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Targeted Disruption of Chlamydia trachomatis Invasion by in Trans Expression of Dominant Negative Tarp Effectors

Chlamydia trachomatis invasion of eukaryotic host cells is facilitated, in part, by the type III secreted effector protein, Tarp. The role of Tarp in chlamydiae entry of host cells is supported by molecular approaches that examined recombinant Tarp or Tarp effectors expressed within heterologous systems. A major limitation in the ability to study the contribution of Tarp to chlamydial invasion of host cells was the prior absence of genetic tools for chlamydiae. Based on our knowledge of Tarp domain structure and function along with the introduction of genetic approaches in C. trachomatis, we hypothesized that Tarp function could be disrupted in vivo by the introduction of dominant negative mutant alleles. We provide evidence that transformed C. trachomatis produced epitope tagged Tarp, which was secreted into the host cell during invasion. We examined the effects of domain specific Tarp mutations on chlamydial invasion and growth and demonstrate that C. trachomatis clones harboring engineered Tarp mutants lacking either the actin binding domain or the phosphorylation domain had reduced levels of invasion into host cells. These data provide the first in vivo evidence for the critical role of Tarp in C. trachomatis pathogenesis and indicate that chlamydial invasion of host cells can be attenuated via the introduction of engineered dominant negative type three effectors.

INTRODUCTION
Chlamydia trachomatis is an obligate intracellular bacterium responsible for many human diseases (Moulder et al., 1984). Distinct serovars are the etiologic agents of endemic blinding trachoma, sexually transmitted disease, and lymphogranuloma venereum (Byrne, 2010). Chlamydiae undergo a unique developmental cycle consisting of two metabolically and morphologically distinct developmental forms adapted for extracellular survival and intracellular multiplication, respectively (Swanson et al., 1975;Szaszak et al., 2011;Omsland et al., 2012). Elementary bodies (EBs) are small, metabolically dormant cell types that actively promote invasion of eukaryotic host cells (Carabeo et al., 2002). Reticulate bodies (RBs) are larger cell types that are metabolically active and undergo replication (Omsland et al., 2012). EBs differentiate into RBs within the first few hours following infection. The RBs then multiply by binary fission until ∼16-24 h post-infection, at which time they asynchronously begin to differentiate back into EBs prior to release from the host cell and initiation of subsequent rounds of infection (Moulder et al., 1984). Like many Gram-negative pathogens, chlamydiae have a type III secretion system (T3SS) which they utilize to translocate various effector proteins into the cytosol of the host cell. Additionally, some secreted effectors localize to the expanding inclusion membrane and are collectively referred to as the Inc., proteins (Coburn et al., 2007). The chlamydial T3SS functions in at least two distinct locations and times during chlamydial development (Muschiol et al., 2006;Betts-Hampikian and Fields, 2010;Case et al., 2010). One pool of early effectors, pre-existing in EBs, is secreted upon contact with a host cell without a requirement for chlamydial protein synthesis (Jamison and Hackstadt, 2008;Valdivia, 2008). Later in the developmental cycle, other effectors are secreted out toward the cytosol from within the inclusion after initiation of protein synthesis (Wolf et al., 2006). The translocated actin-recruiting phosphoprotein (Tarp) is one of the early effectors and is spatially and temporally associated with the recruitment of actin to the site of EB invasion (Clifton et al., 2004). Tarp is phosphorylated upon translocation into eukaryotic cells by host tyrosine kinases (Jewett et al., 2008;Mehlitz et al., 2008). All isolates of pathogenic Chlamydia examined to date harbor the tarP gene (Clifton et al., 2005;Lutter et al., 2010). Biochemical analysis of C. trachomatis Tarp and other Tarp orthologs revealed that Tarp is comprised of an actin nucleating domain which is conserved and a tyrosine-rich repeat domain that is specific to serovars of C. trachomatis (Clifton et al., 2005;Jewett et al., 2006Jewett et al., , 2010. Tarp associates directly with both globular (G-) and filamentous (F-) actin via small alpha helical domains contained within the C-terminal region of the protein (Jewett et al., 2006(Jewett et al., , 2010Jiwani et al., 2013). Tarp's ability to directly bind to actin contributes to two biochemically characterized functions, actin nucleation and actin bundling, which likely lead to cytoskeletal modifications in the target host cell during entry (Jewett et al., 2006;Jiwani et al., 2013). Tarp independently nucleates new actin filaments by forming a large homogenous multimeric protein complex mediated by a conserved proline rich domain (Jewett et al., 2006). Inhibition of the actin binding alpha helix with microinjected antibodies specific for the Tarp actin binding domain blocked Tarp-mediated actin polymerization in vitro and reduced C. trachomatis L2 entry into host cells, suggesting Tarp is a critical virulence factor associated with chlamydial invasion (Jewett et al., 2010).
Although the direct actin-nucleating potential of C. trachomatis Tarp is implicated in bacterial entry of host cells, other actin nucleating pathways involving activation of the Arp2/3 complex are also necessary for entry (Carabeo et al., 2007;Jewett et al., 2010). Interestingly, phosphorylated Tarp may also play a role by indirectly activating the Arp2/3 complex as Tarp immunoprecipitation and peptide array assays have identified host cell signaling proteins such as Eps8, Rac1, Abi1, Sos1, Vav2, and SHC1 that associate with phosphorylated Tarp and promote Arp2/3 activation (Lane et al., 2008;Mehlitz et al., 2010). Biochemically, the actin nucleating properties of Tarp and the Arp2/3 complex work together to rapidly form actin filaments required for internalization (Jiwani et al., 2012). However, the precise details of how these distinct pathways cooperate to promote chlamydial internalization still remains unclear.
Since the Tarp effector has been characterized by various cellular and molecular approaches, and is implicated in chlamydial invasion of host cells, we sought to engineer mutant Tarp effectors that would biochemically interfere with endogenous Tarp function in vivo. In this work we examined C. trachomatis transformants expressing epitope tagged mutant Tarp alleles for their ability to invade host cells. Here, we report that EBs which secrete mutant Tarp effectors harboring specific domain deletions are deficient in bacterial entry of host cells. As hypothesized, those EBs which expressed a mutant Tarp lacking the actin binding domain, required for actin nucleation, were attenuated for bacterial invasion of host cells. Interestingly, the greatest inhibition of chlamydial entry was observed for those EBs which expressed Tarp effectors lacking the phosphorylation domain. These findings strongly support a role for Tarp in pathogen entry of host cells, and suggests that expression and delivery of engineered dominant negative mutant effectors may be employed to attenuate C. trachomatis pathogenesis.

Cloning and C. trachomatis Transformations
In previous studies we had generated a number of in-frame Tarp deletions which were expressed as mutant GST-Tarp fusion proteins from pGEX-6p-1 (GE Health Sciences) plasmids (Jiwani et al., 2013). Tarp domain deletion mutants included: phosphorylation domain deletion ( phos; deletion of D125 to Y424), proline rich domain deletion ( PRD; deletion of S625 to N650), actin binding domain deletion ( ABD; A748 to K758), and F-actin binding domain 1 and 2 deletion ( FAB 1&2; deletion of L871 to G1005). These mutant Tarp alleles were subcloned into the chlamydial shuttle vector pCtSV.1 in a two-step process. First, wild type Tarp sequence was amplified from C. trachomatis (LGV 434) genomic DNA (Qiagen genomic purification kit, Valencia, CA). The forward (5 ′ ACTCC GCGGTATTGCATTTCTTCACAAACGTTACC-3 ′ ) and reverse (5 ′ TATATACAATTGTTACAGGTCCTCTTCAGATATTAGTT TTTGTTCTCCTACGGTATCAATCAGTGAGC-3 ′ ) DNA primers (Integrated DNA Technologies, Coralville, IA) were engineered to amplify 200 bases of putative Tarp promoter sequence and an in frame 3 ′ c-myc epitope tag by PCR with SacII and MfeI linkers. PCR products were purified (Qiagen), digested with restriction enzymes (New England Biolabs, Beverly, MA) and cloned into linearized pCtSV.1. This procedure resulted in the parent pCtSV.Tarp plasmid in which all other plasmids engineered to express Tarp mutants were generated. pCtSV.Tarp mutant derivatives were generated by exchanging the mutant DNA sequence from those pGEX-6p-1 clones described above.
For example, pCtSV.Tarp phos resulted from DNA exchange with digested Tarp DNA sequence flanking the phosphorylation domain with restriction sites BstAP1 and BmgB1 from pGEX-6p-1 Tarp phos. Similarly, the other pCtSV.Tarp mutant clones were generated albeit with unique restriction enzymes which flanked the corresponding domain: The proline rich domain with BmgB1 and Bsm1, the actin binding domain with Bsm1 and Nco1, and the F-actin binding domains 1 and 2 with Nco1 and Mfe1. All engineered vectors were confirmed to be free of extraneous mutations by DNA sequence analysis and all in frame domain deletions were verified. All chlamydial shuttle vectors were purified from E. coli K12 ER2925 cells (New England Biolabs) and transformed into C. trachomatis (LGV 434) (Wang et al., 2011). All C. trachomatis transformants were density gradient purified and the number of infectious (inclusion) forming units (IFUs) was determined by indirect immunofluorescence of infected host cells with serially diluted purified elementary bodies (EBs).

