC and EPC, alone and combined, were seeded on Tutoplast as described above. Approximately 4-5 granules were transferred to a single well of a 96-well plate using sterile forceps and incubated in 100 L medium for a period of 2, 6 or 10 days in a CO 2 incubator at 37 ∘ C. Parallel preparations included the same as above with the addition of substances that promote osteogenic differentiation: Dexamethasone [final concentration 1.0 × 10 −7 M], -glycerolphosphate [final concentration 1.0 × 10 −2 M], and ascorbic acid [final concentration 5 × 10 −5 M] [6]. All substances were obtained from Sigma (Deisenhofen, Germany). In order to detect MSC and EPC, MSC 1 L of DiLDL (CellSystem, St. Katharinen, Germany) or 1 L DAPI [final concentration 1 g/mL] was added to the corresponding wells followed by further incubation for 1 h at 37 ∘ C. After three washes with PBS −/− , the granules were transferred to a new well in order to prevent false-positive results caused by cells adhering to the bottom of the cultivation well. Finally, randomly chosen Tutoplast granules were viewed at 100x magnification using fluorescence microscopy (Zeiss, Axio Observer, Gottingen, Germany), and the DiLDL-stained EPC and DAPI-stained MSC were counted using cell explorer 2001 (BioScieTec, Frankfurt, Germany) software.

Identification of EPC and MSC by Scanning Electron Microscopy (SEM).
Tutoplast granules were seeded with either MSC, EPC, or both. After 2 days the cell-Tutoplast constructs were fixed in 2% glutaraldehyde in PBS (Merck, Darmstadt, Germany) for 30 min and dehydrated by incubation for 15 min in PBS with increasing concentrations of ethanol (25%, 50%, 75%, 96%, and 100%). Subsequently the samples were incubated overnight in 1,1,1,3,3,3hexamethyldisilazane (Merck, Darmstadt, Germany) and slowly air-dried. The granules were fixed with carbon glue to an aluminium carrier and sputtered trice for each 1 min with gold (Agar Sputter Coater, Agar Scientific Ltd., UK) and subjected to scanning electron microscopy. A Hitachi FE-SEM S4500 was used (Hitachi, Düsseldorf, Germany). A voltage of 5 kV was applied. The images were digitally recorded using the Digital Image Processing System 2.6 (Point Electronic, Halle, Germany).

Cell-Tutoplast Adherence (MTT Assay).
For determination of the metabolic activity as a correlate of the number of cells adhering to the Tutoplast scaffold a Cell proliferation Kit I (MTT, Roche Diagnostics, Mannheim, Germany) was used following the manufacturer's instructions. In brief, the MTT assay is based on the cleavage of the yellow tetrazolium salt MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to purple formazan crystals by metabolically active cells. MSC obtained from 5 donors and EPC derived from 5 buffy coats were tested. The following combinations were performed: MSC alone, EPC alone, and MSC and EPC together. All experiments were performed in duplicate.
Cells were seeded on Tutoplast, as described above, and 3 to 5 granules, depending on the granule's size, were transferred to new wells in a 96-well plate (Nunc, Roskilde, Denmark) and cultivated for 2, 6, and 10 days. Before adding the MTT reagent, granules were transferred to an empty well, in order to prevent false positive results caused by cells adhering to the bottom of the well. 90 L of medium and 10 L of MTT labelling reagent were added to each well and cells were incubated for an additional 4 hrs. Next, the cells were incubated overnight with a solubilization solution. The supernatant was collected and transferred to another 96well plate. The absorbance at 570 nm was then measured with an ELISA reader (Ceres UV900c, Bio-Tek Instruments, Windoski, VT, USA). As controls, increasing numbers (1000, 2500, 5000, and 10.000) of MSC and EPC were seeded directly in 96 well plates and assessed separately.

Expression of Osteogenic and Endothelial Marker Genes by Real Time RT-PCR.
In brief, total RNA was isolated using the RNeasy system (Qiagen, Hilden, Germany) following the manufacturer's instructions with the following exception. Approximately 50 L Tutoplast-granules that had been sown with progenitor cells was incubated in RLT buffer for 3 min, the mixture was gently vortexed, and the supernatant was subjected to the RNA isolation procedure. The quality and quantity of RNA was determined using the NanoDrop ND-1000 device (Nanodrop technologies, Wilmington, Delaware, USA). Contaminating genomic DNA was removed by digestion with the RNase-free DNase Kit following the manufacturer's protocol (Qiagen).
Each 250 ng of RNA was reversely transcribed using an Affinity script QPCR-cDNA synthesis kit (Stratagene, La Jolla, CA, USA) following the manufacturer's instructions.
Real time RT-PCR was performed on a Stratagene MX3005P QPCR system (Stratagene, La Jolla, CA, USA). PCR was performed using the primer assays for human collagen-1 (COL1A, NM 000088, catalog number PPH01299F), alkaline phosphatase (ALPL, NM 000478, catalog number PAHS-026), core binding factor-1 (cbfa-1 also known as Runt-related transcription factor 2, RUNX2, NM 004348. 3 A melting curve analysis was applied to ensure the specificity of the PCR reaction. Relative quantification of the mRNA levels of the target genes was determined using the comparative CT (threshold cycle values) method (2 −ΔCT method). The results are presented as fold change to GAPDH gene expression.

Statistics.
Results are presented as mean values and standard error of mean (SEM). Kruskal-Wallis test with Dunn's post hoc test for multiplicity was used for comparisons between the groups and for the analysis of changes during the follow-up period (day 2 versus day 6 and day 10). A value < 0.05 indicates statistical significance.

MSC and EPC Adherence to
Tutoplast. The overall percentage of initially adhering cells ranged from 90% to 95% and did not differ significantly between MSC and EPC.
Scanning electron microscopy images of Tutoplast 2 days after cell seeding revealed a slightly roughened surface lacking sharp edges (Figure 2(a)). Higher magnification revealed a surface composed of a dense layer of fibrils (Figure 2(b)). Both MSC and EPC appeared firmly attached to the Tutoplast surface. While MSC had a bigger size than EPC and showed a flattened and spread-out appearance (Figure 2(d)), EPC appeared elongated and spindle shaped (Figure 2(c)). In the samples containing both MSC and EPC their district shapes made it possible to differentiate between the 2 cell types on the Tutoplast surface (Figure 2(e)).

Number of Adhering MSC and EPC on Tutoplast Surface.
In the samples containing both MSC and EPC, the number of EPC declined significantly ( < 0.05) from day 2 to day 6 and then remained stable until day 10. This was also observed in samples in which EPCs were incubated in the presence of osteogenic supplements. However, on day 2 after seeding, the number of adhering EPCs was significantly higher in cultures supplemented with osteogenic factors (Figure 3(a)).
The number of adhering MSCs also decreased significantly ( < 0.05) from day 2 to days 6 and 10, independent of the presence of osteogenic factors (Figure 3(b)).

MSC and EPC: Metabolic
Activity. The MTT assay demonstrated MSC and EPC metabolic activity over the entire observation period. The number of adhering cells paralleled the metabolic activity of EPC, dropping significantly ( < 0.05, Figure 4(a)) from day 2 to day 6. In the presence of osteogenic supplements the metabolic activity of EPC slightly increased in comparison to controls; however, this increase was not statistically significant (Figure 4(a)).
The metabolic activity of MSC decreased slightly with time, depicting a similar course as described for the number of adhering MSCs. This decrease was not statistically significant (Figure 4(b)). In contrast to EPC, when osteogenic substances were added to MSC, metabolic activity was significantly increased compared to the corresponding controls on day 6 and day 10 (Figure 4(b)).

Endothelial Differentiation (DiLDL Uptake).
The uptake of acetylated low-density lipoprotein is an active process that characterizes endothelial cells. Although a significant ( < 0.05) decline of the DiLDL uptake was noted over the time, a significant ability of EPC to uptake DiLDL was conserved over the entire 10-day observation period (Figure 5(a)). Using an exposure time of 0.25 s the mean pixel brightness decreased from 60.0 ± 5.5 at day 2 to 34 ± 3.8 at day 10 ( < 0.05 versus day 2, Figures 5(a) and 5(c)).

MSC and EPC Differentiation (Gene Expression Analysis).
In order to assess MSC and EPC differentiation expression of osteogenic genes (Collagen-1alpha, RUNX-2, BGLAP, ALP) and endothelial marker genes (vWF, VEGF) were measured on day 10. vWF gene expression significantly ( < 0.05) increased when EPCs were cultured on Tutoplast versus the culture on plastic (reference EPC) (Figure 6(a)). Osteogenic   Figure 4: MTT assay, metabolic activity of EPC (a) and MSC (b) cultivated on Tutoplast. Presence of osteogenic substances lead to a significant increase in MTT conversion in MSC. The metabolic activity was assessed using the MTT assay as described in Section 2. * : < 0.05. stimulation of EPC led to a decrease in vWF gene expression ( = 0.09, Figure 6(a)).
In contrast, VEGF gene expression was significantly ( < 0.05) higher in EPC cultured on plastic as well as on Tutoplast under osteogenic conditions versus EPC cultured with Tutoplast alone (Figure 6(b)).
Collagen-1 and ALP gene expression was limited to MSC whereas a slight gene expression of RUNX and BGLAP was also observed in EPC independent from the culture conditions. Collagen-1 gene expression on Tutoplast was significantly ( < 0.05) reduced in comparison to reference MSC (Figure 6(c)). Osteogenic conditions lead to a further significant reduction of collagen-1 gene expression of MSC cultured on Tutoplast ( < 0.05, Figure 6(c)).
In contrast, a trend towards increased collagen expression was observed in coculture with EPC in comparison to MSC cultured on Tutoplast ( < 0.09). Furthermore, the collagen gene expression in the coculture was significantly elevated to MSC, respectively, and MSC and EPC cultured under osteogenic conditions ( < 0.05, Figure 6(c)). Coculture under osteogenic conditions expressed higher collagen level in comparison to MSC under osteogenic conditions.
The gene expression of ALP (alkaline phosphatase) was significantly increased in reference MSC in comparison MSC cultured on Tutoplast ( < 0.05, Figure 6(d)).
No differences regarding the gene expression of BGLAP (osteocalcin) and RUNX (cbfa1) between the groups were observed (Figures 6(e) and 6(f)).

