extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis

The role of the endothelium in protecting from chronic liver disease and TGFβ-mediated fibrosis remains unclear. Here we describe how the endothelial transcription factor ETS-related gene (ERG) promotes liver homoeostasis by controlling canonical TGFβ-SMAD signalling, driving the SMAD1 pathway while repressing SMAD3 activity. Molecular analysis shows that ERG binds to SMAD3, restricting its access to DNA. Ablation of ERG expression results in endothelial-to-mesenchymal transition (EndMT) and spontaneous liver fibrogenesis in EC-specific constitutive hemi-deficient (ErgcEC-Het) and inducible homozygous deficient mice (ErgiEC-KO), in a SMAD3-dependent manner. Acute administration of the TNF-α inhibitor etanercept inhibits carbon tetrachloride (CCL4)-induced fibrogenesis in an ERG-dependent manner in mice. Decreased ERG expression also correlates with EndMT in tissues from patients with end-stage liver fibrosis. These studies identify a pathogenic mechanism where loss of ERG causes endothelial-dependent liver fibrogenesis via regulation of SMAD2/3. Moreover, ERG represents a promising candidate biomarker for assessing EndMT in liver disease.

C hronic liver disease (CLD) is an increasing global health burden; in the UK, liver disease is the fifth biggest cause of mortality with rates doubling from 1991 to 2007 1 . CLD can be triggered by numerous factors including diet, alcohol, viral infection and genetic disorders, which share common features including excessive inflammation 2 , dysregulated transforming growth factor (TGFβ) signalling 3 and dramatic disruption in the vascular architecture of the liver 4,5 . Recent studies have revealed that disruption of endothelial cell (EC) homoeostasis can initiate tissue damage and fibrosis [6][7][8] . Notably, EC have been shown to lose their lineage-specific markers and morphology and acquire a mesenchymal-like phenotype in a process termed endothelial-tomesenchymal transition (EndMT) 9 . EndMT is associated with human pathologies such as early vein-graft rejection 10 and atherosclerosis 11 , where it correlates with disease severity. EndMT has been shown to be induced by TGFβ in vitro and in vivo 10,12 , and to be enhanced by inflammatory mediators including TNF-α 13,14 .
The ETS transcription factor family plays important roles in vascular development and angiogenesis 21 . The ETS-related gene (ERG) is the most abundant ETS factor in adult ECs and is essential for endothelial lineage identity; indeed, it is one of only three transcription factors required for reprogramming of progenitors to endothelium 22 . ERG is crucial for embryonic development and vascular stability 23,24 , angiogenesis 25 and protection from vascular inflammation 26,27 . ERG exerts its pro-homoeostatic, anti-inflammatory function in ECs by driving expression of homoeostatic genes while repressing pro-inflammatory gene expression and inhibiting cytokine-induced EC activation 21,28 . In turn, pro-inflammatory molecules TNF-α 26 and lipopolysaccharides (LPS) 27 induce a significant loss in ERG expression in ECs, suggesting that regulation of ERG expression is key to control of the balance between endothelial homoeostasis and inflammatory signalling.
In this study, we show that ERG maintains the homoeostatic balance of SMAD-dependent signalling in the endothelium, by promoting the SMAD1 pathway while repressing SMAD2/3 activity. We show that loss of ERG in vitro and in vivo results in spontaneous EndMT which is dependent on enhanced SMAD2/3 activity. TNF-α blockade protects ERG expression and reduces phosphorylation of SMAD2/3 in an acute model of liver fibrosis, but is ineffective in ERG hemi-deficient mice. Finally, we show that ERG expression is lost in liver EC from cirrhotic patients with fibrosis related to alcoholic liver disease (ALD) or primary biliary cirrhosis (PBC) and inversely correlates with increased markers of EndMT. Therefore, this study identifies a central role for ERG in regulating EC canonical TGFβ-SMAD signalling to prevent EndMT and ultimately tissue fibrogenesis. Loss of endothelial ERG expression is an early, causative event during tissue fibrogenesis, linked to inflammatory pathways, which can be targeted therapeutically.