Pyrene Assay
Pyrene actin polymerization assays were performed as previously described (Jiwani et al., 2013). Briefly, monomeric pyrene-labeled actin was prepared by diluting 100 µg of lyophilized pyrene actin (cytoskeleton Inc. Denver, CO) in 2 mL of 5 mM Tris (pH 8.0), 0.2 mM CaCl 2 , 0.2 mM ATP (G buffer) and incubated for 1 h at room temperature, followed by an additional 1 h incubation at 4 • C. Monomeric pyrene actin was obtained by collecting the supernatant after a 2 h 100,000 × g 4 • C spin in a Beckman Optima TLX Ultracentrifuge using a TLA 100.3 rotor (Beckman Coulter). Approximately 20 µg of pyrene-labeled actin was gently mixed with 5 µg of GST fusion proteins in a volume of 500 µL for 10 min before the addition of 1/20th volume of polymerization buffer (500 mM KCl, 20 mM MgCl 2 , 10 mM ATP). The reaction was monitored over 1 h with an LS 55 Luminescence spectrophotometer directed by FL WinLab software version 4.0 (Perkin-Elmer, Beaconsfield, Bucks, United Kingdom) with 2.5-nm bandwidth at 365nm excitation wavelength and 2.5-nm bandwidth at 407-nm emission wavelength.

SDS-PAGE and Immunoblotting
Proteins were separated on SDS 5-15% polyacrylamide gels (BIORAD, Hercules, CA) and either stained with Imperial protein stain (Pierce, Rockford, IL) or transferred to 0.45 µm pure nitrocellulose transfer and immobilization membrane (Schleicher and Schuell, Keene, NH). Immunoblotting employed peroxidase conjugated secondary antibodies (Chemicon International, Temecula, CA) and Supersignal West Pico chemiluminescent substrate (Pierce). The anti-actin C4 monoclonal antibody was purchased from Chemicon International. The anti-actin polyclonal antibody was purchased from Cytoskeleton, Inc. The anti-phosphotyrosine 4G10 monoclonal antibody was purchased from Upstate (Millipore). The anti-chlamydial EB polyclonal antibody, the Momp monoclonal antibody and the GAPDH monoclonal antibody were all purchased from Pierce. The anti-c-myc monoclonal antibody was purchased from Genscript (Piscataway, NJ).

Invasion Assay and Indirect Immunofluorescence Microscopy
Intrinsically fluorescent EBs from C. trachomatis transformants were purified from cell cultures supplemented with CellTracker TM Red CMTPX Dye as previously described (Carabeo et al., 2007). Briefly, CMPTX-labeled C. trachomatis EBs (MOI ∼50) were permitted to attach to HeLa 229 host cells for 30 min at 4 • C. HeLa 229 cells were prepared in 24 well plates with cover slips and grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% L-glutamine for 24 h prior to infection. The cultures were rinsed with cold HBSS and the temperature shifted to 37 • C by the addition of pre-warmed DMEM plus 10% FBS. The cultures were then incubated at 37 • C for 1 h. The cultures were fixed with 4% paraformaldehyde at room temperature for 15 min and rinsed with PBS. The cells were not permeabilized. Extracellular EBs were labeled for 1 h with a monoclonal antibody specific for chlamydial major outer membrane protein (MOMP). After four washes in PBS, secondary antibody conjugated to Alexa 488 was added for 1 h. Coverslips were rinsed and mounted in ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA). Cells were examined with a Zeiss Axio Observer A1 microscope equipped with a phase-contrast and epifluorescence optics. Images were obtained using an AxioCam MRm camera controlled by Axio Vision 4.8.2 and further processed using Adobe Photoshop CS2. The number of green (external) and red (total) EBs was determined for each host cell. These data were then used to determine the percentage of internalized EBs. Twenty fields of view were taken from each cover slip and these percentages were then averaged together to give a final invasion rate.

Subcellular Fractionation and Protein Extraction
C. trachomatis infected cells underwent subcellular fractionation as previously described (Cox and Emili, 2006). Briefly, C. trachomatis infected McCoy or HeLa 229 cells maintained at 37 or 4 • C or host cells alone incubated at 37 • C were removed from flasks and suspended in 100 mM KCl, 10 mM HEPES (pH 7.7), 2 mM MgCl 2 , and 2 mM ATP (Buffer A) and disrupted by sonication delivered in three consecutive 30 s intervals (∼ 2000 joules) using an ultrasonic sonicator processor XL equipped with a microtip (Misonix Incorporated, Farmingdale, NY). All cell lysates underwent subcellular fractionation by sequential centrifugation in which supernatants and pellets were separated. Lysates were initially subject to an 800 × g spin for 15 min at 4 • C. The 800 × g supernatants were then subjected to a 10,000 × g spin for 30 min at 4 • C. The remaining 10,000 × g supernatant underwent a 100,000 × g spin for 1 h at 4 • C. Protein sample buffer was added to all pellets and supernatants and proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting with antibodies specific for c-myc, Tarp, actin, GAPDH, Momp, and chlamydial EBs. Subcellular fractionation experiments were conducted in both McCoy ( Figure 6) and HeLa 229 (Supplemental Figure 1) cells.

C. trachomatis Development
HeLa 229 cells were seeded into 6 well plates (2 × 10 5 cells/well) and grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% L-glutamine for 24 h. Individual wells were infected with wild type C. trachomatis L2 (LGV 434) or C. trachomatis transformants. All host cells and bacteria were collected from select wells (cells scraped off the bottom of each well and collected in 15 mL tubes, and sonicated for 30 s using a microtip equipped Misonix sonicator) at 0, 12, 24, 36, and 48 h. Cell lysates were then frozen at −80 • C until all time points had been collected. Cell lysates were thawed on ice and diluted and then placed onto HeLa cells grown on 16 mm circular cover slips contained within 24 well plates and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Lglutamine. After a 40 h incubation, infected cells were then immunostained and observed under a fluorescent microscope for inclusion formation. Twenty fields of view were taken from each cover slip (the experiment was performed in triplicate) and cover slip counts were averaged. Averages were plotted using GraphPad Prism software.

Mutant Tarp Proteins Diminish the Ability Of Wild Type Tarp to Polymerize Actin Filaments
The Tarp actin binding domain (ABD, amino acids 748-758) and the proline rich polymerization domain (PRD, amino acids 625-650) have previously been found to be required for Tarp mediated polymerization of pyrene actin in vitro (a summary of Tarp protein domains is provided in Figure 1; Jewett et al., 2006). One model for Tarp mediated actin nucleation advocates that Tarp oligomerizes via the proline rich domain. Tarp effectors, which come together following secretion into the host cell cytosol, associate with monomeric actin to form an actin nucleus, affectedly reducing the critical concentration of actin required for actin filament formation (Jewett et al., 2006). Since Tarp is hypothesized to function in a homo-oligomeric complex, we tested whether Tarp PRD and Tarp ABD were able to disrupt wild type Tarp mediated actin polymerization in a dominant negative fashion. To investigate whether mutant Tarps could inhibit actin polymerization induced by wild type Tarp, purified recombinant wild type Tarp proteins and Tarp ABD or Tarp PRD were combined at equal molar ratios and analyzed for actin nucleation in pyrene actin polymerization assays (Figure 2). Similar to our previous findings, when Tarp PRD or Tarp ABD alone was introduced to pyrene actin, an increase in the rate of actin polymerization was not observed compared to actin alone FIGURE 1 | Schematic of Tarp protein mediated signaling pathways implicated in chlamydial entry and development. C. trachomatis Tarp harbors an N-terminal tyrosine rich repeat phosphorylation domain (Y, green box). Tyrosine residues are phosphorylated by members of the Src family kinases (SFKs) such as Src, Yes and Fyn and by other tyrosine kinases, Syk and Abl/Arg kinases. The actin nucleating activity of Tarp results from distinct G-actin binding (red box) and proline rich (blue box) oligomerization domains and from an Arp2/3-dependent pathway resulting from the recruitment and activation of host cell signaling proteins Vav2, Sos1, Eps8, Abi1, Rac1, and WAVE2. Tarp also harbors two F-actin binding domains (FAB 1 and 2: yellow and pink boxes) that are implicated in the formation of actin bundles. Phosphorylated Tarp can also associate with the host cell Src homology 2 domain containing protein 1 (SHC1) and the phosphoinositide 3-kinase (PI3K), which are implicated in altering the activation state of host signaling proteins to create a protective niche for the developing bacterium.
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org controls (Figures 2B,C). The amount of Tarp PRD employed in these experiments was lower compared to previous studies to ensure that Tarp PRD would not appreciably sequester monomeric actin (Jiwani et al., 2013). At higher concentrations, Tarp PRD sequesters monomeric actin and may disrupt native Tarp function by lowering the concentration of available actin in the reaction. As predicted, when (Tarp PRD or Tarp ABD) were mixed with an equal amount of wild type Tarp, the rate of actin polymerization was reduced (Figures 2B,C). Although the rate of pyrene actin polymerization was reduced in the presence Tarp PRD or Tarp ABD, some Tarp mediated actin nucleation was still observed. To confirm that the reduction of actin polymerization was not the consequence of steric hindrance caused by excess Tarp in the reaction, an additional control (Tarp phos) which is not predicted to interfere with actin polymerization was tested. Tarp which lacks the tyrosine rich phosphorylation domain (Tarp phos, harbors the deletion in amino acids 125-424) has previously been shown to nucleate actin to equivalent rates as compared to wild type Tarp (Jiwani et al., 2013). To test whether Tarp mediated actin polymerization might be affected by Tarp phos (Figure 2D), purified Tarp and Tarp phos were mixed at equal molar ratios and tested in pyrene actin polymerization assays. Tarp phos combined with wild type Tarp enhanced the rate of actin polymerization due to the two-fold molar increase of functional actin nucleators ( Figure 2E). These data support the hypothesis that biochemically wild type Tarp may experience reduced actin polymerization kinetics when associated with or competing with defective Tarp PRD or Tarp ABD. Conversely, wild type Tarp mediated actin polymerization is not inhibited by Tarp phos.