Discussion
In the present study we demonstrated that EPC can be cultured successfully on the processed human bone allograft Tutoplast, alone and in combination with MSC.
A decline in the number of EPCs and endothelial function (DiLDL uptake) was observed over the observation period but generally the endothelial properties measured were maintained, but to a reduced level. The addition of osteogenic substances and the coculture with MSC lead to an impairment of endothelial characteristics.
The number of adhering MSCs also declined significantly over time whereas the metabolic activity increased significantly with osteogenic stimulation. This was associated with   a decrease of osteogenic differentiation (reduced collagen-1 gene expression) and an increase of VEGF gene expression.

Clinical Use of Cancellous Bone
Allografts. In a recent study the outcomes of treatment with processed cancellous bone allografts (Tutoplast) versus autologous bone graft obtained from the iliac crest were compared in patients with comminuted distal radius fractures. Over 70% of the patients had good to excellent outcomes in both groups [28]. This study demonstrated that processed human cancellous bone allografts can be as effective as autologous bone grafts for treating distal radius fractures [28]. In another study cranioplasty using the Tutoplast technology for autogenic bone processing was compared to conventional polymethylmethacrylate (PMMA) calvarial reconstruction. In general comparable results were achieved with the autogenic bone but in young patients bone resorption occurred [29].

Role Surface Properties Play in Cell Adhesion and Function.
The favourable results with Tutoplast in clinical settings attributed this effect to its "natural" surface characteristics. Such a surface is thought to promote ingrowth, adherence, and migration of bone forming and other supplementary cells. Studies analyzing the influence of different biomaterials on cellular function demonstrate that cell-to-cell and cell-tomatrix interactions are key to bone formation [30,31].
A recent in vitro study demonstrated that different methods of sterilizing bone grafts significantly alter MSC adhesion, proliferation, and differentiation [24]. In a former study and in the present study we confirm and extend those findings. We found that the number and metabolic activity of MSCs adhering to Tutoplast were superior to the synthetic materials tested [20]. Our SEM analysis revealed that Tutoplast's surface, consisting of a dense mesh of collagen fibrils (Figure 1(b)), is suitable for the adherence of MSC.
Niemeyer and colleagues reported a higher seeding efficiency of MSC on collagen-1 scaffolds coated with hydroxyapatite in comparison to a -TCP matrix. They also observed that MSC cultured on the collagen scaffold displayed a higher gene expression of bone sialoprotein, BMP-2, ALP, and osteocalcin when compared to MSC cultured on uncoated -TCP [32]. We also found a significant improvement in adhesion and metabolic activity of MSC cultured on a collagen scaffold (Tutoplast) versus synthetic scaffolds such as -TCP and -TCP [20].

EPC on Tutoplast.
In the present study we observed a significant decline in EPC adherence to Tutoplast over time. The relative number of adhering cells (as assessed by fluorescence microscopy) was approximately halved over the 10-day observation period. During the same time period EPC endothelial characteristics declined but persisted.
The observed decline of the DiLDL uptake was possibly due to the cultivation medium used. In order to meet the requirements of both EPC and MSC, that is, to maintain endothelial differentiation of EPC and prevent endothelial differentiation of MSC, the medium consisted of one part of EGM + EGM2 singlequots and two parts of MesenCult.
The EGM2 singlequots contains various growth and differentiating factors such as VEGF and insulin-like growth factor (IGF). Due to the addition of MesenCult the concentrations of both factors were reduced to one third. Both factors were necessary to maintain the state of differentiation of EPC and support the DiLDL uptake of EPC (VEGF: [33], IGF: [34]). In previous work we demonstrated that MSC cultured with this mixture did not express endothelial characteristics with the exception of VEGF gene expression [25].
Interestingly, we observed a significant increase of vWF gene expression in EPC on Tutoplast, perhaps due to the collagen surrounding. Usami et al. as well as Schmeisser et al. cultured early EPC in collagen hydrogels for at least 14 days and observed a significant vWF expression. However, no comparisons to other matrices were performed [25,35,36]. To our knowledge no other report is available that describes the regulation of early EPC cultivated on a dense 3D collagen-1 surface.

Effect of Osteogenic Conditions on MSC Cultured on
Tutoplast. In the present study we observed that the number of MSC was equal under osteogenic conditions; however, their metabolic activity was significantly increased. This latter observation could be due to the transformation to an osteogenic phenotype which might be associated with a raised biosynthesis of peptides. Interestingly, the increase in metabolic activity was not accompanied by an elevated gene expression of collagen-1 . It was reported for dermal fibroblasts that the collagen synthesis is negatively regulated by interaction with collagen fibers via a certain collagen receptor, and mesenchymal stem cells also express collagen receptors [37,38]. Thus, it is feasible to assume that the collagen biosynthesis underlies a similar negative feedback loop in MSC seeded onto Tutoplast.

Effect of Osteogenic Conditions on EPC Cultured on
Tutoplast. Osteogenic substances improve the bone forming capacity of MSC but may harm the endothelial differentiation of EPC. Less information is available about the influence of osteogenic differentiation on early EPC. The current study demonstrated that the endothelial differentiation of early EPC on Tutoplast is partly impaired under osteogenic conditions. The DiLDL uptake was decreased under osteogenic conditions, and the gene expression of vWF tended to decline but remained still higher compared to reference EPC cultivated on a fibronectin-coated surface. Gene expression of VEGF tended to increase on Tutoplast under osteogenic conditions. The perpetuation of an endothelial cell-like phenotype under osteogenic conditions underlines the missing potential of early EPC for osteogenic transdifferentiation.
In one study the effect of osteogenic stimuli on CD133 expressing late EPC was evaluated. The authors described a transformation to a osteogenic phenotype [39]. However as described in the introduction those cells differ in many respects from early EPC used in the present work (for review [40]).

Differentiation in Coculture, Gene Expression Analysis.
In coculture a significant decline of the vWF gene expression was observed in comparison to EPC cultured alone on Tutoplast. In coculture experiments a mixture consisting of each 2.5 × 10 5 MSC and 2.5 × 10 5 EPC was seeded; in single culture experiments 5 × 10 5 EPCs were used. Hence, the number of EPCs was halved in comparison to singleculture experiments. This may explain the decline of vWF gene expression observed in coculture experiments. Since the vWF-gene expression of MSC is rather neglectable (10% of vWF gene expression in EPC) [25], it is feasible to assume that EPCs are the main producers of vWF mRNA in our coculture experiments.
The collagen-1 expression was significantly enhanced in coculture experiments. Since our own previous data demonstrated that early EPCs do not express collagen-1 [25], the increase of collagen-1 mRNA could be attributed to MSC.
The increase of collagen-1 gene expression in our coculture experiments might be due to a release of osteogenic substances by EPC. A similar observation was made by Kaigler et al. They described the osteogenic stimulation of MSCs which were cocultured with endothelial cells and identified BMP2 released by the endothelial cells as a responsible factor for the osteogenic differentiation [41].
With regard to our study it is feasible to assume that early EPCs also release osteogenic substances (i.e., BMPs) or substances which foster the collagen synthesis (i.e., TGF-) of MSC. However, if early EPCs release BMP-2 and/or TGF-, this is still not known.

Conclusion
The possibility for cell-based therapy of large bone defects gains increased attention, and it has been demonstrated that the combination of MSC and EPC leads to a superior bone healing response in comparison to single-cell populations in different animal models [5,36]. Additionally, other studies suggest an additional beneficial effect of an osteogenic differentiation of the MSC prior the implantation to the host [6][7][8]. However, less attention has been drawn to the carrier material to immobilize regenerative cells to the defect zone. Recent studies of our group suggest that processed cancellous bone chips are superior to synthetic bone grafts in terms of adhesion and function of MSC [20].
The current study extends those findings. The study provides evidence that the combined cultivation of two types of progenitor cells on a mineralized bone matrix for the purpose of tissue engineering is technically possible. The early EPCs maintain endothelial differentiation even under osteogenic conditions though endothelial activity was found to be reduced. Moreover, our results suggest that concomitant early EPCs stabilize the collagen-1 synthesis of MSC which might be beneficial in bone healing. However, wether MSC and EPC seeded together on a cancellous bone granules improve the bone healing has to be proved in experimental bone healing models.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Role of insulin-like growth factor binding protein-3 in glucose and lipid metabolism

Insulin-like growth factor binding protein (IGFBP)-3 has roles in modulating the effect of IGFs by binding to IGFs and inhibiting cell proliferation in an IGF-independent manner. Although recent studies have been reported that IGFBP-3 has also roles in metabolic regulation, their exact roles in adipose tissue are poorly understood. In this review, we summarized the studies about the biological roles in glucose and lipid metabolism. IGFBP-3 overexpression in transgenic mice suggested that IGFBP-3 results in glucose intolerance, and insulin resistance. IGFBP-3 knockout (KO) mice exhibited normal insulin level and glucose response after glucose challenge. More recent study in IGFBP-3 KO mice with a high-fat diet demonstrated that IGFBP-3 KO mice exhibited elevated fasting glucose and insulin, but normal response to glucose challenge, suggesting that IGFBP-3 KO mice may induce insulin resistance even though preserved insulin sensitivity. In vitro and in vivo studies using 3T3-L1 adipocytes and rat, IGFBP-3 induced insulin resistance by inhibiting glucose uptake. In contrast, the reduced levels of IGFBP-3 in obesity might induce insulin resistance by suppression of IGFBP-3's anti-inflammatory function, suggesting IGFBP-3 has a protective effect on insulin resistance. Also, proteolysis of IGFBP-3 might contribute to the insulin resistance in obesity and type 2 diabetes mellitus. In addition, IGFBP-3 inhibited adipocyte differentiation, suggesting IGFBP-3 may contribute to the insulin insensitivity. Taken together, it is not yet certain that IGFBP-3 has a protective effect or enhancing effect on insulin resistance, and more studies will be needed to clarify the roles of IGFBP-3 in metabolic regulation.