Results
ERG regulates SMAD signalling in EC and protects from EndMT. Gene ontology analysis of transcriptome data from ERG-deficient human umbilical cord endothelial cells (HUVEC) 25 identified 41 target genes associated with TGFβ/BMP canonical signalling as putative ERG targets (Supplementary Table 1; Fig. 1a). qRT-PCR validation of selected hits and other relevant genes is show in Fig. 1b. Inhibition of ERG in HUVEC caused a reduction in gene expression of SMAD1, ACVRL1 (ALK1), endoglin (ENG) and inhibitor of DNA binding 1 (ID1). This response was mirrored by elevated expression of TGFβ isoforms 1 and 2 and of mesenchymal genes collagen A1 (Col1A1), smooth muscle actin (SMA) and smooth muscle calponin (CNN1). Gene expression levels of other signalling SMADs (5 and 8) were not significantly changed in ERG-deficient cells (Supplementary Fig. 2A). A second siRNA (#2) targeting ERG generated a similar mRNA profile ( Supplementary Fig. 2B). Protein levels of SMAD1 and its associated proteins, ENG and ID1, were also significantly reduced (Fig. 1c, d and Supplementary Fig. 2C). Loss of SMAD1 expression was confirmed in vivo, within liver tissue from EC-specific constitutive Erg hemideficient mice 23 (Erg cEC-Het ; Fig. 1e; arrows). Gene and protein expression levels of SMAD3 and its receptor TGFBR1/ALK5 were unaffected ( Fig. 1b-d). However, there was increased expression of the downstream targets SMA (Fig. 1d) and TGFβ2 (Fig. 1f). Furthermore, immunofluorescence microscopy revealed increased TGFβ2 expression in ERG-deficient EC in vitro (Fig. 1g) and in large blood vessels in the liver of Erg cEC-Het mice in vivo ( Fig. 1h; arrows). The profile of ERG-deficient cells, namely marked upregulation of mesenchymal markers and downregulation of endothelial lineage identity markers, together with transition from the typical rounded/cobblestone morphology to a distinct mesenchymal-like shape 25 , suggest that the cells are undergoing EndMT. Thus, ERG reciprocally regulates TGFβ/ BMP canonical signalling in EC, by driving the SMAD1 pathway while repressing the SMAD2/3 pathway to protect EC from EndMT.
ERG promotes SMAD1 whilst repressing SMAD2/3 signalling in EC. To assess whether ERG inversely regulates SMAD1 and SMAD2/3 transcriptional activity, we used a SMAD1-dependent BMP Response Element luciferase reporter (BRE reporter) 29 and a SMAD2/3-dependent SMAD-Binding Element luciferase reporter (SBE reporter) 30 . The SMAD2/3 activator TGFβ2 induced enhanced transactivation activity of the SBE reporter ( Supplementary Fig. 3A), with comparable effects at 1 and 10 ng ml −1 TGFβ2. All following experiments were carried out with 10 ng ml −1 TGFβ2, in line with previous in vitro studies on EndMT 31,32 . Ligand specificity of BMP9 (1 ng ml −1 ) and TGFβ2 (10 ng ml −1 ) was shown by selective induction of their target genes ENG and CNN1, respectively ( Supplementary Fig. 3B). In control siRNA transfected cells, treatment with BMP9-induced a robust BRE-, but no SBE-mediated signal, whilst TGFβ2 induced the opposite response, as expected (Fig. 2a, b). Inhibition of ERG expression caused a significant reduction in SMAD1-dependent BRE transactivation following BMP9 treatment (Fig. 2a); this was paralleled by decreased phosphorylation of SMAD1 in response to BMP9 (Supplementary Fig. 3C) and TGFβ2 (Supplementary Fig. 3D; quantified Supplementary Fig. 3E), suggesting that ERG regulates SMAD1 levels and activity. Notably, ERG overexpression did not further enhance BMP9-induced BRE reporter transactivation activity (Fig. 2c). TGFβ2 induced a modest increase in SBE reporter transactivation, which was significantly enhanced following the deletion of ERG by siRNA (Fig. 2b). Conversely, ERG-overexpression completely inhibited TGFβ2-induced SBE reporter transactivation (Fig. 2d). Basal phosphorylation of SMAD3 was increased in ERG-deficient HUVEC compared to control ( Supplementary Fig. 3D, F, G). These data indicate that ERG promotes SMAD1 expression and activity, while repressing SMAD2/3 activity through a non-transcriptional mechanism (Fig. 2e).
To investigate whether ERG modulation of SMAD2/3 activity was mediated by the formation of a regulatory complex, we assessed protein interaction by co-Immunoprecipitation (Co-IP)   Fig. 2g) isolated from transplant patients with end-stage liver disease. Interestingly, ERG was found to also bind to SMAD2 (Fig. 2f), while no complex formation was observed with SMAD1 in either cell type ( Fig. 2f and Supplementary Fig. 4A). TGFβ2 treatment resulted in the phosphorylation and translocation of SMAD3 into the nucleus of both HUVEC ( Supplementary  Fig. 4B) and HSEC ( Supplementary Fig. 4C). This was paralleled by increased ERG/pSMAD3 complex formation assessed by Co-IP ( Fig. 2f; Supplementary Fig. 4A). PLA confirmed that TGFβ2 treatment induces ERG-pSMAD3 interaction within the nucleus of HUVEC (Fig. 2h) and ERG-SMAD3 in both cell types ( Supplementary Fig. 4D); no co-localisation was observed between ERG and SMAD1 (Supplementary Fig. 4D; controls Supplementary Fig. 4E). Inhibition of SMAD2/3 phosphorylation by pretreatment with SB-431542 prevented ERG-SMAD3 complex formation (Fig. 2f), suggesting that this interaction is dependent on SMAD3 phosphorylation. Selective removal of nucleic acids with benzonase treatment from the protein lysates ( Supplementary Fig. 5A) did not significantly de-stabilise this interaction ( Supplementary Fig. 5B), suggesting that the ERG-SMAD3 interaction can be maintained in the absence of DNA. Notably, SB-431542 normalised the enhanced expression of SMAD3-target genes TGFβ2 and CNN1 in ERG-deficient HUVEC (Fig. 2i) and HSEC (Fig. 2j), indicating that the EndMT phenotype in ERG-deficient EC is dependent on SMAD2/3 activity. Thus, these data demonstrate that ERG inhibits SMAD2/ 3 activity by direct interaction and formation of an inhibitory complex.
ERG regulates SMAD3-DNA binding to repress SMAD3 activity. Bio-informatic analysis of ERG-repressed-SMAD2/3driven target genes TGFβ2 and CNN1 revealed the presence of highly conserved ERG DNA binding motifs, upstream of the transcription start site (TSS), which aligned with histone marks for active promoter regions, namely H3K4Me3, H3K27Ac, H3K9Ac and RNA polymerase II (RNA Pol2) occupancy [from Encyclopaedia of DNA Elements (ENCODE)] (Fig. 3a, b). We investigated binding of ERG and SMAD3 to these regions, which also contain several SMAD consensus motifs ( Supplementary  Fig. 6A, B). Chromatin immunoprecipitation (ChIP)-qPCR showed that in unstimulated HUVEC both ERG (Fig. 3c) and SMAD3 (Fig. 3d) are significantly enriched on the promoters of TGFβ2 and CNN1. TGFβ2 stimulation significantly reduced ERG enrichment ( Fig. 3c) with concurrent increase in SMAD3 enrichment (Fig. 3d). ERG siRNA treatment led to the loss of ERG enrichment, as expected (Fig. 3e) and resulted in a significant increase in the binding of SMAD3 to the promoters of both target genes (Fig. 3f). These data indicate that ERG inhibits SMAD3 binding to DNA at these target gene promoters (model in Supplementary Fig. 6C).