C. trachomatis Transformants are Able to Express Epitope Tagged Wild Type and Mutant Tarp Effectors
In order to determine whether mutant Tarp effectors might disrupt endogenous Tarp function in vivo, we engineered a chlamydial shuttle vector to express epitope tagged wild type and mutant Tarp effectors ( Figure 3A). C. trachomatis genes are temporally regulated so as to match their function with the correct window in the chlamydial developmental cycle. To promote coordinated expression of the mutant Tarp alleles with that of the endogenous Tarp gene, mutant Tarp effectors expressed from the chlamydial shuttle vector were engineered under the control of ∼200 nucleotides of DNA upstream of the annotated Tarp gene, which we have termed the Tarp promoter (tarPp) (Figure 3). Mutant Tarp constructs included deletions of the phosphorylation domain, the proline rich domain, the actin binding domain and a Tarp truncation which resulted in the deletion of the F-actin binding domains 1 and 2 ( Figure 3A). C. trachomatis L2 transformed with each shuttle vector were expanded under antibiotic selection for several passages, density gradient purified and tested for the presence of the c-myc epitope tag by western blot analysis. All of the transformants produced c-myc tagged proteins of the expected size ( Figure 3B). Interestingly, endogenous Tarp in C. trachomatis expressing Tarp phos appears to be lower compared to the relative abundance of Tarp in the other transformants. This may hold true for the other transformants (ABD, PRD, and FAB1 and 2) but cannot be visualized in the western analysis since mutant and endogenous Tarp migrate to the same position on the protein gels.

C. trachomatis Transformants Expressing Mutant Tarp Effectors are Inhibited in Bacterial Invasion of Host Cells
We hypothesized that production of a dominant negative Tarp complex in vivo would have a significant effect on the ability of elementary bodies to invade host cells. To test the invasion potential of the five different C. trachomatis clones expressing FIGURE 4 | C. trachomatis transformants harboring epitope tagged mutant Tarps are deficient in chlamydial entry. (A) Wild type C. trachomatis (L2; circles) or L2 transformants harboring plasmid pCtSV.Tarp (+ pCtSV.Tarp; squares), pCtSV.Tarp phos (+ pCtSV.Tarp phos; triangles), pCtSV.Tarp PRD (+ pCtSV.Tarp PRD; inverted triangles), pCtSV.Tarp ABD (+ pCtSV.Tarp ABD; asterisks), or pCtSV.Tarp FAB1and2 (+ pCtSV.Tarp FAB1and2; "x"), were examined for chlamydial invasion of HeLa 229 cells. Intrinsically fluorescent cell tracker (CMPTX) labeled EBs were used in invasion assays. After allowing 1 h for invasion, extracellular EBs were counterstained by indirect immunofluorescence with a monoclonal antibody to C. trachomatis L2 MOMP and a goat anti mouse antibody conjugated to Alexa 488. The data are represented as the percentage of intracellular EBs relative to the total number of extracellular and intracellular EBs per field of view. The data represented from two biological replicates are shown. Each data point represents a single field of view at 1000× magnification. Data sets were compared with one way ANOVA and Tukey's multiple comparison test of the mean. ***represents a p < 0.001. (B) Development of wild type C. trachomatis L2 (circles) and transformants harboring plasmid pCtSV.Tarp (+ pCtSV.Tarp; squares), pCtSV.Tarp phos (+ pCtSV.Tarp phos; triangles), after normalizing the initial multiplicity of infection for each clone (IFU normalization was confirmed by determining the number of inclusions formed at time zero). Infected cells with antibiotic selection (filled symbols) and infected cells without antibiotic selection (open symbols) were collected at t = 0, 12, 24, 36, and 48 h post infection and mechanically lysed to release infectious EBs. Inclusion forming units (IFUs) were determined for each transformant by serial dilution of released EBs harvested at each time point and reinfection of HeLa cells grown on coverslips to determine the number of inclusion forming units per mL of harvested material. mutant Tarp effectors from the C. trachomatis shuttle vector, we performed invasion assays to quantitate the number of elementary bodies which entered a host cell in a 1 h time period ( Figure 4A). As predicted, C. trachomatis expressing Tarp lacking the actin binding domain (Tarp ABD) demonstrated a significant reduction in host cell invasion compared to wild type C. trachomatis L2 and C. trachomatis harboring pCtSV.Tarp. Surprisingly and in contrast to the in vitro actin polymerization studies, C. trachomatis expressing Tarp phos displayed a significant reduction in invasion relative to all of the clones analyzed, including C. trachomatis expressing Tarp ABD ( Figure 4A). No altered invasion phenotype was observed for C. trachomatis expressing Tarp PRD or Tarp FAB1 and 2. In order to examine if the presence of a dominant-negative Tarp might disrupt chlamydial development, growth curves were performed for C. trachomatis expressing Tarp phos and wild type controls (Figure 4B). No significant changes to chlamydial growth was observed between wild type and C. trachomatis expressing Tarp phos as measured by the number of EBs harvested from infected cells at 0, 12, 24, 36, and 48 h post infection ( Figure 4B and data not shown).

C. trachomatis Expressing Tarp phos Demonstrate Reduced Phosphorylation of Endogenous Tarp
We hypothesized that C. trachomatis expressing Tarp phos might show a reduction in endogenous Tarp phosphorylation following attachment and entry into host cells as Tarp phos is likely to compete with endogenous Tarp for translocation into the host cell. Tarp phos is missing the tyrosine residues known to be phosphorylated by Src family kinases (SFKs) therefore Tarp phos is not expected to be phosphorylated by host cell tyrosine kinases after translocation into the host cell. In order to test this, wild type C. trachomatis or C. trachomatis harboring the plasmids presented in Figure 3 were added to host cells and protein lysates were generated at 0 and 1 h post infection. Tarp has previously been identified as the predominant phosphotyrosine modified protein of ∼150 kDa which is observed in the wild type C. trachomatis infected cells. Interestingly, phosphorylated Tarp was reduced in those bacteria expressing Tarp phos, but not in the other transformants ( Figure 5).

C. trachomatis is Able to Translocate Tarp phos into the Host Cells
C. trachomatis expressing both endogenous Tarp and Tarp phos demonstrated a reduction in host cell invasion and Tarp phosphorylation. These results raised the possibility that type three secretion is altered and/or inhibited in C. trachomatis expressing Tarp phos. To determine whether C. trachomatis expressing Tarp phos was capable of type three secretion we examined whether endogenous Tarp and plasmid encoded epitope tagged Tarp phos could be recovered from the soluble fraction following subcellular fractionation of host cells ( Figure 6A). The fractionation profile of wild type C. trachomatis infected cells is presented as Supplemental Figure 1 for reference. Endogenous Tarp and epitope tagged Tarp phos can be distinguished by their unique molecular weights and the presence or absence of the c-myc epitope tag (Figure 3). Protein supernatant samples sequentially obtained from 800, 10,000, and 100,000 × g centrifugal spins indicated that the endogenous Tarp and Tarp phos effectors co-fractionated and were detectable in fractions that were distinct from intact EBs, which pellet at 10,000 × g ( Figure 6B). The endogenous Tarp and Tarp phos proteins were observed in the 100,000 × g pellet and the 100,000 × g soluble fraction, the latter represents the host cell cytosolic fraction as defined by the presence of the soluble eukaryotic protein GAPDH (Figure 6B). We hypothesize that most of the secreted tarp is restricted to the 100,000 × g pellet due to interactions with actin filaments (short actin filaments pellet at 100,000 × g). The portion of Tarp found in the 100,000 × g supernatant (soluble fraction) may represent those proteins which have not yet associated with host cell molecules of the cytoskeleton. Non-secreted chlamydial antigens identified by the anti-EB and anti-MOMP antibodies were not detected in fractions beyond the 10,000 × g pellet, indicating that these later protein fractions did not contain lysed EBs. It has been shown previously that type three secretion by C. trachomatis is temperature dependent and can be inhibited at low temperatures (Jamison and Hackstadt, 2008). Consistent with this finding, endogenous and mutant Tarp were not detected in the host cell cytosolic fraction of host cells maintained at 4 • C during the course of the C. trachomatis infection. Together these data indicate that C. trachomatis expressing Tarp phos is capable of type FIGURE 6 | C. trachomatis transformants demonstrate secretion of both wild type Tarp and epitope tagged mutant Tarp phos. (A) The schematic of the differential centrifugation steps and the supernatants (sup) and pellets recovered from centrifugation at 800, 10,000, and 100,000 × g. Secreted effector proteins such as Tarp are expected to be detectable in the bacteria-free 100,000 × g pellet (microsomes) and soluble fractions. (B) Subcellular fractionation of C. trachomatis infected cells by differential centrifugation out to 100,000 × g yields a soluble Tarp fraction that is distinct from intact elementary bodies. Total lysates derived from McCoy host cells infected with C. trachomatis serovar L2 transformed with the shuttle vector pCtSV.Tarp phos (L2 pCtSV.Tarp phos) underwent subcellular fractionation by centrifugation. Fractions were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblot analysis with antibodies specific for phosphotyrosine (α Y-PO 4 ), Tarp (α Tarp), c-Myc epitope (α c-myc), elementary bodies (α EB), C. trachomatis major outer membrane protein (α Momp), Glyceraldehyde 3-phosphate dehydrogenase a soluble protein marker (α GAPDH) and actin a protein expected to be present in all fractions (α actin). The infection and subsequent cell fractionation data presented is representative of three repeated experiments.
three secretion mediated delivery of endogenous and mutant Tarp into the host cell cytosol. Although the data presented in Figure 6B do not provide quantitative measures of WT and Tarp phos secretion, the experiment indicates that both endogenous and Tarp phos are actively secreted into the host cell.