Introduction
Insulin-like growth factor binding protein (IGFBP)-3 is the most abundant IGFBP in serum, and comprises 150-kDa ternary complex with insulin-like growth factor (IGF)-I and an acid labile subunit (ALS), thereby prolonging the half-life of IGF-I in circulation. The major function of IGFBP-3 has been proposed to involve IGF binding and modulate the diverse biological actions of the IGFs, such as cell proliferation, differentiation, an increase in metabolic activity, and cell survival by regulating their access to the IGF-I receptor [1][2][3] . Additionally, IGFBP-3 exhibits clear and distinct biological effects, including inhibition of cell proliferation and inducing apoptosis in various cell types in an IGF-independent manner [4][5][6][7][8] . Mechanisms for this IGF-independent actions of IGFBP-3 have been demonstrated that IGFBP-3 increases the ratio of proapoptotic to antiapoptotic proteins 9) , or activates the caspases involved in a death receptor-mediated pathway 10) to induce apoptosis, and arrests cell cycle in G1/S phase to inhibit cell growth 11) .
Recently, issue about the possible role of IGFBP-3 in metabolic regulation has been raised. Apart from conventional concept indicating that IGFBP-3 was used to involve in meta-bolic regulation by modulating IGF bioavailability, several studies have been reported that IGFBP-3 www.e-apem.org may have metabolic role independent of the IGF axis. However, most part of the exact role of IGFBP-3 in glucose and lipid metabolism remains poorly understood. In this review, focus will be placed on the actions of IGFBP-3 in glucose and lipid metabolism.

IGFBP-3-overexpressing transgenic mice study
IGFBP-3 overexpression in transgenic (Tg) mice in early studies exhibited little effect on birth weight, litter size and body weight other than some selective effect on organ growth, such as spleen, liver and heart 12,13) . However, these studies were accomplished in conditions showing only modest increase of IGFBP-3 expression. Use of the mouse phosphoglycerate kinase I and the cytomegalovirus promoters to overexpress IGFBP-3 more effectively demonstrated 4.9-to 7.7-fold increase in serum level of IGFBP-3, and resulted in modest intrauterine and postnatal growth retardation, probably because of reduced IGF-I bioavailability, and showed heavier liver and epididymal fat masses compared with wild-type (WT) mice 14) . This Tg mice also showed dramatic metabolic changes: increased levels of fasting glucose and insulin, increased response to a glucose challenge, attenuated hypoglycemic effects of insulin and IGF-I, and reduced uptake of 2-deoxyglucose in muscle and adipose tissue 15) . These data suggest that overexpression of IGFBP-3 results in fasting hyperglycemia, glucose intolerance, and insulin resistance. However, we have to be cautious to interpret these data on the basis of the finding that environment by overexpression of IGFBP-3 in Tg mice may not be expected in real body.

IGFBP-3-knockout (KO) mice study
It has been reported that body weight of IGFBP-3 KO mice was indistinguishable from WT, demonstrating that pre-natal and post-natal growth was not affected by IGFBP-3 KO 16) . The response to a glucose challenge in IGFBP-3 KO mice was same as that in WT. However, IGFBP-3-4-5 triple KO mice showed lower glucose and higher insulin levels after glucose challenge compared with WT mice, indicating that IGFBP-3-4-5 triple KO mice have increased glucose uptake and enhanced glucose clearance. Another study performed phenotypic comparisons of controls and four mouse lines with genetic mutants: liverspecific IGF-I deficiency (LID), ALS KO (ALSKO), IGFBP-3 KO (BP3), and a triply deficient LID/ALSKO/BP3 line (LAB) 17) . This study demonstrated that LID and LAB mice showed increased levels of serum insulin, probably due to increases in growth hormone (GH) and secondary insulin resistance. ALSKO mice exhibited increased glucose clearance on glucose tolerance test, reflecting increase of β-cell function and insulin sensitivity, while LID and LAB mice showed reduced clearance. In contrast, IGFBP-3 KO mice exhibited normal insulin level and glucose response after glucose challenge. More recent study in IGFBP-3 KO mice with a high-fat diet demonstrated that IGFBP-3 KO mice exhibited heavier weight, decreased resting metabolic rate, and elevated fasting glucose and insulin levels 18) . However, IGFBP-3 KO mice had relatively normal glucose response after glucose challenge, and increased basal hepatic glucose production, but after insulin stimulation during hyperinsulinemic clamps, indicating that IGFBP-3 KO mice preserve insulin sensitivity despite evidence of increased basal glucose turnover.
Data from several studies did not show solid and consistent findings about the effect of IGFBP-3 KO on glucose metabolism. This discrepancy may come from many compounding and complicated factors in these studies. For example, IGF system is comprised of IGFs, type I and II IGF receptors, IGFBPs, and IGFBP proteases, and is working in a very complicated ways affecting influences on each other. Moreover, the prevalence of IGF-I complexes composing with IGF-I, ALS, and IGFBPs in serum and/or in tissues produces different effects on GH secretion and carbohydrate metabolism. Also, there is a possibility that changes in expression and bioactivity of IGFBP-3 in extrahepatic tissues can affect carbohydrate metabolism 17) . Moreover, each experiment was done in different conditions, such as diet (fasting, fed with normal chow, or fed high-fat diet), and age at the time of experiment. Nevertheless, it is of note that IGFBP-3 KO mice with a high-fat diet had heavier weight compared with WT and showed fasting hyperglycemia and hyperinsulinemia, indicating insulin resistance, even not in glucose tolerance test 18) .

In vitro and in vivo study
It has been reported that IGFBP-3 may lead to insulin resistance in adipocytes 19) . In this report, IGFBP-3 reduced insulin-stimulated but not basal glucose uptake in 3T3-L1 adipocytes independent of the IGF axis. IGFBP-3 also reduced insulin-stimulated glucose transporter-4 translocation to the plasma membrane. This inhibitory effect of IGFBP-3 was accomplished through the decrease in threonine phosphorylation of Akt, but not insulin receptor phosphorylation. Another study also reported that IGFBP-3 induces insulin resistance in adipocytes in vitro and in rats in vivo 20) . Kim et al. 20) reported that IGFBP-3 inhibits basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes through the decrease in insulin receptor phosphorylation, and proposed that a possible role for IGFBP-3 in cytokine-induced insulin resistance. They also demonstrated that infusion of IGFBP-3 to rats for 3 hours or 7 days inhibits peripheral glucose uptake and glycogen synthesis suggesting in vivo effect of IGFBP-3.
In contrast, a recent study reported that IGFBP-3 inhibits cytokine-induced insulin resistance and early manifestations of atherosclerosis 21) . Mohanraj et al. 21) demonstrated that IGFBP-3 inhibits tumor necrosis factor (TNF)-α-induced nuclear factor (NF)-κB activity in an IGF-independent manner, thereby restoring the deregulated insulin signaling and negating TNF-α-induced inhibition of glucose uptake. IGFBP-3 further inhibits TNF-α, C-reactive protein and high glucose-induced NF-κB activity in human aortic endothelial cells (HAECs) and subsequently suppresses monocyte adhesion to HAEC through the IGFBP-3 receptor. This study suggested that IGFBP-3 may have a distinct function in pathologic or disease state. This study also exhibited that IGFBP-3 dose not affect insulin-induced glucose uptake even under normal conditions. Another study showing that diet-induced obese rats exhibited a downregulation of IGFBP-3 expression in mesenteric fat suggests that IGFBP-3 may have a protective effect against obesity 22) .
IGFBP-3 may have a regulatory function on lipid metabolism. Chan et al. 23) reported that IGFBP-3 interferes with the peroxisome proliferator-activated receptor (PPAR)γ-dependent process of adipocyte differentiation and maintenance of the gene expression characteristic of mature adipocytes. These changes were associated with a decrease in lipid accumulation 23) and adiponectin expression 20) in cells treated with IGFBP-3 during differentiation. Further, IGFBP-3 activates inhibitory Smad signaling in 3T3-L1 cells and that endogenous IGFBP-3 modulates their adipogenic differentiation by regulating cell sensitivity towards the inhibitory effect of transforming growth factor-β 24) . Since adipocyte differentiation is required to mediate insulin sensitivity in adipocytes 25) , this inhibitory effect of IGFBP-3 on adipocyte differentiation may contribute to progression towards insulin resistance. In contrast, Grohmann et al. 26) reported that IGFBP-3 as well as IGF-I stimulates glycerol-3-phosphate dehydrogenase activity during differentiation of preadipocytes isolated from adipose tissue of children, indicating that IGFBP-3 enhances adipocyte differentiation. It is not yet determined whether IGFBP-3 can be either inhibitory or stimulatory to the cell differentiation according to the cell types or culture conditions, and the mechanisms of such a divergence of the IGFBP-3' s effect are poorly understood 27) .