To determine whether the loss of endothelial ERG expression could influence tissue fibrogenesis in vivo, we assessed tissues from EC-specific constitutive Erg hemi-deficient mice (Erg cEC-Het ) 23 and EC-specific inducible Pdgfb-iCreER-eGFP/Erg fl/fl mice (Erg iEC-KO ; genotype data in Supplementary Fig. 7A, B). Increased collagen deposition and elevated SMA expression was most pronounced in the liver of both transgenic lines (Fig. 4a). We observed that reduction or deletion of EC-Erg in both strains caused disrupted portal tracts (schematic Fig. 4b), with significantly increased peri-portal collagen deposition (Fig. 4c) and SMA expression ( Fig. 4d; quantification Fig. 4e, f; each genotype was compared to Erg fl/fl ). Lineage tracing in Erg iEC-KO mice revealed eGFP + SMA + EC (Fig. 4g, arrow), a sign of spontaneous EndMT, which was confirmed by quantification of CD31 + SMA + double positive cells (representative image Supplementary  Fig. 7C, open arrows; quantified in Fig. 4h).
Activation of SMAD3 signalling was observed in the liver endothelium in Erg-deficient lines, corresponding to the in vitro results. pSMAD3 staining, undetectable in littermate control samples, was increased in both Erg cEC-het and Erg iEC-KO mice ( Fig. 5a, b), in EC as well as surrounding tissue. This was accompanied by proliferation of biliary cells, detected by Ki67 expression, a sign of tissue dysfunction ( Supplementary Fig. 7C, D). These data suggest that loss of EC-ERG induces both autocrine and paracrine responses through SMAD3 activation. In parallel with the in vitro studies (Fig. 2i, j), systemic administration of the ALK5 inhibitor SB-431542 abolished spontaneous SMAD3 phosphorylation in Erg cEC-het mice ( Supplementary  Fig. 8A). Furthermore, in vivo SB-431542 treatment normalised TGFβ2 expression in isolated primary mouse EC (Fig. 5c) and normalised both SMA expression (Fig. 5d, e) and collagen deposition ( Fig. 5f and Supplementary Fig. 8B) compared with vehicle (DMSO)-treatment in Erg iEC-KO mice. These data show that loss of endothelial ERG expression causes enhanced SMAD3 activity in both EC and surrounding tissue, resulting in spontaneous EndMT and a pro-fibrotic microenvironment within the liver.
Loss of ERG in murine and human fibrotic liver disease. The finding of spontaneous EndMT and fibrogenesis in Erg-deficient mice led us to investigate whether endothelial ERG plays a role in the pathophysiology of liver fibrosis, using a carbon tetrachloride (CCL 4 )-induced mouse model. Chronic administration of CCL 4 for 8 weeks resulted in characteristic bridging fibrosis (Supplementary Fig. 9A), increased SMA ( Fig. 6a, b) and pSMAD3 expression ( Supplementary Fig. 9B) in peri-portal areas. Chronic CCL 4 treatment caused loss of ERG expression in all EC, which was partially restored following an additional 3-week recovery period (Fig. 6a, c). ERG expression is downregulated by inflammatory stimuli such as TNF-α 26 and LPS 27 . As elevated inflammatory cytokines are associated with fibrogenesis, we assessed the impact of TNF-α inhibition on ERG expression (Fig. 6d) and Fig. 1 Differentially expression of canonical TGFβ/BMP-SMAD genes in ERG-deficient HUVEC. a Microarray analysis of ERG-dependent genes in HUVEC was performed at 24 and 48 h after ERG depletion, as described (n = 3 biological replicates) 24 ; fold change (log 2) of selected TGFβ/BMP associated genes represented as high (red) and low (blue) expression compared to the median (white). Gene expression data were validated in HUVEC transfected with Control (Con siRNA) or ERG siRNA by b quantitative PCR and c immunoblotting, showing reduction of ERG protein levels following siRNA, d quantification by fluorescence intensity normalised to GAPDH for ERG, SMAD1, ENG, ID1, SMAD3, ALK5 and SMA (n = 5). e Representative images of SMAD1 expression (white; arrows identify expression in the sinusoidal endothelium), VWF (green), SMA (red) and DAPI (blue) in liver tissue from Erg fl/fl and Erg cEC-het mice (Scale bar 50 μm). f Quantitative analysis of TGFβ2 protein expression was performed by ELISA in whole cell HUVEC lysates (n = 7). g Representative image of TGFβ2 expression in HUVEC transfected with control or ERG siRNA by immunofluorescence; nuclei are identified by DRAQ V and cells are co-stained for ERG (green). Scale bar 20 µm. h Representative images of TGFβ2 expression (white; arrows) in large vessel within the liver from Erg fl/fl and Erg cEC-het mice (Scale bar 50 μm). Data were normalised to GAPDH and compared to control siRNA treated (*) by unpaired t-test. All graphical data are mean ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001 SMAD3 activity (Fig. 6e) by co-administration of the TNF-α antagonist etanercept in an acute CCL 4 -induced liver injury. A single CCL 4 injection caused a significant increase in SMA (Fig. 6f), reduction of ERG expression (Fig. 6g) and enhanced phosphorylation of SMAD3 at 48 h ( Fig. 6h and Supplementary  Fig. 9B); co-administration of etanercept significantly reduced CCL 4 -induced SMA expression (Fig. 6f), normalised endothelial ERG expression (Fig. 6g) and reduced phosphorylation of SMAD3 ( Fig. 6h and Supplementary Fig. 9C). However, etanercept was unable to correct the CCL4-induced injury in Erg cEC-het mice (Fig. 6i), with no normalisation of transcriptional target genes (Fig. 6j) or SMA expression in the liver (Fig. 6k).   These data suggest that the TNF-α antagonist protects from CCL 4 -induced tissue injury through the endothelial ERG pathway, and reveal that ERG expression is an early, sensitive marker of vascular inflammation during liver fibrogenesis, which can be modulated by clinically relevant therapies. Finally, to verify the relevance of this pathway in human diseases associated with fibrosis, we obtained liver tissue samples from end-stage liver transplant patients with alcoholic liver disease (ALD), non-alcoholic steato-hepatitis (NASH) or primary biliary cirrhosis (PBC); non-fibrotic tissue from separate donors was used as control. Liver sections were profiled by immunofluorescence microscopy for CD31, SMA and ERG expression (Fig. 7a). In non-fibrotic human liver samples, ERG was expressed in both vascular and sinusoidal EC, with characteristic nuclear localisation ( Supplementary Fig. 10A, B). ERG expression was significantly decreased in CD31 + EC from ALD and PBC patient samples compared to non-fibrotic liver samples; in samples from NASH patient the decrease in EC-ERG expression did not reach significance ( Fig. 7b; additional images provided in Supplementary Fig. 11A-D). EndMT, identified by CD31 + SMA + double positive cells, was identified in liver fibrosis associated with ALD and PBC but not in NASH patients (Fig. 7c). Analysis of CD31 + SMA + co-localisation with ERG expression in all patient samples revealed a significant negative correlation between ERG expression and EndMT (Fig. 7d). Therefore, loss of ERG expression appears to be a sensitive marker of inflammatorydriven fibrogenesis and could represent a novel biomarker for EndMT in human fibrotic liver.