Dissociation of Tarp phos from the Native Tarp Effector is Able to Restore C. trachomatis Invasion of Host Cells
Previous studies with purified recombinant Tarp effectors revealed that Tarp multimerization is mediated by a region of ∼25 amino acids rich with prolines subsequently called the proline rich domain (PRD) (Jewett et al., 2006). This model suggests that plasmid encoded Tarp phos and endogenous Tarp may associate following translocation into the cell perhaps resulting in the observed dominant-negative invasion phenotype. To further test this hypothesis, we sought to disrupt the ability of Tarp phos to form heteromeric complexes with wild type Tarp through genetic deletion of the proline rich domain from the pCtSV Tarp phos construct. According to our model, a Tarp phos PRD double mutant lacking both the phosphorylation domain and the proline rich oligomerization domain would be unable to associate with endogenous Tarp, rescuing the dominate negative phenotype caused by the Tarp phos protein. As predicted, C. trachomatis transformants harboring pCtSVTarp phos PRD demonstrated wild type levels of chlamydial entry after 1 h (80% internalized EBs) and displayed no defect in chlamydial growth (Figure 7 and data not shown).

DISCUSSION
Obligate intracellular pathogens such as C. trachomatis harbor unique tools to hijack host cell processes, promoting bacterial replication and immune evasion. The Tarp effector is a candidate virulence factor that is hypothesized to trigger host cell entry and other host signaling events to promote pathogen invasion. By leveraging our understanding of the biochemical features of the Tarp polypeptide, we now demonstrate that mutant Tarp effectors can be engineered to disrupt pathogen entry of host cells in a dominant-negative manner. The interplay between the host and the pathogen is intricate and involves mechanisms of pathogen-targeted disruption of host cellular processes, which are only partially understood (Ribet and Cossart, 2015). To some degree, C. trachomatis execute cellular override mechanisms by delivering bacterial effector proteins into the host cell cytosol via a type three secretion apparatus (Ferrell and Fields, 2016). Consequentially, a number of studies have focused on how Tarp is able to associate with host cell actin, tyrosine kinases, and SH2 domain containing proteins (Jewett et al., 2006(Jewett et al., , 2008Lane et al., 2008;Mehlitz et al., 2008Mehlitz et al., , 2010Lutter et al., 2010;Jiwani et al., 2012Jiwani et al., , 2013. Tarp is a large protein consisting of 1005 amino acids and is, biochemically speaking, one of the most well characterized C. trachomatis effectors (Mueller et al., 2014). Host cytoskeletal rearrangements are required for bacterial invasion of host cells and it is believed that C. trachomatis actively directs this process, mediated in part by Tarp translocation into the host cell. Recombinant Tarp by itself is a potent nucleator of actin and dramatically increases the rate of actin polymerization compared to actin alone controls in in vitro pyrene actin polymerization assays (Jewett et al., 2006). Domain deletion analysis has revealed the minimum protein sequence required for actin binding and actin nucleation (Jewett et al., 2006). These studies demonstrated that Tarp mediated actin nucleation is predominantly driven by Tarp oligomerization, which could make this biochemical function amenable to interference or disruption if the Tarp complex failed to assemble appropriately in vivo (Jewett et al., 2006). With the advent of a chlamydia transformation system (Wang et al., 2011), it was now possible to genetically manipulate C. trachomatis to express plasmid-encoded mutant Tarp effectors engineered to disrupt endogenous Tarp-mediated actin nucleation. As hypothesized, C. trachomatis producing a mutant Tarp effector, which lacked the actin binding domain required for actin nucleation, resulted in disruption of chlamydial entry into host cells. These findings were consistent with the ability of purified Tarp ABD protein to reduce the actin nucleation activity of purified wild type Tarp protein in vitro. Together these data suggest that C. trachomatis producing both endogenous Tarp and Tarp ABD protein have a diminished ability to nucleate actin resulting in reduced host cell invasion. Conversely, C. trachomatis harboring mutant Tarp effectors lacking the proline rich domain required for Tarp oligomerization did not disrupt bacterial entry. This may not be surprising as Tarp PRD is predicted to lack the ability to associate with endogenous Tarp and therefore is not likely to disrupt the homomeric complex critical for Tarp function. Purified Tarp PRD alone can sequester monomeric actin in a concentration dependent manner in vitro ( Figure 2B; Jiwani et al., 2013). The lower rate of actin polymerization observed for the combination of purified Tarp PRD and purified wild type Tarp in vitro may be the result of reduced monomeric actin available in the pyrene assay due to sequestration of monomeric actin by Tarp PRD protein. Although it remains a possibility that Tarp PRD produced by the C. trachomatis pCtSVTarp PRD clone is able to sequester monomeric actin in vivo, this ability does not result in a measurable change in host cell invasion.
Interestingly, the greatest inhibition of EB entry of host cells was observed for C. trachomatis transformants which expressed Tarp phos. Based on our in vitro pyrene actin polymerization assays it is unlikely that Tarp phos is disrupting the direct actin nucleation activity of endogenous Tarp. Tarp phosphorylation has been implicated in host cell signaling via SH2 domain containing host cell proteins that promote the activation of other host cell actin nucleators such as the Arp2/3 complex (Lane et al., 2008;Mehlitz et al., 2010). Therefore, a heterocomplex between Tarp phos and endogenous wild type Tarp may indirectly disrupt actin nucleation as a result of altered host cell signaling.
Previous experiments have indicated that phosphorylated Tarp is unable to active the Arp2/3 complex directly and that any host cell signaling cascades that may be initiated by phosphorylated Tarp also requires activation of nucleation promoting factors (NPFs) such as WASP/N-WASP (Jiwani et al., 2012). Despite the compelling biochemical evidence for the indirect contribution of Tarp phosphorylation to actin polymerization and host cell entry, the role for Tarp phosphorylation in C. trachomatis invasion remains unclear. In contrast to the invasion phenotype conferred by C. trachomatis expressing Tarp phos, it has been shown that inhibition of Tarp phosphorylation via tyrosine kinase inhibitors such as PP2 does not significantly inhibit C. trachomatis entry into host cells (Jewett et al., 2008). A key difference between these two studies is the chemical inhibition of phosphorylation of full length endogenous Tarp versus phenotypic analysis of C. trachomatis transformants harboring Tarp molecules which are missing the phosphorylation domain. It is therefore possible that the tyrosine rich repeat region is not only important for Tarp phosphorylation, but has an additional undescribed role in EB entry. The data presented herein suggest that the presence of Tarp phos has altered endogenous Tarp function leading to reduced C. trachomatis invasion or that Tarp phos has disrupted EB entry independently. We found no evidence for significantly altered levels of Tarp secretion as our cell fractionation experiments revealed that both endogenous Tarp and Tarp phos were isolated from the soluble fractions containing the host cell cytosol alone. Although it remains to be seen if subtle quantitative differences in Tarp concentration or in secretion relative to wild type may have a profound effect on bacterial invasion. Previous reports have demonstrated that Tarp secretion is mediated by the chlamydial chaperone Slc1 (Brinkworth et al., 2011). Slc1 and Tarp associate via the N-terminal domain of Tarp mapped to amino acids 1-200. Interestingly, Tarp phos lacks amino acids 125-424 and was still capable of translocation into the host cell, therefor Tarp phos further delineates the region required for T3SSmediated secretion of Tarp.
Tarp proteins form oligomeric complexes in vitro which are mediated by the proline rich domain. In support of this model, deletion of the proline rich domain from the Tarp phos protein restored EB invasion to wild type levels. Tarp phosphorylation is implicated in host cell signaling; although these signaling mechanisms have not previously been experimentally defined to be directly associated with entry, our data now suggest that the Tarp tyrosine rich sequence that comprises the phosphorylation domain itself is important for entry. Analysis of Tarp orthologs from different chlamydial species and serovars has revealed conserved biochemical features such as the ability to bind and nucleate actin albeit by engaging slightly different mechanisms (Jewett et al., 2010). Conversely, many unique characteristics, such as C. trachomatis L2 Tarp phosphorylation or C. caviae strain GPIC Tarp's ability to bind to focal adhesion kinase (FAK) reveal that Tarp orthologs may have evolved to serve specific functions for unique human versus animal hosts (Clifton et al., 2005;Thwaites et al., 2014). The ability to introduce Tarp deletion mutants into C. trachomatis is the first step toward defining which protein domains may play a dominant role in vivo. This work may be expanded in the future to include analysis of other Tarp orthologs as well as the generation of a Tarp null or a conditional knockout.
A mechanistic understanding of chlamydial entry of host cells will likely lead to novel interventions that prevent C. trachomatis infections. Many questions still remain, but this work represents the first instance of the in vivo analysis of the Tarp effector and its functional domains within EBs and the utilization of dominant negative mutant alleles to disrupt chlamydial invasion of host cells.

AUTHOR CONTRIBUTIONS
CP: data acquisition, data analysis, data interpretation, revising of the manuscript; RL: data acquisition, data analysis, data interpretation, revising of the manuscript; BN: data acquisition, data analysis, data interpretation; LR: data acquisition, data analysis, data interpretation; TJ: data acquisition, data analysis, data interpretation, writing of the manuscript, revising of the manuscript, principle investigator.