Study about IGFBP-3 proteolysis
Proteolysis of IGFBP-3 may contribute to the insulin resistance of pregnancy, obesity, and type 2 diabetes mellitus. Yamanaka et al. 28) demonstrated that NH 2 -terminal fragment of IGFBP-3 binds insulin with high affinity and blocks insulin action by inhibiting autophosphorylation of the insulin receptor β subunit and phosphorylation of insulin receptor substrate-1. The conserved NH 2 -terminal and COOH-terminal sequences of IGFBP-3 and the appropriated ternary structures formed by disulfide bonds result in high affinity for IGFs but low affinity for insulin. When IGFBP-3 is reduced or fragmented, the insulin binding site at the NH 2 -terminus of IGFBP-3 is exposed, resulting in increased affinity for insulin and diminished affinity for IGFs 28) . Increased proteolysis of IGFBP-3 has been observed in serum from patients with type 2 diabetes mellitus 29) , and simple obesity 21,30) . Mohanraj et al. 21) reported that proteolyzed IGFBP-3 positively correlates with adiposity parameters such as waist circumference, body mass index, fasting insulin, and insulin resistance index such as homeostasis model assessment of insulin resistance in overweight and obesity population. These studies suggest that increased proteolytic fragments of IGFBP-3 in obese individuals probably induces insulin resistance by resulting in reduced serum levels of insulin thereby inhibiting insulin action, and reduced levels of intact IGFBP-3 thereby inhibiting its IGF-independent anti-inflammatory functions.

Conclusions
There is a line of evidence that IGFBP-3 plays an important role in glucose and lipid metabolism. However, previous studies about the roles of IGFBP-3 in metabolic regulation did not provide convincing evidence. It is not yet certain that IGFBP-3 has a protective effect or enhancing effect on inducing insulin resistance. Further, the mechanism of the effect of IGFBP-3 in regulating metabolism has to be elucidated. More clinical, epidemiological, and biochemical studies will be needed to clarify the roles of IGFBP-3 in glucose and lipid metabolism.


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Serum from humans on long-term calorie restriction enhances stress resistance in cell culture.

Calorie restriction (CR) without malnutrition is the most robust intervention to slow aging and extend healthy lifespan in experimental model organisms. Several metabolic and molecular adaptations have been hypothesized to play a role in mediating the anti-aging effects of CR, including enhanced stress resistance, reduced oxidative stress and several neuroendocrine modifications. However, little is known about the independent effect of circulating factors in modulating key molecular pathways. In this study, we used sera collected from individuals practicing long-term CR and from age- and sex-matched individuals on a typical US diet to culture human primary fibroblasts and assess the effects on gene expression and stress resistance. We show that treatment of cultured cells with CR sera caused increased expression of stress-response genes and enhanced tolerance to oxidants. Cells cultured in serum from CR individuals showed a 30% increase in resistance to H2O2 damage. Consistently, SOD2 and GPX1 mRNA, two key endogenous antioxidant enzymes, were increased by 2 and 2.5 folds respectively in cells cultured with CR sera. These cellular and molecular adaptations mirror some of the key effects of CR in animals, and further suggest that circulating factors contribute to the CR-mediated protection against oxidative stress and stress-response in humans as well.

and tumorigenesis of epithelial cells, and by inhibiting stem cell function and antioxidant capacity [8]. It is our working hypothesis that CR by changing factors present in the systemic milieu (i.e. serum) can influence cell function by modulating key signaling pathways.
To test this hypothesis we have evaluated the effects of serum collected from volunteers practicing long-term CR on global gene expression and on stress resistance in cell culture experiments. In these in vitro experiments, we compared the molecular and cellular effects on human primary fibroblasts cultured of media supplemented with human sera obtained from: 1) healthy lean and weightstable volunteers who were consuming CR diets, with more than adequate intake of protein and vitamins, for an average of 7 years, and 2) age-, sex-matched, non-obese individuals eating Western diets (WD).

Gene expression analysis shows up-regulation of genes involved in NRF2-mediated oxidative stress response
Mammalian fibroblasts have been extensively used to assess global transcriptional changes induced by circulating factors (i.e. serum) on cell function [9]. To test the hypothesis that the CR-induced changes in circu-lating factors can influence cell function, we compared the transcriptional profiles of human primary fibroblasts treated with CR or WD serum. Differentially expressed genes were selected using a moderate t-test approach [10] and multiple test correction [11], and these genes were used to identify transcriptionally altered pathways. Ingenuity Pathways Analysis software (IPA) showed a significant association of a subgroup of 83 genes (Supplementary Table S1) with many oxidative response-related pathways (Fig. 1). In particular, NF-E2-related factor 2 (Nrf2)-mediated oxidative stress response contained 30 genes that were dysregulated in the treated fibroblasts (BH p-value 3.33E-02), and many of them were direct expression effectors of Nrf2 activity (Fig. 2). Meta-analysis performed using a competitive gene set test [12] pinpoints 11 molecular signatures significantly downregulated in a 'self-contained' gene test (Table 2 red Highlights). To further investigate the result of microarray data and confirm the association with oxidative response-related pathways, we performed quantitative real-time PCR for the following genes which were shown by microarray to be detectable and significantly dysregulated: GSTK1, GPX1, SOD2, GSTT1, and IDH2. As shown in Figure 3, relative abundance of message for these genes was significantly higher in the CR group than the control group (p<0.0005) with a fold change difference ≥ 2.2-fold.    www.impactaging.com

Incubation with human CR serum increases stress resistance to hydrogen peroxide in vitro
To confirm the gene expression findings with a functional test, human primary fibroblasts cultured for 48 hours with CR or WD sera were exposed to increasing concentrations of the powerful oxidizing agent, hydrogen peroxide (H 2 O 2 ) for 24 hours. As shown in Figure 4, treatment with H 2 O 2 produced a dose-dependent decline in survival, but fibroblasts cultured with CR serum were significantly protected from the cytotoxic effect of the oxidant. At 600 µM about 67% of cells pretreated with CR serum were viable, while only 37% of cells pretreated with WD serum survived (p< 0.001), and even at higher H 2 O 2 concentrations (800 µM) 32% of the cells pretreated with CR serum survived, while only 10% of the cells exposed to WD serum were still alive (p< 0.01).

DISCUSSION
We report here that some molecular and functional adaptations of CR, such enhanced stress resistance, can be mimicked in vitro by culturing cells with serum from individuals practicing long-term CR without malnutrition. Our findings show that the incubation of human fibroblasts with CR sera induces a transcriptional activation of several anti-oxidant genes along the Nrf2 pathway. In addition, we found that the incubation of fibroblasts with human CR serum is sufficient to increase stress resistance to hydrogen peroxide in vitro. These molecular and cellular adaptations are consistent with findings in CR rodents and monkeys, and may contribute at least in part to the protective anti-aging effects of CR [1,3].
In particular, accumulating data indicate that aging is associated with a down-regulation of stress-response genes and pathways [3]. To test the hypothesis that CR can counteract some of these age-associated detrimental effects in vitro, we first sought to determine if circulating factors of CR sera modify gene expression profiles of fibroblasts, which are the most abundant cell type in connective tissues and form the structural framework of tissues through their secretion of extracellular matrix components. To this end, we cultured human primary fibroblasts with sera from 12 middle-aged (57.8±8.4 yrs), weight-stable very lean (BMI=19.1±1.2 kg/m 2 ) individuals who have been practicing ~30% CR with adequate nutrition (at least 100% of RDI for each nutrient), for 3-20 years, and a control group of 13 non-obese (BMI=25.7±2.3 kg/m 2 ) age-matched sedentary controls eating a typical Western diet. Remarkably, we found that culturing cells with human CR sera for 48-hrs was sufficient to trigger a rapid transcriptional up-regulation of important genes and pathways that control stress resistance. In particular, we found that a highly significant number of transcripts along the Nrf2 pathway were altered by CR, and genes encoding important antioxidant enzymes, such as GSTK1, GSTT1, IDH2, GPX1 and SOD2, were up-regulated at least two fold in the CR group. Nrf2 is a key transcription factor that binds to the antioxidant response element (ARE) in response to environmental carcinogens or low levels of insulin, and increases the transcription of a variety of antioxidative and carcinogen-detoxification enzymes that boost protection against cellular and molecular damage and cancer [14][15][16]. It might be also possible that CR causes oxidative stress and therefore induce a NRF2-dependent response, supporting the hypothesis that oxidative damage is not a cause of aging [17][18][19].
In order to further investigate the role of CR sera in modifying the resistance of cells to oxidative stress, we next performed a series of experiments in which we exposed human primary fibroblasts to increasing concentrations of H 2 O 2, a powerful oxidizing agent. Our data indicate that incubating fibroblasts with CR sera exerts a rapid and profound protection against hydrogen peroxide-induced cytotoxicity. These findings are consistent with data from studies in mice, suggesting that these selected cellular adaptations induced by circulating factors are conserved among species [4]. Indeed, most of the metabolic and hormonal parameters examined in CR humans are similar to those reported in CR rodents [20][21][22].
In conclusion, our findings reported here show that enhanced stress responsiveness known to occur in animals on CR following oxidative stress can be reproduced in vitro by culturing cells using serum from CR patients. Our findings provide support for a possible neuroendocrine based mechanism of CR and offer a model for the screening and investigation of CR mimetic agents. We anticipate that this novel in vitro model will be instrumental in the elucidation of biochemical, molecular and cellular mechanisms underlying the anti-aging effects of CR in humans.