Discussion
In this study, we report that the endothelial transcription factor ERG controls the homoeostatic balance of TGFβ/BMP canonical signalling in vitro and in vivo; loss of endothelial ERG results in EndMT and spontaneous liver fibrosis in mouse models and inversely correlates with EndMT and liver fibrosis in patients.
Expression profiling of ERG-deficient HUVEC identified dysregulation of genes involved in SMAD1 and SMAD2/3 signalling. Expression of EC-restricted genes SMAD1 and its co-receptors ACVRL1 and ENG was reduced in ERG-deficient HUVEC, in line with the previous report of ENG as a direct ERG target 33 . Mutations in ACVRL1 and ENG cause hereditary haemorrhagic telangiectasia (HHT), a disease characterised by vascular malformations 34,35 , in agreement with the model that this arm of the BMP/TGFβ pathway is required for vascular homoeostasis. Our data reveal that loss of ERG expression significantly affects SMAD1 phosphorylation and transcriptional activity. Co-IP and PLA assays revealed that ERG does not interact with SMAD1, suggesting that ERG is primarily required for the expression of key proteins in the SMAD1 pathway.
In ERG-deficient EC, loss of SMAD1-dependent transcription was accompanied by enhanced expression of SMAD2/3 target genes, indicating an inverse relationship between ERG and SMAD2/3-dependent targets. This shift in SMAD signalling is consistent with an EndMT phenotype, characterised by the loss of EC identity and the emergence of a mesenchymal gene expression profile 9, 10, 13 . We demonstrate that ERG directly represses SMAD2/3 transcriptional activity, independently of expression of SMAD2/3 or its receptor ALK5. We find that ERG can interact with SMAD2/3 in HUVEC and in HSEC in agreement with studies that show overexpression of exogenous ERG and SMAD3 results in protein interaction 36,37 . ERG and SMAD3 both bind to the promoters of target genes, indicating that ERG acts as a co-repressor for SMAD3. These data are in line with a study mapping SMAD2/3 global DNA binding in keratinocytes, which revealed that ETS motifs are represented in~50% of SMADenriched genes 38 , suggesting that ETS/SMAD dual motifs may be important in the regulation of TGFβ/SMAD signalling.
Previous work investigating the regulation of TGFβ/BMP signalling by ERG in vivo has focused on developmental biology. ERG was shown to be involved in endocardial-mesenchymal transition in cardiac valve morphogenesis 39 and for TGFβmediated differentiation of sclerotome cells during vertebrae bone development 36 . Here we utilised two EC-specific Erg-deficient mouse strains to assess the role of ERG in regulating SMAD signalling in post-natal liver physiology; this is the first study to identify ERG expression in liver vascular and sinusoidal EC. Both strains displayed deformation of portal tracts with increased collagen deposition, SMA and pSMAD3 expression confirming a pro-fibrotic phenotype. Furthermore, analysis of CD31 + SMA + double positive EC and lineage tracing indicates that Erg-deficient EC undergo spontaneous EndMT in vivo, which was dependent on SMAD3, as shown by rescue of the phenotypes with SB-431542. Interestingly, endothelial ERG deficiency caused enhanced pSMAD3 not only in the endothelium, but also in the surrounding tissue. Thus, these data suggest that ERG is central to EC-specific regulation of SMAD2/3 and promotes tissue homoeostasis by preventing a paracrine TGFβ2-SMAD3 positive feedback loop, resulting in a pro-fibrotic tissue microenvironment.
In a murine model of acute and chronic CCL 4 -induced liver fibrosis, we observed a rapid, marked and persistent reduction in ERG expression. Notably, ERG expression was partially restored in recovering tissue (3 weeks after terminating chronic CCL 4 administration) and protected by co-administration of the TNF-α inhibitor etanercept. By profiling ERG expression in human liver, we found marked loss of ERG expression in end-stage liver tissues from ALD and PBC patients. These diseases have been associated with inflammatory pathways 40,41 . Loss of ERG expression could be due to chronic vascular inflammation; a similar observation Fig. 2 ERG differentially regulates transcriptional activity of SMAD1 and SMAD3. ERG-dependent regulation of SMAD1 and SMAD3 activity were assessed by transactivation assays using either SMAD1 reporter pGL3-BRE (BRE reporter, a and c) or SMAD3 reporter pBV-SBE4 (SBE reporter, b and d). HUVEC were either co-transfected with Control siRNA (Con siRNA) or ERG siRNA for 24 h (a and b) or with pcDNA or ERG3 overexpression construct (ERG3) (c and d) prior to treatment with TGFβ2 (red) and BMP9 (blue) or PBS (dashed line) for 18 h (data from pooled HUVEC in triplicate experiments). The ratio of luciferase to renilla from each transfection was normalised to PBS-treated control or to groups co-transfected with (a and b) Con siRNA or (c and d) pcDNA (n = 3). e Schematic of the inverse regulation of SMAD1 and SMAD3 by ERG. f Protein−protein interactions between ERG, SMAD1, SMAD2 and SMAD3 were assessed by Co-IP assay in whole cell lysate from HUVEC. SB-431542 (SB; 10 μM) treatment was performed for 1 h and TGFβ2 (10 ng ml −1 ) or PBS treatments were performed 30 min prior to lysis. Lysates were immunoprecipitated with mouse IgG or mouse α-ERG and then immuno-blotted for α-ERG, α-SMAD1, α-SMAD2, α-SMAD3 and α-pSMAD3 (Ser423/425). Images representative of four experiments. g ERG-SMAD3 interaction was assessed by Co-IP in untreated HSEC. h Cellular localisation of the interaction between ERG with pSMAD3 was investigated by Proximity Ligation Assay (PLA) following 30 min TGFβ2 treatment in HUVEC (n = 2). i HUVEC or j HSEC were pre-treated with SB-431542 or DMSO prior to transfection with either control siRNA or ERG siRNA for 48 h and analysed by qPCR (n = 3). Data were normalised to GAPDH and compared to control siRNA treated (*) or to ERG siRNA treated with DMSO (#) by unpaired t-test. All graphical data are mean ± s.e.m., * or # P < 0.05, ** or ## P < 0.01, *** or ### P < 0.001 was reported in the endothelium overlaying human atherosclerotic plaques 26 . Anti-TNF therapy has been effective in preclinical liver fibrosis models 42, 43 ; furthermore, etanercept has shown some efficacy in a case study of PBC associated with rheumatoid arthritis 44 . Since ERG expression can be downregulated by inflammatory agents, it is likely that inflammation plays a key role in the loss of ERG expression in these patients. Interestingly, etanercept could restore ERG expression and reduce SMAD3 activity in the in vivo model of CCL 4 -induced liver fibrosis in wild-type mice, but was ineffective in Erg cEC-het mice, suggesting that anti-TNF therapies exert a beneficial effect by restoring endothelial homoeostasis through the ERG pathway.
In conclusion, our findings identify ERG as a novel EC-specific regulator of canonical TGFβ/BMP, promoting EC-dependent tissue homoeostasis. We provide evidence that loss of ERG expression is an early, causative event during liver fibrogenesis. We describe EndMT in human liver disease, which showed pathological differences between ALD, PBC and NASH patients. Therefore, we propose that ERG may represent a sensitive tissue biomarker to monitor vascular dysfunction and influence therapeutic strategies.