FUNDING
This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R21AI117013. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.


s2fieldsofstudy: Biology Medicine

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Erythropoietin does not activate erythropoietin receptor signaling or lipolytic pathways in human subcutaneous white adipose tissue in vivo

Background Erythropoietin (Epo) exerts direct effects on white adipose tissue (WAT) in mice in addition to its erythropoietic effects, and in humans Epo increases resting energy expenditure and affect serum lipid levels, but direct effects of Epo in human WAT have not been documented. We therefore investigated the effects of acute and prolonged Epo exposure on human WAT in vivo. Method Data were obtained from two clinical trials: 1) acute Epo exposure (rHuEpo, 400 IU/kg) followed by WAT biopsies after 1 h and 2) 10 weeks treatment with the erythropoiesis-stimulating agent (ESA) Darbepoietin-alpha. Biopsies were analyzed by PCR for Epo receptor (Epo-R) mRNA. A new and highly specific antibody (A82, Amgen) was used to evaluate the presence of Epo-R by western blot analysis in addition to Epo-R signaling proteins (Akt, STAT5, p70s6k, LYN, and p38MAPK), activation of lipolytic pathways (ATGL, HSL, CGI-58, G0S2, Perilipin, Cidea, Cidec, AMPK, and ACC), and mitochondrial biogenesis (VDAC, HSP90, PDH, and SDHA). Results No evidence of in vivo activation of the Epo-R in WAT could be documented despite detectable levels of Epo-R mRNA. Conclusion Thus, in contradiction to animal studies, Epo treatment within a physiological relevant range in humans does not exert direct effects in a subcutaneous WAT.

Background
Erythropoietin (Epo), a glycoprotein produced in the kidneys and known for its erythropoietic effects, is used to treat nephrogenic anemia. Moreover, Epo receptors (Epo-R) of alleged functionality have been identified in numerous tissues [1]. In particular, data in mice suggest high expression of Epo-R mRNA in spleen and in white adipose tissue (WAT) [2].
Studies in end-stage renal disease (ERSD) patients show that serum levels of triglycerides, total cholesterol, and LDL decrease to normal levels after Epo treatment [3][4][5], but acute administration of rHuEpo in healthy subjects do not significantly affect lipid levels, although a tendency to increased free fatty acid (FFA) levels have been observed [6]. However, prolonged treatment to healthy humans with an erythropoiesis-stimulating agent (ESA) significantly increases serum FFA levels and hepatic lipid content [7].
In mice overexpressing the Epo gene, increased fat oxidation and an up-regulation of genes involved in lipid metabolism in skeletal muscle has been shown [8]. On the other hand, mice with a lack of Epo-R, except for the bone marrow, have a significantly higher body weight, increased fat mass and increased serum TG levels [2,9]. Furthermore, a reduction in body weight due to reduced WAT mass in obese mice is found after Epo treatment [2,[8][9][10][11].
The mechanism by which Epo regulates fat metabolism and body weight is currently unknown. One explanation could be an increase in resting energy expenditure including fat oxidation [2], but only the former has been documented in healthy human subjects [6,7]. As regards direct peripheral actions of Epo, there is no evidence to support such effects in human skeletal muscle [12,13]. The effect of Epo treatment on WAT in humans has so far not been evaluated, but Epo-R protein and mRNA has been detected in WAT from mice in addition to a suppressive effect of Epo on fat accumulation and preadipocyte differentiation and expansion in vitro [2,14]. Epo administration in mice is also associated with induction of brown adipose tissue (BAT) like features in WAT with increased mitochondrial content and uncoupling [9]. Furthermore, adipocyte specific deletion of the Epo-R in mice lead to obesity and decreased insulin sensitivity [9]. In contradiction, specific knockdown of the adipocyte Epo-R showed that Epo signaling at physiological levels was not essential for WAT metabolism [15].
The aim of the current study was to evaluate both the acute and prolonged effects of Epo treatment on human subcutaneous WAT on; 1) Epo-R expression, 2) activation of Epo-R related signaling pathways, and 3) direct and indirect effects on lipolysis and WAT metabolism.

Acute study
Ten healthy young men (23 (20-28) years, 179 (173-192) cm, and 77 (68-90) kg, median (range)) participated after providing written informed consent, in adherence to the declaration of Helsinki. The Local Ethical Committee of Central Denmark Region (M-2008-0016) approved the study and it was further registered at clinicaltrials.gov (M-20080035). Results regarding whole body and skeletal muscle metabolism from the same study have previously been published [6].

Study design Acute study
The subjects were in a single-blind, crossover design randomized to receive placebo and rHuEpo on two occasions with a 14-day wash-out period in-between. On the experimental days, the subjects arrived fasting (from 10 pm the evening before, water allowed) at the clinical research unit in the morning. Placebo (saline) or rHuEpo (Epoietin alpha, Eprex, 400 IU/kg) were administered i.v. and subcutaneous fat biopsies were collected 1 h post administration from the abdomen by liposuction under local anesthesia. Fat biopsies were cleaned from blood, frozen in liquid nitrogen, and stored at −80°C until further analysis. The experiments were performed under thermo neutral conditions (21-23°C).

Prolonged study
Only subjects fulfilling the following criteria were included in the study: maximal oxygen uptake (VO 2 max) below 50 ml/min/kg, age between 18 and 35, body mass index (BMI) between 18 and 29 kg/m 2 , normal blood pressure <135/85, and a hematocrit <45 %. The subjects were randomly assigned to either a placebo group (saline, n = 9) or an Epo group (n = 9). The subjects were in a single blinded manner treated with either an erythropoiesis-stimulating agent (ESA) (Darbepoietin-α, Aranesp, Amgen, Thousand Oaks, CA, USA) or placebo (saline) for 10 weeks. ESA/placebo were administrated subcutaneously once weekly and the ESA dose was 40 μg for the first 3 weeks and 20 μg for the remaining 7 weeks as previously detailed [7]. Subcutaneous fat biopsies were collected from the abdomen with liposuction under local anesthesia, before and after the 10 weeks treatment. The biopsies were cleaned for blood, frozen in liquid nitrogen, and stored at −80°C until further analysis. The procedure was performed under thermo neutral conditions (21-23°C). The participants were instructed to refrain from hard physical activity, alcohol intake, or dietary changes 3 days prior to collection of the biopsies. On the day of biopsy collection the subjects arrived at the clinical research unit after an overnight fast (water intake allowed). During the study period the participants were informed not to change their level of physical activity or dietary habits.

Epo-R mRNA analysis
Subcutaneous WAT from the subjects in the acute study (placebo treatment), bone marrow, and K-562 cells were used for RNA extraction using TRIzol (Gibco BRL, Life Technologies, Roskilde, Denmark) and homogenized with 1 tungsten bead (Qiagen, Germantown, MD, USA) using a Mixer Mill. RNA was quantified by measuring absorbance at 260 and 280 nm using a NanoDrop 8000 Spectrophotometer (Thermo Scientific Pierce, Waltham, ME, USA). Integrity of the RNA was checked by visual inspection of the 2 ribosomal RNAs on an agarose gel.
cDNA was synthesized using a Verso cDNA kit (cat# Ab-1453, Thermo Fischer Scientific, Waltham, MA, USA) with random hexamer primers. The levels of Epo receptor mRNA was investigated, b2microglobolin were used as housekeeping gene. For Epo receptor PCR a RealTime ready Assay (Roche Diagnostics Corporation, 9115 Hague Road, Indianapolis, IN 46250-0414 USA) was used. In this assay the following primers were used: Epo-R: 5′-GCATTGCTGATTTTGTCTGC-3′ and 5′-AAATACTGCAAGGTTGTGGTTTC-3′ together with a small Universal hydrolysis probe substituted with Locked Nucleic Acids from Roche (no152). No amplification was observed in negative control tubes (without reverse transcription or with only water). The b2microglobulin PCR was performed as a SYBR-Green assay using KAPA SYBR® FAST qPCR Kit (Kapa Biosystems, Inc. Woburn, MA, USA) with primers: 5′-GAGGCTATCCAGCG-TACTCC-3′ and 5′-AATGTCGGATGGATGAAACCC-3′.
The PCR-reactions were performed in duplicate in a LightCycler 480 (Roche Applied Science) using the following protocol: EPO-receptor assay: One step at 95°C for 3 min., then 95°C for 10 s., 60°C for 30 s., and 72°C for 11 s. b2microglobulin assay: One step at 95°C for 3 min., then 95°C for 10 s., 60°C for 20 s., and 72°C for 10 s, and finally a melting curve analysis was performed (for the b2microglobulin assay). The increase in fluorescence was measured in real time during the extension step. The relative gene expression was estimated using the default "Advanced Relative Quantification" mode of the software version LCS 480 1.5.0.39 (Roche Applied Science).
In short, western blotting was performed as follows; 10 μl homogenate was loaded onto a 4-15 % SDS gel (Criterion TGX stain-free gels, Bio-Rad, Hercules, CA, USA), followed by electro blotting onto a PVDF membrane. The stain-free technology was used to ensure equal loading [18]. Membranes were blocked with 2.5 % skimmed milk for 2 h before the primary antibody was added and incubated overnight at 4°C. The following primary antibodies were used: From Cell signaling, Danvers, MA, USA; phospho-

Haematoxylin/Eosin staining
To evaluate adipocyte morphology, selected WAT biopsies from the prolonged study was fixed in cold (4°C) 4 % formaldehyde (pH 7.0) for 2 days and embedded in paraffin, after which sections of 3 μm were obtained. After de-waxing and rehydration, the sections were stained with Haematoxylin and Eosin and examined under an Olympus light microscope (Olympus BX50).

Statistics
Due to a low sample size and non-normally distributed data, a Wilcoxon signed-rank test was used to test for treatment effect on intracellular signaling in the acute study. Results are shown as median and 25 % and 75 % percentiles. A two-way ANOVA was used to analyze results from the prolonged study, QQ-plots and plots of residuals vs. the fitted values checked normality, and data were log-transformed when not normally distributed. The level of significance was set to p < 0.05. Results are presented as means ± SE. Statistical analyses were made in STATA version 12 (StataCorp, Collage Station, TX, USA) and graphical presentations were made in Sigmaplot version 11.0 (Systat Software, San Jose, CA, USA).