MATERIALS AND METHODS
Human subjects. Human serum samples were collected in the morning after an overnight fast from twelve individuals of the Calorie Restriction Society who had been practicing severe CR with adequate nutrition for an average of 7 years (range 3-20 years). Thirteen healthy, non-obese and sedentary (regular exercise < 1 h per week) individuals eating typical Western Diets (WD) were used as a sedentary comparison group; they were matched with the CR group in terms of age, sex and height. Whole blood was collected into sterile vacutainer tubes, allowed to clot for 30 minutes and then centrifuged. The serum was then stored at -80 o C. The characteristics of the participants are shown in Table 1. All of the study participants were weight stable, i.e. < 2 kg weight change in the preceding 6 months. None of the participants had evidence of chronic disease (including cardiovascular, lung, gastrointestinal, type 2 diabetes and cancer). None were smokers. This study was approved by the Human Studies Committee of Washington University School of Medicine, and all participants gave informed consent before their participation.
Cell culture. The BJ human fibroblast cell line was obtained from ATCC and used in these experiments. BJ cells were cultured in 75 cm 2 culture flasks containing basic culture medium: 86% of E-MEM medium supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 1% Sodium Pyruvate, 1% non-essential amino acids, 1% GlutaMAX and 1% antibiotics (GIBCO, Carlsbad, CA, USA) until the time of treatment. Cells were cultured in a humidified incubator at 37 °C and 5% CO 2 . For the experiments, FBS was replaced with human serum from CR and WD participants. Both FBS and human serum were heat inactivated for 30 minutes at 55 °C.
Microarray analysis. BJ cells were seeded at a concentration of 4.000 cells/cm 2 in 25 T-25 flasks; when they reached 60% of confluence, the cells were starved for 24h without FBS. After starvation, 10% of human serum from 12 CR and 13 WD individuals was added to the cultures for 48h. Whole cells were detached from the flask with trypsin/EDTA (Sigma-Aldrich, St. Louis, MO, USA) and Total RNA was extracted using the mirVANA (Ambion, Inc, Austin, TX, USA) according to manufacturer's protocol. Integrity of total RNA was evaluated using capillary electrophoresis (Bioanalyzer 2100, Agilent technologies, Santa Clara, CA, USA) and quantified using a Nanodrop 1000 (Nanodrop, Wilmington, DE). Aliquots of RNA (400 ng) samples were amplified according to the specifications of the Illumina® TotalPrep™ RNA Amplification Kit (Ambion, Austin, TX, USA) to produce a pool of biotin-labeled RNA corresponding to the polyadenylated (mRNA) fraction. The cRNA samples were applied to the arrays of Illumina whole-genome HumanHT-12 v 3.0 (Illumina, San Diego, CA, USA) and hybridized according to the manufacturer's specification. Each array on the BeadChip targets over 25.000 annotated genes using 3micron beads bearing covalently attached 50-base oligonucleotide probes. Each probe interrogates a single gene, and each bead type is represented with an average 15-fold redundancy on every array. The BeadChips were scanned with the Illumina's Beadarray system scanner (Illumina, San Diego, CA, USA). The hybridization images signal intensity was extracted and background subtracted using Illumina Inc. BeadStudio software version 3.3.7. The produced data were checked for the Illumina internal quality control and loaded into Bioconductor software [23,24] for statistical analysis using lumi package [25] (see below). The data discussed in this publication have been deposited in www.impactaging.com NCBI's Gene Expression Omnibus [26] and are accessible through GEO Series accession number GSE41790.(http://www.ncbi.nlm.nih.gov/geo/query/acc .cgi?token=nnonbsicqouiilw&acc=GSE41790).
Real Time PCR. Real-time PCR was used to verify microarray data. Total RNA was DNase I treated and reverse transcribed using the High Capacity RNA-to-cDNA kit (Applied Biosystems, Carlsbad, CA, USA). The cDNA was then used to evaluate the relative expression of select genes including SOD2, GPX1, IDH2, GSTK1 and GSTT1 using specific TaqMan Gene Expression Assays (Applied Biosystems, Carlsbad, CA, USA), which are pre-designed and preoptimized gene-specific probe sets. DNA amplification was performed using the Applied Biosystems 7500 Fast Real-Time PCR system according to the manufacturer's protocol. The relative amount was calculated from the threshold cycles with the instrument's software (SDS 2.0, Applied Biosystems, Carlsbad, CA , USA), according to the manufacturer's instructions. We used the comparative cycle threshold (Ct) method (2 -CT) to calculate relative gene expression under experimental and control conditions normalized to GAPDH. The results are expressed either as the abundance of the gene of interest relative to the house keeping gene (2 -CT), or as fold change over control values (2 -CT).
Cell Proliferation assays in response to Hydrogen Peroxide. To determine the Differential Stress Resistance to hydrogen peroxide of serum from CR and WD individuals, human fibroblasts were seeded in 96-well plates at a concentration of 3000 cells/well in 86% D-MEM low glucose w/o phenol red supplemented with 1% Sodium Pyruvate, 1% nonessential amino acids , 1% GlutaMAX and 1% antibiotics (GIBCO, Carlsbad, CA, USA). After cells had seeded, media was removed and replaced with media containing 10% of human serum from 11 CR and 11 WD individuals was added in triplicate to the plates for 48hs. After incubation, freshly prepared hydrogen peroxide was added to the plates at a concentration of 200, 400, 600, 800, 1000 µM and cells were challenged for 24hs.
Cell viability was determined by the addition of 20 µl of the ready-to-use WST1 reagent (Roche diagnostics, Mannheim, Germany) to each well; the cells were incubated for approximately 3 hours at 37 °C and 5% CO 2 in a humidified incubator. After this incubation period, the absorbance of the formazan dye formed was measured at a wavelength of 450 nm using a microplate spectrometer. The measured absorbance directly correlates to the number of viable cells.
Statistical analysis. Microarray data produced were quality checked with arrayQualityMetrics [24] package under R version 2.15.1 Bioconductor software [23] and undetectable and low detection value probes were filtered out from further analysis accordingly to the lumi package standards [25]. To identify differentially expressed genes, based on a moderate t-test, the Limma package [10] was used and genes were selected based on a p-value cut-off <0.05 (after FDR adjustment) and absolute fold change >1 following the Macroarrays Quality Control Program criteria [11]. IPA software was then used (www.ingenuity.com) to detect the enrichment of biofunctions and networks in the resulting list. Core analysis was performed using the following settings: Reference set: HumanHT-12 v 4.0 [v3 is mentioned in the methods]; Relationship to include: Direct and Indirect; Includes Endogenous Chemicals; Filter Summary: Consider only molecules and/or relationships where (species = Uncategorized (e.g. chemicals) OR Human) AND (confidence = Experimentally Observed). Canonical pathways analysis has been performed using as scoring method B-H Multiple Testing Correction p-values set to a threshold value of 0.05.
Meta-analysis was performed using the CAMERA function implemented in the Bioconductor Limma package for a 'competitive gene set test' [12]. The resulting top Molecular signatures have been further analyzed using a 'focused' gene set test ROAST [27]. All the list of genes organized as Signature and present in The Molecular Signature Database C2 collection (MSigDB) (www.broadinstitute.org/gsea/msigdb/) were updated to official symbols using the illumina annotation file HumanHT-12_V3_0_R2_11283641_A, while the CR signatures were created according to the Table2 of 3 and M.musculus to H.sapiens conversion obtained using the NCBI HomoloGene tool (http://www.ncbi.nlm.nih.gov/homologene).
Representation of differentially expressed genes in their Genomic localization and visual representation of their relationships with the different pathways were produced using CIRCOS software [28]. Statistical analysis of real time PCR and cell proliferation data was performed using Student's t test to compare the transcripts relative abundance in the CR group and WD group. P values <0.05 were considered statistically significant.


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Role of SUMO-1 and SUMO interacting motifs in rhesus TRIM5α-mediated restriction

Background TRIM5α is a member of the tripartite motif family of proteins that restricts retroviral infection in a species-specific manner. The restriction requires an interaction between the viral capsid lattice and the B30.2/SPRY domain of TRIM5α. Previously, we determined that two SUMO interacting motifs (SIMs) present in the B30.2/SPRY domain of human TRIM5α (huTRIM5α) were important for the restriction of N-tropic Murine Leukemia Virus. Here, we examined whether SUMO expression and the SIM1 and SIM2 motifs in rhesus monkey TRIM5α (rhTRIM5α) are similarly important for Human Immunodeficiency Type 1 (HIV-) restriction. Results We found that mutation of SIM1 and SIM2 of rhTRIM5α abolished the restriction of HIV-1 virus. Further, knockdown of SUMO-1 in rhTRIM5α expressing cells abolished restriction of HIV-1. These results may be due, in part, to the ability of SUMO-1 to stabilize rhTRIM5α protein expression, as SUMO-1 knockdown increased rhTRIM5α turnover and the mutations in SIM1 and SIM2 led to more rapid degradation than the wild type protein. The NF-κB signaling ability of rhTRIM5α was also attenuated by SUMO-1 knockdown. Finally, upon inhibition of CRM1-dependent nuclear export with Leptomycin B (LMB), wild type rhTRIM5α localized to SUMO-1 bodies in the nucleus, while the SIM1 and SIM2 mutants did not localize to SUMO-1. Conclusions Our results suggest that the rhTRIM5α B30.2/SPRY domain is not only important for the recognition of the HIV-1 CA, but it is also important for its association with SUMO-1 or SUMO-1 modified proteins. These interactions help to maintain TRIM5α protein levels and its nuclear localization into specific nuclear bodies.