Methods
Cell culture. Primary HUVEC were collected from umbilical cords using 0.5 mg ml −1 Collagenase dissolved in HBSS warmed to 37°C. Isolated cells were cultured in M199 media supplemented with Endothelial cell growth supplement (Sigma) and 20% bovine calf serum 22 . Hepatic sinusoidal endothelial cells (HSEC) were isolated from ∼30 g human liver tissue obtained from explanted livers collected from patients in the Liver Unit at Queen Elizabeth Hospital in Birmingham with informed consent and ethics committee approval. HSEC were then cultured in medium composed of human endothelial basal growth medium (Invitrogen), 10% AB human serum (HD Supplies), 10 ng ml −1 vascular endothelial growth factor (VEGF), and 10 ng ml −1 hepatocyte growth factor (HGF) (PeproTech). Human ERG expression was repressed by transfection of cells with 20 nM siRNA against ERG exon 6 (Qiagen; 5′-CAGATCCTACGCTATGGAGTA-3′) or a second siRNA (#2) targeting exon 7 (Invitrogen; 5′-ACTCTCCACGGTTAATGCATGCTAG-3′)   c d e f Fig. 5 Peri-portal ERG-deficient mouse fibrogenesis phenotype is SMAD3-dependent. a, b SMAD3 activity in portal tract regions was assess by immunofluorescence for pSMAD3 (white), VWF (green), SMA (red) and DAPI (blue) in Erg cEC-het and Erg iEC-KO mice, respectively, aged 8-10 weeks. Quantification of pSMAD3 area (three fields per mouse, n = 3). SB-431542 (10 mg kg −1 , i.p) was administered in Erg cEC-het mice three times a week for 2 weeks. Scale bar 20 μm. c mRNA was isolated from CD31 + murine EC isolated from lung tissue and analysed by qPCR (n = 4). Data were compared to EC from Erg fl/fl littermate controls (*) or to DMSO treated Erg cEC-het (#) by one-way ANOVA and Bonferroni multiple comparison post-test. d Representative images of portal tract VWF (grey scale; green in merge), SMA (grey scale; red in merge), ERG (white), DAPI (blue) and merged panels (Scale bar 50 µm). e Quantification of SMA expression (5 fields per mouse, n = 4). f Quantitative analysis of Picro-sirius red positive tissue, using polarised light (more representative images in Supplementary Fig. 8B). Data were compared to Erg fl/fl littermate controls (*) or to DMSO treated Erg cEC-het (#) by one-way ANOVA and Bonferroni multiple comparison post-test. All graphical data are mean ± s.e.m., * or # P < 0.05, ** or ## P < 0.01, *** or ### P < 0.001 of the ERG locus, both are denoted in the text as ERG siRNA. In parallel, an AllStars Negative Control siRNA (Qiagen) was used.
Pharmacological growth factor in vitro cell treatments. SB-431542 was purchased from Sigma (UK). SMAD3-dependent SMAD-Binding Element (SBE) reporter (SBE4-Luc) was a gift from Bert Vogelstein (Addgene plasmid #16495) 30 . pBV-Luc empty vector, lacking a promoter sequence, was a gift from Bert Vogelstein (Addgene plasmid #16539) and was used as a control 45 . BMP response element (BRE) luciferase reporter (pGL3-BRE Luciferase) was a gift from Martine Roussel and Peter ten Dijke (Addgene plasmid #45126) 29  Human liver tissue sections. Fibrotic human liver tissue was collected from patients undergoing liver transplantation surgery at the Queen Elizabeth Hospital in Birmingham for end-stage liver diseases including non-alcoholic steatohepatitis (NASH), primary biliary cholangitis (PBC) and alcohol related liver disease (ALD). Non-fibrotic tissues were used as control. All tissues were accessed with informed written patient consent and research ethics committee approval provided by the Human Biomaterials Resource Centre at the University of Birmingham, UK. Tissue was formalin fixed upon collection from patients and paraffin embedded for immunohistochemical analysis.
Mice and breeding. A Cre/LoxP strategy was used to develop a constitutive EC-specific heterozygous deletion of Erg using Tie2-Cre-Erg fl/+ (Erg cEC-Het ) 23 or an inducible homozygous deletion of Erg using Pdgfb-iCreER-eGFP/Erg fl/fl mice (Erg iEC-KO ). All experiments with Erg-deficient mice were conducted with age and gender matched animals at Imperial College London in accordance with the UK Animals (Scientific Procedures) act of 1986. All animals used were retained on a C57BL/6 background. Both male and female mice were used for experiments and were 8-10-weeks old. All experiments were conducted using littermate controls denoted in the text as Erg fl/fl .
Isolation of mouse lung endothelial cells. Primary mouse lung endothelial cells were isolated from the lungs of control Erg fl/fl and Erg cEC-het mice. Lungs were dissociated using GentleMACS C tubes and GentleMACS Dissociator (Miltenyi Biotec), digested with 0.1% collagenase type I (Invitrogen), and sieved through a 70 μm-pore cell strainer (BD Falcon). Magnetic immunosorting (Dynabeads; Invitrogen) was used to select EC by a negative sort for FcγRII/III receptor-positive macrophages followed by a positive sort for CD31-positive endothelial cells. Cells were plated in flasks precoated with a mixture of 0.1% gelatin (Sigma) and human plasma fibronectin (Chemicon) and cultured with EGM-2 media (Lonza).
Carbon Tetrachloride CCL 4 liver injury model. C57BL/6 J mice were obtained from Harlan OLAC LTD or from in-house colonies. Acute hepatic inflammation was induced using intraperitoneal injections of CCl 4 (carbon tetrachloride) (Sigma-Aldrich). CCl 4 was diluted 1:4 with mineral oil, and injected intraperitoneally (i.p) at a concentration of 1 ml kg −1 (control animals were treated with i.p mineral oil alone). For chronic models, mice were injected with CCl 4 IP bi-weekly for 8 weeks. Indicated mice were allowed to recover for a further 3 weeks before the livers were collected. Experiments requiring TNF-α blockade were conducted using etanercept (10 mg kg −1 ) (Amgen). Mice were injected with etanercept intravenously (i.v.) 30 min prior to single CCl 4 administration, control animals were given PBS alone and liver tissues collected 48 h post-injection. All CCL 4 experiments were conducted at University of Birmingham with experimental procedures undertaken in 6-8-week-old mice and used under procedure in accordance the Animals (Scientific Procedures) Act of 1986.
Immunofluorescence analysis of tissue and HUVEC. Tissues were fixed in 4% paraformaldehyde for 2 h at room temperature, then transferred to 70% ethanol prior to being embedded in paraffin. Sections were de-paraffinised with Histoclear, re-hydrated for hematoxylin and eosin and picro-sirius red or heated to 95°C for 10 min with DAKO antigen retrieval solution for immunofluorescence staining. All sections were blocked with 2% BSA prior to incubation with primary antibodies (listed in Supplementary Table 2). HUVEC for immunofluorescence and proximity ligation assay (PLA) were grown on gelatinised coverslips in 12-well plates. Cells were fixed with 4% paraformaldehyde for 15 min prior to blocking with 3% BSA for 1 h. PLA was performed by manufacturer's instructions (Duolink Sigma). Primary antibodies listed in Supplementary Table 2. All tissue sections and HUVEC were mounted with Prolong Diamond (Molecular Probes) and allowed to dry overnight before imaging. Confocal microscopy was carried out on a Carl Zeiss LSM780. Images were analysed with ImageJ (NIH) and Volocity software (PerkinElmer).
Immunoblotting analysis. CelLytic reagent (Sigma), supplemented with 1 mM phenylmethylsufony fluoride (PMSF) protease inhibitor cocktail and phosphatase inhibitor cocktail 2 and 3 (Sigma), was used to obtain whole cell protein lysates. Immunoblots were probed with the primary antibodies detailed in Supplementary  Table 2. Primary antibodies were detected using fluorescently labelled secondary antibodies: goat α-rabbit IgG DyLight 680 and goat α-mouse IgG Dylight 800 (Thermo Scientific). Odyssey CLx imaging system (LI-COR Biosciences) and Odyssey 2.1 software was used for detection and quantification of fluorescence intensity. HRP-conjugated secondary antibodies were used in some instances for chemiluminescence detection and protein levels were quantified by densitometry and normalised against loading controls. Supplementary Fig. 12 for the uncropped immunoblots.
Reporter assays. Reporter assays in HUVEC were performed with the Dual-Luciferase Reporter Assay System (Promega) and a GloMax-Multi + Microplate Multimode Reader (Promega). Twenty-four hours after co-transfection with the luciferase reporters and expression plasmids and 18 h after stimulation with human recombinant BMP9 (1 ng ml −1 , Peprotech) or TGFβ2 (10 ng ml −1 , Peprotech), HUVEC were lysed and reporter assays were performed in triplicate. For siRNA experiments, cells were transfected with 20 nM ERG or control siRNAs and after 24 h transfected with the luciferase reporters and activated with the ligands. The ratio of luciferase signal to Renilla signal from each transfection was determined to control for well-to-well variation in transfection efficiency.
Real-time polymerase chain reaction. RNA was extracted from tissues and HUVEC using the RNeasy kit (Qiagen). Supercript III Reverse Transcriptase (Invitrogen) was used for first strand cDNA synthesis. Quantitative real-time PCR was carried out using PerfeCTa SYBR Green Fastmix (Quanta Biosciences) on a Fig. 6 Etanercept prevents ERG loss during murine liver injury. a C57/B6 mice were injected with mineral oil (vehicle; bi-weekly i.p) or with chronic CCl 4 administration (8 weeks, bi-weekly i.p); a third group was subjected to a 3-weeks recovery period following CCL 4 administration. Representative immunofluorescence of liver tissue samples stained for VWF (grey scale, green in merge), SMA (grey scale, red in merge) and ERG (white). Scale bar 50 μm. Quantification of b SMA expression and c ERG expression (three fields per mouse, n = 3). Data were compared to mineral oil controls (*) or to chronic CCL 4 administration (#) by one-way ANOVA and Bonferroni multiple comparison post-test. d Representative immunofluorescence following acute (48 h) mineral oil, CCL 4 or co-administration of etanercept and CCl 4 . Staining for ERG (white); merged images showing VWF (green), SMA (red) and DAPI (blue). e pSMAD3 (white; stars); other colours as in d; scale bar 20 μm. f-h: Quantification of f SMA, g ERG and h pSMAD3 expression (three fields per mouse, n = 3). Data were compared to mineral oil controls (*) or to 48 h CCL 4 administration (#) by one-way ANOVA and Bonferroni multiple comparison post-test. i Co-administration of etanercept and CCL 4 for 48 h was able to significantly reduce SMA expression in Erg fl/fl mice while it was ineffective in Erg cEC-Het mice. Representative immunofluorescence of liver tissue, colour scheme as above. Scale bar 50 μm. j Quantification of SMAD2/3 target gene expression by qPCR in liver tissue of Erg fl/fl mice injected with etanercept and CCL 4 compared to CCL 4 alone (*) or Erg cEC-Het treated with etanercept and CCL 4 compared to CCL 4 alone (#). k Quantification of SMA expression (three fields per mouse, n = 3). Data analysed by one-way ANOVA and Bonferroni multiple comparison post-test. All graphical data are mean ± s.e.m., * or # P < 0.05, ** or ## P < 0.01, *** or ### P < 0.001 Bio-Rad CFX96 system. Oligonucleotides used for human and mouse are listed in Supplementary Tables 3, 4, respectively.
ChIP-qPCR. Chromatin immunoprecipitation (ChIP) was carried out using ChIP-IT express kit (Active Motif). HUVEC were transfected with ERG or control siRNA (20 nM, for 48 h) or treated with PBS or TGFβ2 (10 ng ml −1 , for 30 min), and cross-linked for 10 min with formaldehyde (final concentration of 1%). Chromatin was sheared using a Bioruptor UCD-200 ultrasound sonicator (Diagenode), resulting in DNA fragments of 500-1000 bp in size. Chromatin was immunoprecipitated with 2 μg antibody to ERG (sc-354X, Santa Cruz Biotechnology), SMAD3 (#9523, Cell Signalling) or negative control rabbit IgG (PP64, Chemicon, Millipore) using protein G magnetic beads (Active Motif). Immunoprecipitated DNA was then used as template for qPCR using primers specific for genomic loci. Oligonucleotide sequences are listed in Supplementary Table 5.
Bioinformatics analysis. Gene ontology analysis was performed using Database for Annotation, Visualisation, and Integrated Discovery (DAVID), to identify overrepresented gene ontology categories from microarray data obtained from control and ERG-deficient HUVEC 24 . Functional clustering tool within DAVID assigned group gene associated with TGF signalling with an enrichment score of 1.3 (which corresponds to P < 0.05). Microarray data are deposited in GEO database: GSE32984 24 .  Fig. 7 Loss of ERG expression correlates with EndMT in end-stage human liver disease. a Representative immunofluorescence of human liver sections stained for SMA (green), CD31 (grey scale; red in merge), ERG (white) and DAPI (blue) were captured from non-fibrotic donor patients with chronic endstage NASH, PBC and ALD with enlarged merge panel. Filled white arrows identify CD31 + ERG + EC whereas empty white arrows show CD31 + ERG − EC (Scale bar 50 µm). b Quantification of SMA, CD31 and ERG expression (five fields per section, n = 4-5). Scale bar 20 μm. Data were compared to non-fibrotic group (*) by one-way ANOVA and Dunnett's comparison post-test. c Representative images for CD31 (Red) and SMA (green) staining, showing co-localisation indicative of EndMT (arrows) and their pixel co-localisation profile in ALD, PBC and NASH patient samples. d Quantification of pixel co-localisation of CD31 (Red) and SMA (green) by overlap co-efficient was correlated with ERG expression for all individual human section data, non-fibrotic (black circles), NASH (purple squares), PBC (blue triangle down) and ALD (red triangle up). Correlation was calculated by linear regression, resulting in a significant negative correlation. All graphical data are mean ± s.e.m., *P < 0.05