Epo-R mRNA and protein in subcutaneous WAT
Epo-R mRNA was detected in WAT from all subjects (average ct level of 29.0 cycles), as well as in the positive K-562 cells (ct level of 29.6 cycles). A slightly lower level of Epo-R mRNA was observed in bone marrow (average ct level of 29.3 cycles) (Fig. 1a).
No band corresponding to the Epo-R (59kD) was identified in any of the fat biopsies or the negative control 769-P cells (Amgen, Thousand Oaks, CA, USA), however a band was identified in the two positive controls, K-562 (Abcam, Cambridge, UK) and UT-7/Epo cells (Amgen, Thousand Oaks, CA, USA) (Fig. 1b + c).

Alterations in lipolysis and markers of metabolism after prolonged ESA treatment
Changes in both phosphorylation (Fig. 4) and total protein levels (Fig. 5) of proteins involved in lipolysis in adipose tissue after prolonged treatment with ESA were evaluated. We did not record changes in any of the proteins analyzed (two-way ANOVA, interactions p > 0.05, time effects p > 0.05, ESA effects p > 0.05).

Haematoxylin/Eosin staining of WAT after prolonged ESA treatment
Selected WAT sections after prolonged treatment with ESA were stained with haematoxylin/Eosin in order to evaluate WAT morphology and visually evaluate adipocyte size. Biopsies from 2 subjects in each group were evaluated (Fig. 6), the morphology appeared normal and

Alterations in mitochondrial proteins from WAT after prolonged ESA treatment
Different mitochondrial proteins (VDAC, HSP90, PDH, and SDHA) were measured before and after prolonged ESA treatment in order to evaluate mitochondrial protein content, as an indirect measure of mitochondrial biogenesis. None of these proteins changed in response to prolonged ESA treatment (Fig. 7). The level of uncoupling protein 1 (UCP1) was also measured as a marker of BAT in the human subcutaneous adipose biopsies. A band corresponding to UCP1 was detected in rat BAT but not in our human WAT biopsies (data not shown).

Discussion
Previous studies in mice have shown significant effects of Epo treatment on adipose tissue lipolysis and total body weight. In human studies, FFA levels is increased both after acute and prolonged rHuEpo/ESA treatment [6,7]. However, the current study did not confirm either acute or prolonged effects of rHuEpo/ESA treatment on adipose tissue lipases and signaling pathways involved in the regulation of lipolysis.

Epo-R mRNA and protein and activation of signaling pathways in WAT
Mouse WAT contains a sizable amount of Epo-R mRNA [2], and Epo treatment associates with phosphorylation of Akt [9]. Moreover, mice lacking the Epo-R in nonhematopoietic tissues exhibit significantly reduced phosphorylation levels of p38MAPK, ERK42/44, and PPARγ [2]. Furthermore, phosphorylation of ERK42/44, p38MAPK, and PPARγ in preadipocytes does not depend on Epo-R expression under normal culture conditions, but during adipocyte differentiation the expression of Epo-R increases in concomitance with increased phosphorylation of ERK42/44, p38MAPK, PPARγ, Akt, and STAT5 [2,19]. In our human study, we detected higher Epo-R mRNA levels in WAT than in bone marrow and than previously found in human skeletal muscle [13]. However, we were not able to measure any Epo-R . Two subjects were omitted from the analysis due to inadequate homogenization, thus, n = 8. No statistical significant differences in phosphorylation levels were detected between treatments using a Wilcoxon signed-rank test protein in subcutaneous WAT and no evidence of pertinent signaling cascades related to the Epo-R after acute Epo exposure. Species and dosing differences could be an explanation for these conflicting results.

Direct and indirect effects of Epo on lipolysis
The direct effects of Epo on lipolysis in WAT have not previously been investigated. Adipose triglyceride lipase (ATGL) converts triglycerides (TG) to diacylglycerol, and hormone sensitive lipase (HSL) hydrolyses diacylglycerol to monoacylglycerol, in the process of converting TG to FFA [20]. HSL and ATGL were not affected by rHUEpo/ESA treatment in the current studies.
ATGL is primarily activated by protein-protein interaction with the co-activator comparative gene identification-58 (CGI-58) [21]. G 0 /G 1 switch gene 2 (G0S2) is identified as an inhibitor of ATGL. Inhibition by G0S2 appears to be dominant to activation by CGI-58 [22,23]. In the current study, prolonged ESA treatment did not affect protein levels of CGI-58., nor did G0S2 change after acute or prolonged rHuEpo/ESA treatment.
Different lipid droplet proteins are involved in the regulation of lipolysis. Perilipin (PLIN1) regulates the activity of ATGL and HSL and their access to lipid substrates in the lipid droplet. Cidea and Cidec are negative regulators of lipolysis and promote lipid droplet stabilization and inhibit lipolysis by providing a barrier around the lipid core [20]. No alteration in PLIN1 or Cidea/Cidec was found in the current study.
Acetyl CoA Carboxylase (ACC) is involved in the regulation of beta-oxidation. In its active state ACC catalyzes the conversion of acetyl-CoA to malonyl-CoA, which inhibits beta-oxidation. Phosphorylation levels of ACC were not altered in the current studies. Insulin also affects lipolysis, with increased insulin levels inhibiting lipolysis and stimulating lipogenesis. Insulin levels were not altered in response to either acute or prolonged rHuEpo/ESA treatment [6,7]. In addition, ESA treatment did not result in a visual reduction in adipocyte morphology evaluated from haematoxylin/eosin staining. It cannot be excluded that Epo can affect lipolysis in other indirect ways not evaluated in the current study. . Two subjects were omitted from the analysis due to inadequate homogenization, and 1-2 subjects because of shortage of homogenate, thus, n = 6-7. No statistical significant differences in phosphorylation levels or total protein levels were detected between treatments using a Wilcoxon signed-rank test Fig. 4 Phosphorylation levels of pertinent proteins involved in lipolysis after prolonged ESA treatment. Western blot analysis of phosphorylation levels of pertinent proteins related to lipolysis in adipose tissue. Biopsies were collected before (black bars) and after 10 weeks (gray bars) of treatment with either placebo or ESA. Phosphorylation levels were normalized to the total protein level for the given protein. Furthermore, the relative increases were measured and the placebo-baseline measurements were set to average 100 %. No effect of ESA treatment or time was found for any of the measured phosphorylation levels (n = 9) Fig. 5 Total protein levels of proteins involved in lipolysis after prolonged ESA treatment. Western blot analysis of pertinent proteins related to lipolysis in adipose tissue. Biopsies were collected before (black bars) and after 10 weeks (gray bars) of treatment with either placebo or ESA. Data were normalized to total protein level in the gel. Furthermore, the relative increases were measured and the placebo-baseline measurements were set to average 100 %. No effect of ESA treatment or time was found for any of the measured proteins (n = 9)

Epo and metabolic activity in white adipose tissue
High-dose Epo treatment to mice leads to pronounced reductions in body weight and knock-down of the Epo-R in non-hematopoietic tissues leads to dramatic elevations in WAT amount [2,9]. In contradiction, specific knock-down of the Epo-R in mice adipose tissue did not affect either body weight or body composition [15]. In support hereof, in the current prolonged human study we did not observe any changes in weight or body composition [7].
Recently it was shown that Epo treatment leads to increased AMPKα phosphorylation both in preadipocytes and in WAT from diet-induced obese mice [24]. The increased AMPKα activity changed the NAD+/NADH ratio resulting in activation of Sirt1 followed by increased deacetylation of PGC-1α. Thus, Epo could through activation of the AMPKα-Sirt1-PGC-1α pathway acts as an energy sensor and modulate cellular redox state and energy homeostasis in adipocytes [24]. In the current study, AMPKα phosphorylation was not altered 1-h post rHuEpo administration.
Alterations in body weight and the amount of WAT could also be due to alterations in energy expenditure. In a mouse model, Epo was shown to affected activity levels, total oxygen consumption, and respiratory quotient (RQ) [2]. The authors speculated that Epo could directly stimulate the proopiomelanocortin (POMC) neurons in the hypothalamus to induce α-melanocytstimulating hormone production that exerts direct effects on food intake and energy homeostasis [2]. In support of this, adipose tissue specific deletion of Epo-R in mice did not significantly alter whole-body energy expenditure or RQ values [15]. Resting energy expenditure significantly increased in the current studies, and fat oxidation tended to be increased in relation to rHuEpo treatment [6,7]. The effect of Epo on POMC was not Fig. 6 Haematoxylin/Eosin staining of WAT after prolonged ESA treatment. Haematoxylin/eosin staining of selected WAT samples from 2 subjects treated with placebo and 2 with ESA for 10 weeks measured in our human studies, hence, we cannot conclude on the neurological effects.

Epo and mitochondrial content in white adipose tissue
Diet-induced obese mice treated with EPO exhibit increased CytC, Cpt1, and PGC-1α mRNA levels indicating an increased mitochondrial biogenesis [9]. In addition, Epo stimulation increases citrate synthase activity and fatty acid oxidation in both adipocytes and WAT from diet-induced obese mice [9]. Thus, Epo seems to increase cellular mitochondrial respiration and oxidative metabolism beyond its effects on increased oxygen transport in these animal studies. However, prolonged ESA treatment to humans did not support the hypothesis that ESA should induce alterations in mitochondrial content in humans.
In contradiction to WAT, BAT does not respond to Epo treatment in mice models [9]. However, Epo treatment seems to promote a BAT-like phenotype in WAT in mice with increased gene expression of e.g. Cidea, UCP1, UCP3, PPARα, and PGC-1α, resulting in mitochondrial biogenesis and increased uncoupled respiration [9]. In the current study, UCP1, Cidea and Cidec protein levels were not significantly altered after 10 weeks of ESA treatment, thus, ESA treatment does not seem to induce browning of WAT in humans.