Small ubiquitin-related modifier (SUMO) proteins are conjugated to cellular substrates and regulate diverse cellular processes (for review see [9,10]). SUMO proteins are transferred to lysine residues within the UBC9 binding site of the target protein. This binding site has a consensus sequence ΨKXE (where Ψ is a hydrophobic residue, K is the lysine to which SUMO-1 is conjugated, X is any amino acid and E is glutamic acid) [11,12]. Conjugation of SUMO proteins to a substrate mediates distinct protein-protein interactions in vivo. These non-covalent interactions with SUMO modified proteins are mediated by SUMO interacting motifs (SIMs) [11,13,14]. The best-characterized SIMs have the consensus sequence V/I/L-x-V/I/L-V/I/L or V/I/ L-V/I/L-x-V/I/L (where x is any amino acid) [14,15].
We have previously identified that SUMO conjugation system is involved in huTRIM5α-mediated restriction of N-MLV [16]. Three SIMs were identified in the huTRIM5α B30.2/SPRY domain. Two of them, SIM1 and SIM2, were responsible for the enhanced restriction of N-MLV in human cells observed upon SUMO-1 overexpression. Specifically, mutation of SIM1 and SIM2 resulted in a dramatic decrease in the ability of huTRIM5α to restrict N-MLV infection. The SIMs are conserved among TRIM5α orthologs, as the same SIM mutations also affected the rhTRIM5α restriction of N-MLV [16]. As such, we hypothesized that rhTRIM5α restriction of HIV-1 was similarly dependent on SUMO-1 expression and the SIM1 and SIM2 motifs of rhTRIM5α.
Here we report that SIMs are important for HIV-1 restriction, and that knockdown of SUMO-1 in rhTRIM5α expressing cells drastically reduces HIV-1 restriction. We hypothesize that the presence of SUMO-1 stabilizes proteins levels of rhTRIM5α, as knockdown of SUMO-1 decreases steady state levels of rhTRIM5α and NF-κB activation. Additionally, mutation of the SIMs abolishes the co-localization of rhTRIM5α with promyelocytic leukemia protein (PML, also known as TRIM19)/SUMO-1 in the nucleus upon LMB treatment. Our results suggest that the rhTRIM5α B30.2/SPRY domain is not only important for the recognition of the HIV-1 CA, but that it is also important for its association with SUMO-1 or with SUMO-1 modified proteins. This association helps maintain TRIM5α protein levels, as well as its ability to mediate NF-κB, and nuclear localization into specific nuclear bodies.

Results and discussion
Mutations in rhTRIM5α SIM1 and SIM2 motifs abolish HIV-1 restriction To explore the importance of rhTRIM5α SIMs in restriction of HIV-1, we generated CRFK, HeLa, and TE671 cell lines stably expressing comparable levels of FLAG-tagged wild type rhTRIM5α or the rhTRIM5α variants with  interacting motifs are important for HIV-1  restriction by rhesus macaque TRIM5α. CRFK, TE671 and HeLa  cells were transduced with a retroviral vector encoding wild type  rhTRIM5α, SIM1mut, SIM2mut and SIM3mut or an empty vector (EV) control. A. Western blot assay showing the expression of the FLAGtagged rhTRIM5α, using an anti-FLAG antibody, GAPDH was used as a loading control. B-C. Cells stably expressing the various rhTRIM5α proteins or an EV control were infected with an HIV-1 firefly luciferase reporter virus pseudotyped with VSV-G envelope. 48 hours after infection luciferase activity was measured. Representative experiment of four independent experiments for CRFK (B), HeLa (C) and TE671 (D) cells. E. The CRFK cells were infected with an N-MLV firefly luciferase reporter virus pseudotyped with VSV-G envelope. Representative experiment of four independent experiments. Error bars show standard deviation between triplicates. mutations in SIM1 (376-379), SIM2 (405-408) and SIM3 (430-433) ( Figure 1A). These cell lines were infected with VSV-G pseudotyped HIV-1 carrying a firefly luciferase reporter gene to assess retroviral restriction. The wild type and SIM3 rhTRIM5α efficiently restricted HIV-1 infection when compared to the empty vector (EV) in CRKF ( Figure 1B) and HeLa cells ( Figure 1C). Conversely, mutation of SIM1 and SIM2 of rhTRIM5α completely abolished the restriction activity in these cell lines ( Figure 1B, C). Similar results were observed in TE671 cells ( Figure 1D), although the expression of the SIM1 and SIM2 mutants was noticeably reduced compared to the expression of wild type rhTRIM5α ( Figure 1A). In all cell lines, mutation of rhTRIM5α lysine 10 to arginine (K10R), a predicted SUMOylation site had minimal effect on restriction, consistent with our previous observations of huTRIM5α and rhTRIM5α N-MLV restriction [16]. Similarly, in CRFK cells, which do not express a functional TRIM5 gene [17], the restriction of N-MLV by rhTRIM5α required a functional SIM1 and SIM2, recapitulating the restriction profile observed for HIV-1 ( Figure 1E). Therefore, SIM1 and SIM2 present in rhTRIM5α are important for its antiviral activity against both N-MLV and HIV-1. Consistent with this observation, another group has recently reported that the SIM1 and SIM2 mutations disrupt the binding of rhTRIM5α to the HIV-1 capsid [18].

Restriction of HIV-1 by rhTRIM5α is reduced following SUMO-1 knockdown
To determine if the ability of rhTRIM5α to restrict HIV-1 infection was dependent on interactions with SUMO-1, we stably knocked down SUMO-1 (SUMO-1 KD) using a SUMO-1 specific shRNA in TE671 cells expressing FLAG-rhTRIM5α or empty vector. A non-silencing shRNA was used as a control. To confirm SUMO-1 KD, we performed quantitative PCR (qPCR) and found that these cells had 70% SUMO-1 KD ( Figure 2B). Control cells transduced with empty vector did not show appreciable differences in HIV-1 infection following SUMO-1 KD (Figure 2A). Cells transduced to express FLAG-rhTRIM5α showed considerable restriction of HIV-1. Notably, following SUMO-1 KD the restriction activity of cells expressing FLAG-rhTRIM5α was reduced to levels similar to cells expressing empty vector ( Figure 2A). This demonstrates that rhTRIM5α restriction of HIV-1 is sensitive to SUMO-1 depletion.

SUMO-1 enhances rhTRIM5α stability in cells
As noted earlier, TE671 cells expressing rhTRIM5α SIM mutants showed reduced expression compared to TE671 cell lines expressing wild type or K10R forms of rhTRIM5α ( Figure 1A). This suggests that disrupting interactions with SUMO-1 may increase the turnover of rhTRIM5α. It was previously reported that knockdown of host cellular proteins which interact with rhTRIM5α increased rhTRIM5α turnover [19]. We therefore examined rhTRIM5α expression levels by Western blot following SUMO-1 siRNA treatment in a HeLa cell line stably expressing HA-rhTRIM5α [8]. In these cells, SUMO-1 KD reduced HA-rhTRIM5α expression (Figure 3), although this reduction did not correlate with the degree of restriction observed ( Figure 2B). Other studies have noted that small alterations in TRIM5α expression do not dramatically affect restriction activity at non-saturating amounts of virus [4,20]. These observations make it difficult to separate the contribution of reduced protein expression and the relief of restriction observed in studies of this type. Therefore, it remains possible that the effects of SUMO-1 KD on restriction are not entirely due to reduced expression of rhTRIM5α.