text:
Variability in P1 gene redefines phylogenetic relationships among cassava brown streak viruses

Background Cassava brown streak disease is emerging as the most important viral disease of cassava in Africa, and is consequently a threat to food security. Two distinct species of the genus Ipomovirus (family Potyviridae) cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). To understand the evolutionary relationships among the viruses, 64 nucleotide sequences from the variable P1 gene from major cassava producing areas of east and central-southern Africa were determined. Methods We sequenced an amplicon of the P1 region of 31 isolates from Malawi and Tanzania. In addition to these, 33 previously reported sequences of virus isolates from Uganda, Kenya, Tanzania, Malawi and Mozambique were added to the analysis. Results Phylogenetic analyses revealed three major P1 clades of Cassava brown streak viruses (CBSVs): in addition to a clade of most CBSV and a clade containing all UCBSV, a novel, intermediate clade of CBSV isolates which has been tentatively called CBSV-Tanzania (CBSV-TZ). Virus isolates of the distinctive CBSV-TZ had nucleotide identities as low as 63.2 and 63.7% with other members of CBSV and UCBSV respectively. Conclusions Grouping of P1 gene sequences indicated for distinct sub-populations of CBSV, but not UCBSV. Representatives of all three clades were found in both Tanzania and Malawi.

Background
Cassava (Manihot esculenta Crantz, Family: Euphorbiaceae) is an important staple food crop for over 800 million people across the globe [1]. Although cassava is known to be vulnerable to at least 20 different viruses, the two most economically damaging viral diseases in Africa are cassava mosaic disease and cassava brown streak disease (CBSD). The diseases have been associated with production losses worth more than US$1 billion every year [2]. Recent developments in cassava research have shown that CBSD is emerging as the most important viral disease of cassava in Africa, and is consequently a threat to food security [1]. Two distinct species of the genus Ipomovirus (family Potyviridae), Cassava brown streak virus [3] and Ugandan cassava brown streak virus (UCBSV [4,5]) cause the disease. In this paper, both viruses are collectively called CBSVs. The characteristic symptoms of CBSVs include typical 'feathery' chlorosis and yellow patch symptoms along secondary and tertiary veins of older leaves of cassava, brown streaks on the stems, constriction in storage roots, and brown spots in the tuber visible when it is cut [6,7]. Previously, CBSD was reported only from the coastal lowlands of East Africa, but recently it has spread throughout the Great Lakes region of East and Central-Southern Africa [8][9][10][11][12][13][14].
Potyviridae is a family of plant viruses with a single stranded, positive sense RNA genome and flexious, filamentous particles [10]. The monopartite +ssRNA genomes of the members of Potyviridae share similar genomic organization, with levels of amino acid identity in their polyproteins ranging from 42 to 56% among different species of the same genus and from 25 to 33% among viruses from different genera [11]. However, conservation of individual mature proteins varies. P1, a serine protease that self-cleaves at its C terminus and acts as an accessory factor for genome amplification (reviewed in [12] is the first protein of the polyprotein and the most variable in length and amino acid sequence [11]. Other roles of P1 include boosting the activity of the helper component protease (HCPro) to suppress RNA silencing [13] and to enhance the pathogenicity of heterologous plant viruses during coinfection [14,15]. The genomes of CBSVs lack HCPro and the short P1 gene has RNA silencing suppression function [16]. Significantly divergent P1 gene sequences of CBSV have been found and recent studies have suggested that the P1 gene of CBSV (together with NIa, 6 K2, and NIb) have evolved more rapidly compared to other genes [17].
Genetic variability is an intrinsic feature of RNA viruses because of high mutation rates resulting from the lack of proofreading activity of their RNA-dependent RNA polymerases [14,18]. RNA recombination events can additionally shape the diversity of populations of RNA viruses [15] which can lead to new phenotypes such as host range expansion [19]. Diversity among CBSV isolates was initially assessed using sequences at the conserved 3′terminus of the RNA genome comprising the coat protein gene and parts of NIb [16] while comparative studies with complete viral genomes [5,9,20], have revealed more pronounced and distinctive features among virus isolates. In one previous study [5], sequence analysis of 7 virus isolates revealed two distinct CBSV sequence clades that were separated to the species level. Different biological features of members of these two clades provided justification for CBSVs to be assigned to two species: UCBSV and CBSV [5]. In that same study an isolate from coastal Tanzania (CBSV-Tan70, FN434473) was identified which was very similar to CBSV isolates throughout much of its genome, but with a strikingly different P1 gene which was equidistantly related to both CBSV and UCBSV isolates. As this divergent P1 region was found only in one CBSV isolate which otherwise had similar biological features than other CBSVs, further species delineation was not possible because of lack of similar isolates. The recent analyses of additional CBSV genome sequences from Tanzania [9] and from Uganda [16] revealed further diversity and also indicate the potential for an additional species or subspecies within the CBSVs.
In the study presented here, a total of 64 P1 gene sequences of CBSV isolates from major cassava producing areas of east and central-southern Africa were analysed. We sequenced a portion of the P1 gene from 31 isolates (from Malawi and Tanzania) and analyzed them with those previously reported from Uganda, Kenya, Tanzania, Malawi and Mozambique and present substantial evidence for the widespread occurrence of a distinct Cassava brown streak virus clade tentatively named CBSV-Tanzania (CBSV-TZ).

Source of virus isolates, amplification and sequencing
Cassava cuttings collected from CBSD-symptomatic plants in Malawi and Tanzania (Table 1) during national surveys in 2013 (under the auspices of each country's agricultural research institutes). The plants were classified by having symptoms that were consistent with CBSD (feathery chlorosis along veins in leaves and brown streaks/lesions along the plant stem), or potentially were coinfected with agents causing both CBSD and CMD (mosaic, mottling, misshapen and twisted leaflets) and were taken to The Leibniz Institute -Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ) Plant Virus Department, where they were maintained under greenhouse conditions. Total RNA was extracted from the virus-infected leaves of the cassava plants using the cetyl trimethyl ammonium bromide method [21] with modifications described previously [22] or using an RNeasy Mini kit (Qiagen). Nucleic acids were quantified using a Nanodrop spectrophotometer, and about 2.0 × 10 −5 μg/mL nucleic acid was used for virus detection by RT-PCR as detailed in Winter et al. [5]. A cDNA fragment, the partial sequence of the P1 gene, was amplified using virus specific primer sets designed by Winter et al. [5]. The reactions were performed in a GeneAmp 9700 PCR thermal cycler (Applied Biosystems, Foster City, CA, USA) set with the following conditions: 42°C for 30 min for reverse transcription, followed by heat denaturation at 94°C for 5 min; and then 35 cycles of amplification comprising the following: denaturation at 94°C for 1 min, annealing at 52°C for 1 min, extension at 72°C for 1 min, followed by a single cycle of final extension at 72°C for 10 min. All RT-PCR products were purified using a Qiagen gel extraction kit, ligated into the pDrive U/A cloning vector (Qiagen) and subsequently electroporated into Escherichia coli DH5α cells. The clones were Sanger sequenced in both orientations. A single consensus sequence for each isolate was verified to be CBSV by blastn searches of GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The resulting nucleotide sequences were submitted to GenBank (pending accession numbers, Table 1).

Nucleotide similarity and putative recombination breakpoint analysis
Percentage nucleotide identities were computed in Geneious Software v10.0.5 [23]. A matrix of nucleotide identities was produced using the Sequence Demarcation Tool v1 [24]. Putative recombination events were detected using nine recombination detection programs within the RDP4 package (http://darwin.uvigo.es/rdp/ rdp.html): RDP, GENECONV, MaxChi, Chimaera, Bootscan, Siscan, PhylPor, LARD, and 3Seq [25]. Analyses were carried out using default settings (except sequences were set to linear) and the Bonferroni correction P-value cut-off of 0.05. Only breakpoints supported by at least three methods were considered further [26].

Phylogenetic analysis
Phylogenetic relationship among P1 regions of CBSV isolates (Table 1) was determined. The sequences were aligned using ClustalW [27] in MEGA 7 [28] and edited manually. The alignment was trimmed to give all sequences uniform length. MEGA 7 was used to construct maximum likelihood (ML) phylogenetic trees, and editing was done in FigTree v1.4.2 (http://tree.bio.ed.ac.uk/ software/figtree/). The trees were created using a GTR nucleotide substitution model, and the best tree was bootstrapped with 1000 replicates [29].