Conclusion
In contrast to alluring data in mice model, the present human studies do not support direct effects of Epo on adipose tissue lipases and signaling pathways involved in the regulation of lipolysis. Although Epo-R mRNA was detected in human WAT samples, this did not translate into detectable Epo-R protein, which is compatible with the absence of evidence of Epo-R signaling. However, it has to be acknowledged that the sample size in the current study is small and the variation substantial. It remains to be tested whether prolonged administration of Epo in supra-physiological amounts to human subjects may affect adipose tissue similar to what has been observed in animal studies. Fig. 7 Mitochondrial protein levels after prolonged ESA treatment. Westernblot analysis of selected mitochondrial proteins in adipose tissue. Biopsies were collected before (black bars) and after 10 weeks (gray bars) of treatment with either placebo or ESA. Data were normalized to total protein level in the gel. Furthermore, the relative increases were measured and the placebo-baseline measurements were set to average 100 %. No effect of ESA treatment or time was found for any of the measured proteins (n = 9)


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Transarterial chemoembolization of hepatocellular carcinoma in a rat model: the effect of additional injection of survivin siRNA to the treatment protocol

Background Transarterial chemoembolization is one of the most widely accepted interventional treatment options for treatment of hepatocellular carcinoma. Still there is a lack of a standard protocol regarding the injected chemotherapeutics. Survivin is an inhibitor of Apoptosis protein that functions to inhibit apoptosis, promote proliferation, and enhance invasion. Survivin is selectively up-regulated in many human tumors. Small interfering RNA (siRNA) can trigger an RNA interference response in mammalian cells and induce strong inhibition of specific gene expression including Survivin. The aim of the study is to assess the effectiveness of the additional injection of Survivin siRNA to the routine protocol of Transarterial Chemoembolization (TACE) for the treatment of hepatocellular carcinoma in a rat model. Methods The study was performed on 20 male ACI rats. On day 0 a solid Morris Hepatoma 3924A was subcapsullary implanted in the liver. On day 12 MRI measurement of the initial tumor volume (V1) was performed. TACE was performed on day 13. The rats were divided into 2 groups; Group (A, n = 10) in which 0.1 mg mitomycin, 0.1 ml lipiodol and 5.0 mg degradable starch microspheres were injected in addition 2.5 nmol survivin siRNA were injected. The same agents were injected in Group (B,=10) without Survivin siRNA. MRI was repeated on day 25 to assess the tumor volume (V2). The tumor growth ratio (V2/V1) was calculated. Western blot and immunohistochemical analysis were performed. Results For group A the mean tumor growth ratio (V2/V1) was 1.1313 +/− 0.1381, and was 3.1911 +/− 0.1393 in group B. A statistically significant difference between both groups was observed regarding the inhibition of tumor growth (P < 0.0001) where Group A showed more inhibition compared to Group B. Similarly immunohistochemical analysis showed significantly lower (p < 0.002) VEGF staining in group A compared to group B. Western Blot analysis showed a similar difference in VEGF expression (P < 0.0001). Conclusion The additional injection of Survivin siRNA to the routine TACE protocol increased the inhibition of the hepatocellular carcinoma growth in a rat animal model compared to regular TACE protocol.

Background
Being one of the most common malignancies in the world Hepatocellular carcinoma (HCC) is estimated to be responsible for about one million deaths per year. Owing to its rapid infiltrating growth and complicating liver cirrhosis HCC usually have a poor prognosis [1]. Transarterial chemoembolization (TACE) is a widely accepted therapeutic option for HCC [2]. Survivin is an inhibitor of Apoptosis protein that functions to inhibit apoptosis, promote proliferation, and enhance invasion [3]. Under normal conditions Survivin is expressed in embryonic and fetal tissues and is barely detectable in most differentiated normal adult tissues [4]. In addition, it has been shown that Survivin expression is present in some normal adult tissue cells like T-Lymphocytes, polymorphonuclear-neutrophils, primitive hematopoietic cells and vascular endothelial cells [5,6].
Survivin is selectively up-regulated in many human tumors, an overexpression of Survivin correlates with poor outcome, treatment resistance [3] reduced disease free survival and overall survival of cancer patients [7]. It has been shown to increase resistance of tumor tissue to apoptotic stimuli mainly through a Caspase-dependent mechanism [5]. Small interfering RNA (siRNA) can trigger an RNA interference response in mammalian cells and induce strong inhibition of specific gene expression hence they can be used to inhibit cancer-related genes including Survivin [8].
In-spite of the wide acceptance of TACE a meta-analysis [9] has demonstrated that 4 of the 6 randomized controlled studies included in the analysis comparing TACE with untreated controls failed to show any impact of TACE on patient survival. Keeping into consideration that there is a great controversy regarding the best chemotherapeutic agents for TACE [10] the need for introducing new chemotherapeutic agents targeting HCC is critical.
Thus, the aim of the current study was to assess the therapeutic efficacy of the additional transarterial injection of Survivin siRNA to the regular TACE protocol compared to the regular TACE protocol in an animal model of hepatocellular carcinoma, and its effect on tumor growth, angiogenesis and vascular endothelial growth factor expression (VEGF) levels.

Animals and tumor cells
All of the experiments on animals were approved by the Regional Administrative Authority in Darmstadt, Germany.
Twenty inbred male ACI-rats (Harlan Winkelmann; Borchen, Germany) weighing 220-240 g were used. The animals were kept under conventional conditions with a temperature of 22 ± 2°C, a relative humidity of 55 ± 10 %, a dark-light rhythm of 12 h, and they were fed with standard laboratory chow and tap water ad libitum. The hepatoma cell line used in the current study was obtained from the German Cancer Research Center (DKFZ; Heidelberg, Germany). The injected cell line (Morris hepatoma 3924A) represents a rapidly growing, poorly differentiated hepatocellular carcinoma.
Survivin siRNA was provided from Ruibo Gentech Co (Wuhan, PR. China). The target sequence of Survivin siRNA is as follows: CCGAGAATGAGCCTGATTT. A dose of 2.5 nmol Survivin siRNA was stable at 2-8°C for 10 min before administration.

Tumor implantation (day 0)
The technique for tumor implantation was basically similar to that described by Yang et al. [11] with minor modifications [12]. The Morris Hepatoma 3924A tumor tissue, recovered from the passaged animals 12 days after subcutaneous implantation (corresponding to 5 × 10 6 tumor cells), was cut into small cubes about 2 mm 3 . A small sub-capsular incision on the left lateral lobe of the liver was made in the recipient ACI-rats under anesthesia. The tumor fragment was gently placed into created pocket with a small cotton swab on the liver surface and the abdominal wall was then closed.

Interventional therapy (day 13)
For interventional studies a second laparotomy was performed. By using a binocular operative microscope (M651, Leica; Wetzler, Germany), a PE-10 polyethylene Micro-catheter (inner diameter 0.28 mm, outer diameter 0.61 mm; Wenzel; Heidelberg, Germany) was retrogradely inserted into the Gastro-duodenal artery and pushed forward to the hepatic artery. Different agents were then injected into the hepatic artery using the sandwich technique (subsequent injection of Mitomycin +/− Survivin siRNA + Lipiodol + degradable starch Microspheres) within 20 min. Each group of animals received treatment according to the following protocols: MRI imaging before (on day 12) and after (on day 25) treatment was performed using a 3.0 Tesla MRI unit (Magnetom, Siemens; Erlangen, Germany) by using a wrist coil (Small field of view). T1-weighted (SE: TR/TE, 500/12 ms) and T2-weighted (TSE: TR/TE, 3870/80 ms) transverse images with a section thickness of 2 mm and 184 × 256 matrix were acquired. There was no gap between sections and no contrast medium was administered. The tumor volume was determined and evaluated in the T2-weighted image according to the formula [13]: V = 0.5 × d1 × d2 2 , where d1 is the maximum diameter of the tumor and d2 is the minimum diameter perpendicular to d1. Image evaluations and size assessments were performed by a single radiologist with more than 15 years' experience in abdominal MRI imaging and who was blinded to the group assignment of the animal in the study.

Western blot (day 25)
Western blot analysis was carried out to determine the expression level of the VEGF in the two groups. After the MRI examination, all the rats were sacrificed using an over-dose of intravenous Sodium Pentobarbital. To homogenize the tumors, Precellys Homogenizer IV (Peqlab Biotechnologie GmbH; Erlangen, Germany) at 4°C was used in a lysis buffer which is composed of 50 mM HEPES, 200 mM NaCl, 0.2 mM MgSO4, 0.4 mM Phenylmethylsulfonyl fluoride, 2 % Triton-X-100, 10 μg/mL Leupeptine, 10 μg/mL Aprotinine, 0.02 % soybean trypsin inhibitor and 0.2 mM Orthovanadate (Sigma-Aldrich; Taufkirchen, Munich, Germany). The resulting Cell lysates were centrifuged for 10 min at 12,000 × g at 4°C. Coomassie Plus protein assay kit (Pierce; Rockford, IL, USA) was used to measure the protein concentration in the supernatants. The protein concentration results were obtained Spectrophotometrically by Tecan Infinite® M 200 microplate reader (Tecan-Deutschland; Crailsheim, Germany) at 595 nm. Protein was then denatured in Laemmli sample buffer (Bio-Rad Laboratories; Munich, Germany) with β mercaptoethanol (Sigma; Taufkirchen, Germany), boiled for 5 min, and transferred on ice. Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) (50 μg per lane) was then conducted. The molecular weight standards used were PeqGold prestained protein markers IV (Peqlab Biotechnologie GmbH; Erlangen, Germany). After separation using gel electrophoresis, protein was blotted onto a Polyvinylidene Difluoride membrane (Hybond P; GE Healthcare; Munich, Germany). Blots were then blocked with 10 % low-fat milk for 1 h at room temperature followed by overnight incubation at 4°C with primary antibody from Santa Cruz Biotechnology (Rabbit polyclonal VEGF 1:200; Rabbit polyclonal MMP-9 and mouse monoclonal β Actin, (clone AC-15, 1:1000; Sigma). Blots were then washed 3 times with Towbin buffer with 0.5 % Tween 20 followed by incubation for 30 min at room temperature with secondary antibody from Millipore GmbH; Schwalbach/Ts, Germany (Polyclonal goat Anti-rabbit IgG, 1:5000; goat Anti-mouse IgG, 1:5000, both HRP conjugated). All antibodies were diluted in Towbin Buffer with 0.5 % Tween 20 and 0.5 % bovine serum albumin. Blots were then washed and incubated withEnhanced Chemiluminescence detection kit (GE Healthcare; Munich, Germany). Signal intensity was finally detected and captured by Fusion FX-7 (Vilber Lourmat, Marnee la Vallee, France), documented and analyzed by Bio1D software (Vilber Lourmat). β Actin was used as the loading control.