NF-κB activation by rhTRIM5α is sensitive to SUMO-1 expression
Recent studies have shown that TRIM5 proteins can activate intracellular signaling pathways that culminate in AP-1 and NF-κB activation [21,22]. In order to understand the role of SUMO-1 and SIMs in rhTRIM5α-mediated signaling, we transiently expressed wild type and rhTRIM5α mutants along with an NF-κB luciferase reporter. Both the wild type and SIM3 mutant form of rhTRIM5α were able to activate NF-κB. On the other hand, the SIM1 and SIM2 mutants did not induce significant signaling above background in this context ( Figure 4A, top panel). However, following transient transfection, the protein expression levels of the SIM1 and SIM2 mutants were reduced compared to wild type and SIM3 mutants, possibly explaining the loss of NF-κB activation ( Figure 4A, bottom panel). To assess NF-κB signaling by rhTRIM5α SIM mutants at comparable protein levels, we generated 293A cell lines stably expressing wild type rhTRIM5α and the SIM mutants and measured NF-κB activation in these cells. Under these conditions, when the SIM mutants were expressed at comparable levels to wild type rhTRIM5α, they elicited similar levels of NF-κB activation ( Figure 4B). Consistent with a Figure 4 The role of SIMs and SUMO-1 expression on NF-κB activation by rhTRIM5α. A. 293T cells plated in triplicate were transfected with empty vector (EV), ΔRING/SPRY rhTRIM5α (ΔRS), wild typerhTRIM5α, SIM mutants or RIG-I along with NF-κB-responsive firefly luciferase construct. A renila luciferase construct was used as an internal transfection efficiency control. 48-hours post transfection, cells were lysed and luciferase activity was measured. NF-κB luciferase readings were normalized to renilla luciferase, and plotted as an average fold increase over empty vector. Upper panel, representative luciferase activity in the presence of various constructs.Error bars represent the SEM between the triplicates. Lower panel,representative Western blot. Representative of 4 independent experiments. B. 293A stably expressing wild typerhTRIM5α, K10R mut, SIM mutants with a FLAG-tag or an EV. Cells weretransiently transfected with an NF-κB firefly luciferase and renila luciferase plasmids as in A. Upper panel, NF-κB luciferase activity in the presence of rhTRIM5α mutants. Error bars represent the SD between 3 independent experiments. Lower panel, representative Western blot at the time of data acquisition. C. 293T cells were transfected with Control siRNA or SUMO-1siRNA for 48 hours. Cells were then seeded in a 96-well plate in triplicate and transfected with empty vector or wild type rhTRIM5α. NF-κB activity was measured as in A. Data were plotted by dividing SUMO-1 siRNA activation by control siRNA activation x 100. Inset, representative Western blot. P <0.004 by Student's t-test. Error bars represent the SEM between the triplicates. Data is representative of 3independent experiments. D. 293T cells were transfected with empty vector, and wild type rhTRIM5α constructs in presence and absence of SUMO-1. NF-κB activity was measured as in A. Data were plotted as in C. Error bars represent the SEM between triplicates. Representative of 3 independent experiments.
previous report that showed normal oligomerization of these mutants [18], and the data here that demonstrate the ability of these mutants to activate NF-κB, we conclude that the defect in restriction by rhTRIM5α SIM mutants is not due to gross misfolding of the protein.
We next asked how SUMO-1 depletion affected the ability of wild type rhTRIM5α to induce NF-κB activation. We co-transfected 293T cells with wild type HA-rhTRIM5α, SUMO-1 or control siRNA and an NF-κB driven luciferase reporter. We measured NF-κB activation by rhTRIM5α in SUMO-1 siRNA treated cells compared to rhTRIM5α cells treated with control siRNA. As shown in Figure 4C knocking down SUMO-1 had little effect on NF-κB activation when transfected with EV (~10%, black bar). However, depletion of SUMO-1 significantly reduced (~60%, p<0.004, Student's T-test) NF-κB activation by wild type rhTRIM5α ( Figure 4C, grey bar). The reduction of NF-κB activation in the presence of SUMO-1 siRNA was not due to reduced rhTRIM5α protein levels ( Figure 4C inset) as SUMO-1 knockdown does in cells stably expressing rhTRIM5α (Figure 3). Conversely, overexpression of SUMO-1 increased NF-κB activation following transfection with empty vector or vector expressing wild type rhTRIM5α ( Figure 4D). This increase was more pronounced when rhTRIM5α was present, although this result was not statistically significant (p=0.187, Student T-test). These experiments demonstrate that the ability to associate with SUMO-1 or SUMOylated proteins is relevant to rhTRIM5α-mediated NF-κB signaling.
The SIM1 and SIM2 mutations disrupt rhTRIM5α trafficking to nuclear bodies containing PML and SUMO-1 We next analyzed the association of rhTRIM5α and SUMO-1 by immunofluorescence to verify that SIM1 and SIM2 do not interact with SUMO-1 or SUMO-1 modified proteins. In HeLa cells stably expressing YFP-rhTRIM5α, we examined the co-localization of rhTRIM5α and endogenous SUMO-1. We used two antibodies to SUMO-1 to examine both the cytoplasmic and nuclear fractions of SUMO-1. The first antibody (GMP1, clone 21C7) recognized nuclear SUMO-1 as well as numerous cytoplasmic puncta. However, the cytoplasmic SUMO-1 did not colocalize with YFP-rhTRIM5α ( Figure 5). When these cells were stained with an antibody to PML, we did not observe significant localization of SUMO-1 to PML (data not shown). As SUMO-1 positive structures in the nucleus are well characterized and known to contain PML [23,24], we used a second antibody (clone Y299) that recognized nuclear structures that were PML positive (Data not shown). This antibody detected primarily diffuse and punctate nuclear SUMO-1. We used this antibody in subsequent experiments to examine SIM1 and SIM2 localization with SUMO-1. A recent study demonstrated that while steady state rhTRIM5α is excluded from the nucleus, it can transiently enter and exits the nucleus, where it associates with PML bodies. This nuclear localization of rhTRIM5α to PML bodies is observed when the nuclear export of rhTRIM5α is inhibited with Leptomycin B (LMB), which is an inhibitor of CRM1 mediated nuclear export [25].  Inhibiting the nuclear export of rhTRIM5α using LMB revealed that wt and all three SIM mutants localized to the nucleus ( Figure 6A), contrary to a recent report by another group which found that the SIM1 and SIM2 mutants did not localize to the nucleus under these conditions [18]. However, when we quantified the localization of these mutants to nuclear SUMO-1 bodies, SIM1 and SIM2 mutants of rhTRIM5α failed to localize to SUMO-1 positive bodies when nuclear export is inhibited, while the SIM3 mutant associated with these bodies to an intermediate degree ( Figure 6B). These SUMO-1 positive rhTRIM5α nuclear bodies were also positive for PML (data not shown).

Potential mechanisms of SUMO-1 regulation of rhTRIM5α
Collectively, the data presented here demonstrate that SIMs in rhTRIM5α and SUMO-1 are important for rhTRIM5αmediated retroviral restriction. rhTRIM5α is sensitive to changes in SUMO-1 expression as SUMO-1 knockdown relieves rhTRIM5α restriction ( Figure 2) and increases rhTRIM5α protein turnover in stable cells (Figure 3). Additionally, SUMO-1 modulates rhTRIM5α-dependent activation of NF-κB ( Figure 4). Upon SUMO-1 knockdown, NF-κB activation is decreased in a rhTRIM5α-dependent manner ( Figure 4C). We also observed decreased localization of SIM1 and SIM2 to SUMO-1 puncta following LMB treatment ( Figure 6).
The mechanism by which SUMO-1 stabilizes rhTRIM5α expression or affects retroviral restriction is unclear. It is possible that rhTRIM5α interacts with a protein that stabilizes its expression in a SUMO-dependent manner. It is also possible that the expression of another cellular protein that is stabilized by SUMOylation, stabilizes rhTRIM5α expression. Consistent with this, it is known that SUMOylation can protect some proteins from ubiquitin dependent degradation [26]. Our observation that the SIM1 and SIM2 mutations, which abrogate restriction, are also turned over more rapidly than wild type rhTRIM5α are consistent with all of these possibilities. Interestingly, a recent study by Brandariz-Nunez et al. suggests that these residues are not present on the surface of the SPRY domain [18], as modeled using an NMR structure recently described for the PRY/SPRY domain [27]. It may be that conformational changes induced by the binding of other cellular factors conditionally expose the SIM1 and SIM2 motifs. These authors also observe that SIM1 and SIM2 mutants oligomerize normally [18], suggesting these proteins are not misfolded. While we cannot exclude the possibility that SIM1 and SIM2 mutations prevent proper folding of the B30.2/SPRY domain, our own results demonstrate that alterations in SUMO-1 expression can influence the stability of wild type rhTRIM5α.
The data presented here may begin to explain the mechanistic of rhTRIM5α nuclear trafficking. It is possible that the recruitment of TRIM5α to SUMO-1/PML bodies has direct relevance to the restriction process, as both the SIM1 and SIM2 mutants that do not localize to SUMO-1/PML bodies are completely unable to restrict retroviral infection. Considering that PML protein undergoes SUMOylation on three lysine residues [28][29][30] and rhTRIM5α has a rapid turnover [31], we can speculate that the steady state levels of rhTRIM5α protein are maintained by its interaction via SIMs with SUMOylated-PML in the nucleus. When SIMs or SUMO-1 are not present rhTRIM5α is less retained in the nucleus and therefore is degraded faster in the cytosol, reducing the amount of protein available to restrict incoming viruses.
Interestingly, owl monkey TRIM-Cyp, which contains a C-terminal cyclophilin A domain instead of a B302/SPRY domain (the B30.2/SPRY domain contains all three putative SIMs) [32], does not traffic to the nucleus [25]. Therefore, given the strong homology between the RBCC domains of owl monkey TRIM-Cyp and rhTRIM5α, it is possible that the SPRY domain contains the determinants that govern nuclear trafficking. Consistent with this hypothesis, our data demonstrate that wild type rhTRIM5α, but not the SIM1 and SIM2 mutants, localizes to nuclear SUMO-1 bodies ( Figure 5) support this idea. Although the results we have obtained using the SIM1 and SIM2 mutants were consistent with the effect of SUMO-1 knockdown on wild type rhTRIM5α activity, the results obtained with these mutants should be cautiously interpreted [18].
Future studies are needed to determine if this association is merely correlative or the association of rhTRIM5α with nuclear SUMO-1/PML bodies potentiates specific steps in the restriction process.

Conclusions
Our results demonstrate that rhTRIM5α restriction and expression is governed by SUMO-1 expression. SUMO-1 depletion, or mutations that are expected to disrupt (See figure on previous page.) Figure 6 The SIM1 and SIM2 mutations disrupt rhTRIM5α localization to nuclear bodies containing PML/TRIM19 andSUMO-1. A. HeLa cells stably expressing wild typeYFP-rhTRIM5α or SIM mutants were treated with LMB for 4 hours. Following treatment the cells were fixed and imaged. Z-stack images were collected with a DeltaVision microscope equipped with a digital camera using a 1.4-numerical aperture (NA) 100× objective lens, and were deconvolved with SoftWoRx deconvolution software. Individual channel images were superimposed to create the merged panels. B. HeLa cells expressing wild type YFP-rhTRIM5a or SIM mutants were treated with LMB for 4hours. Following treatment the cells were fixed and stained with an anti-SUMO-1 antibody. Z-stack images were collected as described in A. Deconvolved images were analyzed for YFP-rhTRIM5a maximum fluorescence intensity (MFI) in SUMO-1nuclear bodies by the use of the Surface Finder function in the Imaris software (Bitplane). For each SUMO-1 puncta, the MFI of YFP-rhTRIM5a mutants was determined and the data was plotted in GraphPad Prism 5 W software.
SUMO-1 binding, abrogate restriction and increase the rate of rhTRIM5α turnover. The ability of rhTRIM5α to activate innate immune signaling pathways is also sensitive to SUMO-1 expression. Additionally, the ability to traffic to nuclear SUMO-1 bodies is abrogated by mutation of the SIM1 or SIM2 motifs present in rhTRIM5α. As rhTRIM5α does not appear to itself be SUMOylated, future studies are needed to identify the cellular factor or factors that are likely regulating rhTRIM5α in a SUMO-1 dependent fashion.