Results
To examine the genetic diversity of CBSVs, field surveys and extensive sampling were performed in Malawi and Tanzania in 2013, yielding a total of 31 newly sequenced isolates (16 from Tanzania and 15 from Malawi). Thirty-three other previously published P1 sequences of CBSVs were retrieved from GenBank and aligned with these new sequences and a sister taxon, Sweet potato mild mottle virus [3]. The alignment (510 nt) was found to be free of detectable recombination. A phylogenetic tree generated from these 64 partial P1 sequences confirmed significant genetic variability among CBSVs and unambiguously resolved three clades. Seven isolates; five from Tanzania (TZ-Nal:07, TZ_Mari_1_13, TZ:Kor6:08, TZ-19-1, Tan_70) and two from Malawi (MW16, MW40) formed a clade which is significantly divergent from other CBSV isolates (we term this clade CBSV*) and the UCBSV isolates respectively (Fig. 1). We have tentatively named this group CBSV-Tanzania as it is more closely related to CBSV than UCBSV isolates and contains sequences predominantly from Tanzania. The clade includes the CBSV isolate Tan_70 from coastal Tanzania which was previously reported [5]. Isolates belonging to the CBSV-TZ clade were closely related, sharing P1 gene sequences very different from those in the CBSV* and UCBSV clades (Fig. 2). P1 sequences in the CBSV-TZ clade have low sequence identity with P1 gene sequences of isolates in the CBSV* (63.2 to 70.9%) and UCBSV (62.0 to 65.4%) clades.

Discussion
As CBSD continues to threaten subsistence cassava production in east, central and southern Africa, there is a  [32] need to understand dynamics of viral diversity as this has implications on evolution and emergence of new species or strains. This is especially critical in light of the rapid spread of the disease from the Great Lakes region of east and central-southern Africa [8,9,[11][12][13]20]. We present here an analysis of 64 partial P1 sequences of cassava brown streak viruses from cassava growing regions of Africa where CBSVs are known to occur. Considerable variance of gene size and sequence within P1 genes of the family Potyviridae has been previously reported [17,14] indicating that P1 is an ideal region to reveal population differentiation and incipient speciation within cassava ipomoviruses. Further, whole genome analyses of CBSVs had previously identified unusual sequence diversity in P1 [5]. Our phylogenetic analysis revealed that the CBSVs sequences formed three distinct clades ( Figure 1). In addition to the previously characterized species UCBSV and CBSV, the novel clade which includes the Tan_70 isolate [5] presents a major sub-group of CBSV, for which we propose the tentative name CBSV-Tanzania.
Another study on variation of CBSVs, based on short coat protein fragments (~230 nt) revealed a number of viruses that are intermediate between the two CBSV and UCBSV species subgrouping, and consequently presented a hypothetical possibility of a new novel species or sub-species associated with CBSVs [30]. Recent whole genome analyses of UCBSV isolates [9] suggested further speciation among isolates of UCBSV from Tanzania. Our results, concentrating on the analysis of the variable P1 gene and additional virus isolates from east and centralsouthern Africa, confirm the diversity observed with the in other studies and provides evidence from P1 gene analysis for subdivision of CBSV and the presence of the clade CBSV-TZ. Our results also show that the Malawi and Tanzania viruses are more diverse than those found in Kenya, Uganda, and Mozambique ( Figure 1). That Tanzania has qualitatively higher diversity of CBSVs may not just be due to increased surveillance and sampling there; while UCBSV is distributed all over Malawi, CBSV* and sub-group CBSV-TZ are localized in northern Malawi, bordering Tanzania [30]. Movement of cultivars between the two countries could help to explain the shared diversity of CBSVs, which could be due to either purely geographical reasons or unique adaptations of circulating CBSV-TZ to locally popular cassava cultivars.
While the region around Lake Malawi was where CBSD was first observed [6] the higher prevalence and wide distribution of UCBSV compared to CBSV throughout Malawi, Tanzania and surrounding countries suggest that UCBSV was likely the virus implicated in the first finding of CBSD. Comparisons of full genome sequences of Malawian CBSVs with those of CBSVs obtained from  (Table 1). Sequences are from Tanzania (green), Mozambique (yellow), Kenya (red), Uganda (purple) and Malawi (blue). Bootstrap values higher than 70% are shown. The scale is in substitutions/site CBSD-affected areas of neighboring countries (Tanzania and Mozambique) would likely clarify questions about the evolutionary history and biogeography of the viruses in the region. Regardless, it is clear that the CBSVs do not have geographically distinct distributions as was previously hypothesized [4].
Studies by Ndunguru et al. [9] showed that a previously described CBSV Tanzanian isolate TZ-Nal 07 had a recombination event in the 5′ end in the P1 gene. The P1 region is known to harbor obvious recombination in several potyviruses [31] and contributes to its overall variability. Although our final dataset did not statistically support recombination breakpoint (s) within P1, when diverse isolates from Kenya [32] were left out of the analysis, the isolate (TZ-Nal 07) was identified by two methods as a putative recombinant between a member of the CBSV-TZ clade (TZ:Kor6:08) and CBSVMo_83 (data not shown). This recombination event may be better supported from the full genome dataset [9] but the finding is consistent with the phylogenetic placement of TZ-Nal 07 as basal to the CBSV-TZ clade. However, we have no evidence for recombination being the origin for this well-supported subgroup and hence the diversification of P1 in the genomes of CBSV-TZ isolates still requires further investigation.

Conclusions
Our in-depth look at CBSVs from Malawi and Tanzania has revealed that the divergent Tan_70 isolate is in good company, and that the CBSVs have three separable groups of diverse P1 gene sequences. Further research will establish if the variable P1 region is an accurate bellwether for overall population divergence, and future phenotypic characterization will determine whether CBSV-TZ represents a novel strain or subspecies of CBSV.  Fig. 2 Pairwise identity matrix generated from CBSV partial P1 gene sequences. Each colored key represents a percentage to the identity score between two sequences


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Antimicrobial resistance profiles and molecular characterization of Escherichia coli strains isolated from healthy adults in Ho Chi Minh City, Vietnam

In this study, we attempted to isolate Escherichia coli from healthy adults in Ho Chi Minh City, Vietnam, and characterized its antimicrobial resistance profile, extended-spectrum β-lactamase (ESBL) genotype, phylogenetic grouping and virulence gene profile. A total of 103 E. coli isolates were obtained, and most of them were antimicrobial resistant such to streptomycin (80.6%), tetracycline (67.0%), ampicillin (65.0%), sulfamethoxsazole/trimethoprim (48.5%), nalidixic acid (43.7%), chloramphenicol (34.0%), cefotaxime (15.5%), ciprofloxacin (15.5%), kanamycin (12.6%), ceftazidime (10.7%), fosfomycin (4.9%) and gentamicin (2.9%). However, all these E. coli strains were susceptible to imipenem. Surprisingly, of 103 strains, 74 (71.8%) and 43 (41.7%) strains showed resistance to more than 3 and 5 classes of antimicrobials, respectively. Furthermore, 10 E. coli strains were ESBL-producers and positive for blaCTX-M genes (7 for blaCTX-M-9 and 3 for blaCTX-M-1), while five were additionally positive for blaTEM genes. S1-nuclease pulsed-field gel electrophoresis analysis revealed that 7 and 3 strains of E. coli carry blaCTX-M genes on their large plasmid and chromosome, respectively. Phylogenetic analysis exhibited that majority of the E. coli strains was grouped into A (44.7%), followed by B1 (23.3%), B2 (18.4%) and D (13.6%). Virulence genes associated with diarrheagenic E. coli, such as astA, EAF, eaeA, elt and eagg were also detected in ESBL-producing E. coli as well as antimicrobial resistant strains. These data suggest that commensal E. coli of healthy human could be a reservoir for antimicrobial resistance determinants and some of them might be harmful to human.

doi: 10.1292/jvms.  investigated in only children [14], raw food samples [49], household and small chicken farms [31]. However, comprehensive studies regarding analysis of antimicrobial resistance, ESBL type and virulence gene profile of commensal E. coli in adults were not conducted.
The study of the commensal E. coli population from healthy individuals is of great significance to understand and monitor the prevalence, distribution and carriage of pathogenic traits and antimicrobial resistance. The potentially pathogenic microbes with antimicrobial determinants may spread to other individuals not only from human but also animal and animal originated foods. On the other hand, the antimicrobial determinants may be transferred to more pathogenic bacteria in this niche. Keeping these factors in mind, this study was designed to characterize the commensal E. coli strains isolated from healthy adults of Ho Chi Minh City, Vietnam, for the presence of virulence gene, antimicrobial resistance profile and ESBL detection.