Statistical analysis
The mean tumor growth ratio (V2/V1, V2 tumor volume after treatment and V1 tumor volume before treatment) by MRI and the mean expression ratio (VEGF/β-actin) level of VEGF by Western blot from each group were measured and the significance of differences between the two groups were analyzed using the paired-t-test, the statistical software used was GraphPad Prism (version 3.02, La Jolla, CA, USA). Immunohistochemical staining of VEGF was evaluated using descriptive and semi-quantitative methods. The differences between both groups in the Western-Blot analysis and Immunohistochemical analysis were tested for statistical significance using the unpaired-ttest and the Wilcoxon signed rank test respectively. Differences with a p value less than 0.05 were considered statistically significant.

MRI examination
Tumor implantation was successful in 100 % of the rats. Most tumors appeared homogeneous and were hypointense on T1-weighted images and hyperintense on T2weighted images prior to treatment, after treatment the tumors appeared inhomogenous. After different interventional treatments, intrahepatic metastases developed in two of the 10 rats in group B. The means of the volume ratios (V2/V1) were 1.1313 ± 0.1381 in group A, and 3.1911 ± 0.1393 in group B. Compared to group B, group A showed a statistically significant reduction of the tumor growth within the period of observation (p < 0.0001) (Fig. 1).

Western blot analysis
VEGF expression level was lower in group A (TACE + Survivin siRNA) than in group B (TACE alone). There was a statistically significant difference between group A and group B regarding the VEGF expression by Western blot analysis (p < 0.0001) (Fig. 2).

Immunohistochemical assay
The angiogenesis of the tumor was evaluated using the Anti-VEGF antibodies. VEGF were expressed in all specimens. The Immuno-expression of the protein was confirmed by the presence of brown stained cytoplasm in tumor cells. Higher expression of VEGF in hepatocellular carcinoma was observed in group B (TACE alone) with a median histological score of 4.250 compared to group A (TACE + Survivin siRNA) with a median histological score of 2.450. The difference between both groups was statistically significant (p = 0.0020) (Table 1) (Fig. 3).

Discussion
Since the introduction of TACE as a palliative treatment in patients with unresectable HCC, it has become one of the most common forms of interventional therapy [2,10]. TACE reduces the maximum plasma concentration, lengthens the half-life, and increases the average concentration of chemotherapeutic agents in the tumor [14]. However, post-interventional metastasis and recurrence of tumors have hindered the curative effect of interventional therapeutic procedures and the long-term survival rates [2]. Moreover, a study by Kobayashi et al. [15] showed that the serum concentration of VEGF was markedly increased in patients following embolization.
Biologically, Survivin has been shown to inhibit apoptosis, enhance proliferation and promote angiogenesis. High expression levels of Survivin correlate with an increased rate of tumor recurrence and resistance to chemotherapy [16]. Several in vitro and in vivo studies have indicated that Survivin down-regulation is able to sensitize human tumor cells of different histologic origins to conventional chemotherapeutic drugs [17]. Another important point to notice is that Survivin plays an important role in response to Radiotherapy too; a high level of Survivin has been shown to increase both the resistance to Radiotherapy and the incidence of local recurrence in rectal cancer patients [18] and is associated with worsened survival in patients treated with definitive Radiotherapy for cervical cancer [7,19]. Similarly, it might be suggested that Survivin expression might have a similar effect on patient response to Radioembolization of hepatic malignancy and that inhibiting Survivin expression using Survivin siRNA might have a favorable effect on patient response to Radioembolization; similar to what was previously reported by Yang et al. [20] where the authors reported an enhanced radiosensitivity in human hepatoma cells both in vitro and in vivo in response to Survivin downregulation by Survivin siRNA. Hence combining Survivin siRNA and Radioembolization might be associated with an improved response to Radioembolization, still the medical literature is lacking such a study.
As Survivin is not a cell surface protein and does not have an intrinsic enzymatic activity, targeting of Survivin for therapeutic purposes might be expected to be difficult. In addition, crystallographic data has revealed few potential drug able sites on Survivin protein [21]. Because of the up-regulation of Survivin in malignancy and its key role in apoptosis, proliferation and angiogenesis, Survivin is currently attracting considerable attention as a new target for anti-cancer therapies. Strategies under investigation to target Survivin include antisense oligonucleotides, small interfering RNA (siRNA), ribozymes, immunotherapy and small molecular weight molecules [22].
Recently, siRNA technology holds a great promise as a therapeutic intervention for targeted gene silencing in cancer. RNA interference (RNAi) is a biological mechanism whereby the presence of double-stranded RNA (dsRNA) interferes with the expression of a particular gene that shares a homologous sequence with the dsRNA. Recent studies have provided insights into the molecular mechanisms of RNAi, in which dsRNA induces the silencing of homologous mRNA. In the cytoplasm of mammalian cells, an enzyme known as Dicer initiates RNA silencing by the breakdown of long dsRNA to generate siRNA of about 21-23 nucleotides in length. The resulting siRNAs are incorporated into an RNAinduced silencing complex (RISC) and unwound into a single-stranded RNA (ssRNA), which is followed by the degradation of ssRNA [23]. siRNA is 10-100-fold more  potent for gene silencing, making it attractive tool for silencing of target genes in cancer [24]. An Adenovirusmediated siRNA expression vector was reported to decrease Survivin expression of the established HCC tumor in nude mice. In vitro study showed that stable Survivin-knockdown inhibited cancer cell proliferation, enhanced apoptotic susceptibility, arrested cell cycle in the G1 phase and resulted in an apparent mitotic catastrophe. An additional in vivo study showed that intratumoral injection of Adenovirus-delivered Survivin siRNA suppressed tumor growth by spontaneous apoptosis of cancer cells and significantly prolonged animal survival [25]. However, in vivo systemic delivery of siRNA-based therapeutics to tumor tissues/cells remains a challenge. The major limitations against the use of siRNA as a therapeutic tool are its degradation by serum nucleases, poor cellular uptake, nonspecific immune stimulation and rapid renal clearance following systemic administration [24,26]. The current study was designed to reduce tumor progression by using a combination of transarterial administration of Survivin siRNA and TACE using the sandwich technique in an animal model of HCC. We did not include intravenous delivery approach because the transarterial route was much more efficient in the treatment of HCC. In the current study however we did not evaluate the expression of Survivin directly in the Hepatoma 3924a in addition we did not assess the degree of suppression of Survivin expression in response to treatment for several reasons. First the expression of Survivin has been reported to occur in several HCC cell lines and in human HCC tissue [27], it has been reported to be highly expressed in the vast majority of human cancers including HCC [28]. Second we relied on the radiologic oncologic assessment of the therapeutic effect of Survivin siRNA rather than on its direct assessment. A direct assessment of the suppression of Survivin was rather outside the scope of the current study. Finally Survivin expression has been shown to significantly correlate with VEGF expression in HCC [29], hence we relied on this correlation to indirectly assess Survivin suppression (through VEGF assessment). Still we regard this as one Fig. 3 The immunohistochemical staining of VEGF in hepatocellular carcinoma. a VEGF staining in hepatocellular carcinoma in the group A (TACE + survivin siRNA) (×100). b Significantly higher VEGF staining in hepatocellular carcinoma was observed in group B (control group, TACE alone) than group A (×100). c Median histological score of VEGF (N = 10) of the limitations of the study and recommend the subject for further evaluation in future studies assessing not only the response based on radiologic evidence but based on direct assessment of Survivin. Another limitation of the current study is the lack of a negative control group (receiving si-control RNA).
Survivin has been shown to play an important role in the regulation of expression of VEGF in breast cancer lymphatic metastases [30]. In addition it has been shown to significantly correlate with the expression with VEGF in hepatocellular carcinoma and promote the expression of VEGF [28,29]. Our experimental results demonstrated that groups A (TACE + Survivin siRNA) showed a significant reduction of tumor growth in the period of observation compared to the control group (TACE alone). Higher Immunohistochemical expression of VEGF in hepatocellular carcinoma was observed in group B (TACE alone) than that of group A (TACE + Survivin siRNA) (P < 0.01). The invasive progression of tumor cells in group A was noticeably inhibited compared with group B. This complex targeting treatment method seemed to overcome the insufficiency of TACE and improve the overall therapeutic effects.

Conclusion
In conclusion, the additional injection of Survivin siRNA to the routine protocol of TACE has shown to be safe and effective in an animal model of HCC in rats. This combined therapeutic therapy noticeably inhibited the growth of the hepatic carcinoma in rats compared with TACE alone. Further experimental studies will be required to fully understand the benefits and risks of this strategy for treating HCC before transferring it to human studies.

Ethics approval and consent to participate
The current animal study was approved by the Regional Administrative Authority in Darmstadt, Germany.

Consent for publication
The current study does not contain any personal materials that require patient approval.

Availability of data and materials
Due to organizational restrictions the data will not be available.