Cell lines and plasmids
Human embryonic fibroblast 293T, 293A, human medulloblastoma cell line TE671, HeLa and Crandall feline kidney (CRFK) fibroblast were maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 100 UI/ml penicillin and 100 mg/ml streptomycin. All cells were cultured at 37°C in 5% CO 2 .
The rhTRIM5α constructs encoding the FLAG tagged wild type and mutant versions were identified and cloned as previously described [16]. YFP tagged wild type and mutant versions were cloned into a retroviral vector previously described [33]. RIG-I construct was kindly donated to us by Dr. Susan Baker (Loyola University Chicago).

Analysis of SUMO-1 knock down
In the case of the TE671 cells where SUMO-1 was stably knocked down, the cells were harvested and total RNA was extracted using TRIZOL reagent (Invitrogen). 2 μg of total RNA per cell line were used to produce cDNA using random hexamers and SuperScript III kit (Invitrogen). 2 μl of each cDNA were used for quantitative PCR analysis of SUMO-1 and GAPDH transcript levels. Fold change was calculated using the relative standard curve method.
For the transient knockdown of SUMO-1, HeLa cells stably expressing HA-rhTRIM5α were transfected with siRNA specifically targeting SUMO-1 or a non-targeting siRNA (Santa Cruz Biotechnology). Cells were transfected with Lipofectamine2000 (Invitrogen) using a 2-day transfection protocol followed by 24-hours or 48-hours of rest to gain maximum knockdown efficiency. Cells were collected following the resting period and analyzed by western blot using a monoclonal anti-SUMO-1 antibody (Abcam clone Y299). Densitometry analysis was performed on the western blots using ImageJ software.

Image analysis
20 Z-stack images were acquired using identical acquisition parameters. Surfaces for cytoplasmic bodies in all samples analyzed were defined by using a fluorescence threshold for YFP-rhTRIM5α, and all YFP-rhTRIM5α bodies over a volume of 0.011 μm 3 were used in the analysis. Deconvolved images were analyzed for SUMO-1 maximum fluorescence intensity (MFI) in cytoplasmic bodies using the Surface Finder function of the Imaris software package (Bitplane) and the data was plotted in Prism (Graphpad Software Inc) for statistical analysis.

Dual-luciferase reporter assay SIMs
293T cells seeded in a 96-well plate were transfected with empty vector, ΔRING/SPRY rhTRIM5α (ΔRS, negative control), wild type rhTRIM5α, SIM1 mut, SIM2 mut, SIM3 mut or RIG-1 (positive control) in triplicate. Transfection was carried out using polyethylenimine (PEI) protocol in which the constructs were added at a 9 (EV/ rhTRIM5α/RIG-I): 3 (NF-kB-responsive firefly luciferase construct): 1 (Renilla luciferase construct for transfection efficiencies) ratio. Cells were lysed 48-hours post transfection with Passive lysis buffer (Promega) and the luciferase activity was measured using a Dual-Glo luciferase assay system (Promega) in a Veritas Microplate luminometer. Firefly luciferase data were normalized to Renilla luciferase readings in each well. Data were plotted by determining the fold increase over empty vector.

SUMO-1 overexpression
293T cells seeded in a 96-well plate were transfected with empty vector, and rhTRIM5α constructs in presence or absence of SUMO-1 in triplicate. Transfection was carried out using PEI protocol in which the constructs were added at a 5 (rhTRIM5α): 4 (SUMO-1 or EV): 3 (NF-κB-responsive firefly luciferase construct): 1 (Renilla luciferase construct for normalization of transfection efficiencies) ratio. Cells were lysed 48-hours post transfection with Passive lysis buffer (Promega) and the luciferase activity was measured using a Dual-Glo luciferase assay system (Promega) in a Veritas Microplate luminometer. Firefly luciferase data were normalized to Renilla luciferase readings in each well. Data were plotted by dividing SUMO-1 siRNA NF-κB activation by Control siRNA NF-κB activation x 100.

SUMO-1 knockdown
293T cells seeded in a 12-well plate were transfected with Control siRNA or SUMO-1 siRNA (Santa Cruz Biotechnology, Inc) following a Lipofectamine2000 (Invitrogen) protocol for two days. On the third day the cells were seeded in a 96-well plate in triplicate and transfected with empty vector or wild type rhTRIM5α using PEI in which constructs were added at a 9 (EV/rhTRIM5α): 3 (NF-kB-responsive firefly luciferase construct): 1 (Renilla luciferase construct for transfection efficiencies) ratio. Cells were lysed 48-hours post transfection with Passive lysis buffer (Promega) and the luciferase activity was measured using a Dual-Glo luciferase assay system (Promega) in a Veritas Microplate luminometer. Firefly luciferase data were normalized to Renilla luciferase readings in each well. Data were plotted by dividing SUMO-1 siRNA NF-κB activation by Control siRNA NF-κB activation x 100.


s2fieldsofstudy: Biology Medicine Chemistry

text:
NK cell-released exosomes

We have recently reported that human natural killer (NK) cells release exosomes that express both NK-cell markers and cytotoxic molecules. Similar results were obtained with circulating exosomes from human healthy donors. Both NK-cell derived and circulating exosomes exerted a full functional activity and killed both tumor and activated immune cells. These findings indicate that NK-cell derived exosomes might constitute a new promising therapeutic tool.

Exosomes are nanovesicles naturally released by almost all the cells of our body that-in both physiological and in pathological settings-deliver several molecules including proteins, lipids and nucleic acids to target cells. They are able to interact with target cells located in the close proximity or at distance using different mechanisms, including ligand-receptor interactions 1,2 and plasma membrane fusion, leading to the transfer of their contents to the target cell cytoplasm. 3 Thus, exosomes appear as a vectorized signaling system operating between the cytoplasm of a donor cell and either the extracellular compartment or potentially all the internal compartments of a target cell. These observations place exosomes at the center of current interests in translational research and point to exosomes as potential self-nanovectors for future nanomedicine approaches. Moreover, exosomes are attracting great consistent attention as tools for the identification of novel disease biomarkers. In fact, new tests offering the possibility to simultaneously characterize and quantify exosomes in human body fluids have been recently developed. 4 Such a dual potential of exosomes suggest that they might consistute an ideal tool for "theranostic." This new discipline of nanomedicine focuses on a multi-disciplinary research approach to build new systems NK cell-released exosomes Natural  for various nano-biomedical applications, ranging from the medical use of nanoplatform-based diagnostic agents to the development of therapeutic interventions, alone or combined to each other (therapy + diagnostic = theranostic).
We have recently shown that human natural killer (NK) cells release exosomes in both resting and activated condition. 5 Moreover, we found that NK cell-derived exosomes not only express both typical NK markers (i.e., CD56) and killer proteins (i.e., FASL and perforin) but also exert antitumor and immune homeostatic activities. These findings demonstrate that-at odds with T and B cells 6,7 -NK cells secrete exosomes in a constitutive way and independently from their activation status. This may suggest that NK cellderived exosomes are involved in the control of the immune response without the need for specific stimuli. In fact, NK cellderived exosomes contain both FASL and perforin when they are released by resting NK cells. However, NK-cell derived exosomes are functional exclusively against activated immune cells, suggesting that they have a control on the immune cell expansion only upon activation via cellintrinsic or cell-extrinsic stimuli.
Our in vitro results were strongly supported by ex vivo findings on circulating exosomes obtained from healthy donors, showing expression of NK markers, such as CD56 and perforin, and exerting exosome-induced cytotoxicity. Thus, it appears conceivable that NK cell-derived exosomes control immune responses both in a paracrine fashion and sistemically. Together with CD56, NK cellderived express detectable amounts of the activating receptor NKG2D, whereas natural cytotoxicity receptors (NCRs), the other NK-cell receptors that mediate cytotoxic functions (i.e., NKp30, NKp46 and NKp44), are less expressed. Intriguingly, we detected perforin in exosomes purified from both NK-cell culture supernatants and the plasma of healthy individuals, whereas FASL was undetectable in plasmatic exosomes. 5 Moreover, perforin-containing plasmatic exosomes were exclusively associated with NK-cell but not CD8 + T-cell marker. This is in line with a previous report showing that perforin is highly expressed by resting NK cells, but not by resting CD8 + T lymphocytes. 8 Of note, perforin is one of the most conserved proteins involved in immune responses, with a potential ancestral role in the immune system of heterogeneous organisms. 9 The role of perforin in the control of tumor initiation, growth, and dissemination is well established and perforin-deficient mice are profoundly immunocompromised and more prone to develop spontaneous tumors. 10 Hence, it appears conceivable that we have recently reported that human natural killer (NK) cells release exosomes that express both NK-cell markers and cytotoxic molecules. similar results were obtained with circulating exosomes from human healthy donors. Both NK-cell derived and circulating exosomes exerted a full functional activity and killed both tumor and activated immune cells. these findings indicate that NK-cell derived exosomes might constitute a new promising therapeutic tool. exosomes may interact with target cells, [1][2][3] it is likely that NK cell-derived exosomes may interact with target cells with either a exosome-to-membrane fusion or a receptor-to-ligand interaction (Fig. 1).

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. and delivery of exosomes, as they are attracted by low pH and these conditions promote membrane fusion. 3 Thus, paradoxically, differences in the electrostatic charges between exosomes and the plasma membrane of tumor cells might be more important than the specificity of ligandreceptor interactions. This said, based on the different mechanisms through which exosome-associated perforin may constitute a new target for the development of therapies against cancer and other diseases.
In this context, it should be noted that the tumor microenvironment is acidic, which, on one hand, may impairs the effectiveness of chemotherapeutics and possibly of the antitumor immune response, but, on the other hand, may favor the accumulation Figure 1. Mechanisms through which NK-cell derived exosomes may interact with target cells. Natural killer (NK) cell-derived exosomes express two molecules involved in cytotoxicity: perforin, which may exert its action upon fusion of exosomes with the plasma membrane of target cells, and FAsL, which mediates cytotoxic effects by interacting with FAs on the plasma membrane of target cells.