Bacterial isolates
A total of 103 stool specimens were collected from healthy adults whose ages ranged from 20 to 70 years in 3 urban districts (73 adults in 3, 6 and Tan Phu) and 1 suburban district (30 adults in Cu Chi) in Ho Chi Minh City, Vietnam, during March to November 2013. It was confirmed that none of the study participants had history of taking antimicrobials at least 3 months before the sample collection. All people whose samples were collected gave their informed consent for the participation in this study. Stool specimens were inoculated onto CHROMagar Orientation (CHROMagar Paris France, Paris, France). After incubation at 37°C for 22 ± 2 hr, a dark pink to reddish colony was selected and then identified to be E. coli by the IMViC tests [19]. One E. coli strain was collected from each sample and used for further analysis.

Determination of antimicrobial susceptibility
Antimicrobial susceptibility of the E. coli strains against ampicillin (AMP), cefotaxime (CTX), ceftazidime (CAZ), imipenem (IPM), nalidixic acid (NAL), ciprofloxacin (CIP), gentamicin (GEN), kanamycin (KAN), streptomycin (STR), fosfomycin (FOS), tetracycline (TET), trimethoprim/sulfamethoxazole (SXT) and chloramphenicol (CHL) was examined by disk diffusion method [11] on Mueller-Hinton agar (Becton Dickinson and Co., Sparks, NV, U.S.A.) using commercially available disks (Becton Dickinson and Co.). Charaterization of E. coli strains as susceptible, intermediate resistant or resistant was based on the size of the inhibition zones according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [10]. E. coli strain ATCC 29522 was used as a control strain in the susceptibility test. MDR was defined as resistance to at least one antimicrobial agent in three or more classes of antimicrobial agents [6].

ESBL phenotype and genotype confirmation
ESBL phenotype was analyzed by a double-disk diffusion test using CTX, CAZ, CTX/clavulanic acid and CAZ/clavulanic acid [10]. ESBL (bla CTX-M , bla TEM and bla SHV ) genes were determined by a multiplex PCR as described previously [27]. Genotype of bla CTX-M genes was further analyzed by PCR using four primer sets targeting the CTX-M-1, CTX-M-2, CTX-M-8 and CTX-M-9 groups [40].

Plasmid profile and location of bla CTX-M gene
Plasmid profiles of ESBL-producing E. coli strains were examined by S1-nuclease-PFGE with some modifications [3]. Briefly, a single genomic DNA block was incubated at 37°C for 40 min with 4 units of S1 nuclease (Thermo Fisher Scientific, Waltham, MA, U.S.A.) in 200 µl of 30 mM sodium acetate buffer (pH 4.6) including 50 mM NaCl, 1.0 mM zinc acetate and 5% (v/v) glycerol. The digested DNA was electrophoresed on a 1.0% pulsed-field certified agarose (Bio-Rad Laboratories Inc., Hercules, CA, U.S.A.) in 0.5X TBE (45 mM Tris, 45 mM boric acid and 1 mM EDTA) buffer at 6 V/cm 2 , with a switch time of 2-60 sec for 21 hr using the CHEF Mapper machine (Bio-Rad Laboratories Inc.). XbaI-digested Salmonella serotype Braenderup H98121 was used as a molecular size marker.
Location of bla CTX-M genes was analyzed by Southern hybridization. The resolved plasmids and genomic DNA by S1-PFGE gels were transferred onto nylon membranes (Perkin Elmer, Waltham, MA, U.S.A.). The membrane was hybridized with a specific 32 P-labeled DNA probe as described below. The bla CTX-M-1 and bla CTX-M-9 gene-probes were prepared by PCR using primers and conditions as described elsewhere [40] and labeled by random priming method using MultiPrime DNA Labeling System (GE Healthcare UK, Buckinghamshire, U.K.) with [α-32 P]-dCTP (111 TBq/mmol) (Perkin Elmer). Radioactivity was visualized by the BAS FLA-3000 system (GE Healthcare, Tokyo, Japan).

Phylogenetic characterization
Phylogenetic group of E. coli strains was determined by using a multiplex PCR as reported previously [9].

Antimicrobial susceptibility
A total of 103 E. coli strains including 73 from urban districts and 30 from suburban district were successfully isolated from 103 healthy adults in Ho Chi Minh City, Vietnam, and further analyzed for their antimicrobial susceptibility to 13 different drugs including AMP, CTX, CAZ, IPM, NAL, CIP, GEN, KAN, STR, FOS, TET, SXT and CHL. In this study, E. coli strains showing intermediate antimicrobial resistance were interpreted as resistant. As shown in Table 1, most of the E. coli strains were resistant to STR (80.6%), TET (67.0%), AMP (65.0%), SXT (48.5%) and NAL (43.7%), although all E. coli strains were susceptible to IPM. Moreover, 96.1% of the E. coli strains (99/103) were resistant to one or more antimicrobial agents. Notably, MDR was detected in 71.8% (74/103) of the tested strains. In addition, extensive MDR, which was defined to be resistant against at least five different classes of antimicrobial agents, was detected in 41.7% (43/103) strains. There was no significant difference in rate of MDR of E. coli strains isolated from healthy adults in urban (54/73; 74.0%) and suburban districts (20/30; 66.7%).

ESBL characterization and its genotype
Since 51, 5 and 11 E. coli strains were resistant to AMP alone, AMP and CTX, AMP, CTX and CAZ, respectively, these strains might produce ESBL. Indeed, 10 out of the 67 strains were confirmed to be ESBL-producing E. coli. Among 10 ESBLproducing E. coli strains, 9 were isolated from healthy adults in urban districts, while 1 was from healthy adult in suburban district. Subsequently, ESBL genes were characterized for these 10 ESBL-producing E. coli strains, and the result is summarized in Table  2. The bla CTX-M genes were detected in all the ESBL-producing E. coli. Moreover, five CTX-M-positive E. coli strains additionally possessed bla TEM genes, but none of the strains were positive for the bla SHV genes. Analysis of genotype for the bla CTX-M genes Table 1. Antimicrobial resistant E. coli strains isolated from healthy adults fecal samples (n=103)

Plasmid profiles and location of bla CTX-M genes
In general, the ESBL genes are predominantly located on plasmids and are easily disseminated. To see if ESBL genes are located on plasmid or chromosome, plasmid profile and location of bla CTX-M genes were analyzed for the 10 ESBL-producing E. coli strains by PFGE with S1-nuclease digestion and Southern hybridization. As shown in Fig. 1, there was at least one large plasmid ranging from 50 to 244 kb in size and a variation of plasmid profiles in ESBL-producing E. coli.
The Southern hybridization of S1-PFGE gel demonstrated that most of the ESBL-producing E. coli (7/10 isolates) harbored the bla CTX-M genes on a large plasmid (ranging from 50 to 200 kb in size) as shown in Fig. 1. Other 3 strains, however, carried the bla CTX-M genes on chromosome. Moreover, among the 5 strains harboring both bla CTX-M and bla TEM genes, 3 strains carried these genes on the same plasmid (data not shown).

Virulence gene profile
Distribution of virulence genes associated with DEC was examined, and the result is shown in Table 2. Of 103 E. coli strains, none of the strains were positive for bfpA, est, stx1, stx2, invE and daaD genes. However, astA was most prevalent and detected in 15 strains, followed by EAF (n=5), elt (n=3), eagg (n=2) and eae (n=1), indicating that some of the E. coli strains isolated from healthy adults are potentially virulent to human. In addition, 7 astA genes were detected in combination with EAF (n=2), elt (n=3), eagg (n=1) and eaeA (n=1). Fifteen out of 18 E. coli strains harboring virulence genes were MDR. Thus, the strains carrying both virulence and antimicrobial resistance genes may pose a serious threat to public health. Besides, 8, 7 and 3 E. coli strains harboring virulence genes belonged to the phylogenetic groups A, B1 and D, respectively (Table 2). Furthermore, 2 out of these 18 E. coli strains harboring virulence genes were isolated from healthy adults in suburban districts.

DISCUSSION
Emergence of antimicrobial resistant bacteria has been increasingly recognized as a serious problem in the world. Recently, a number of studies regarding prevalence of antimicrobial resistant E. coli including ESBL producer in not only hospitalized patients but also healthy people have been reported in many parts of the world [1, 4, 15, 21-23, 32, 42, 47]. However, there was less report regarding the comprehensive analysis of antimicrobial resistant E. coli strains isolated from healthy children in Vietnam [14].
In the present study, therefore, we attempted to isolate E. coli from healthy adults in Ho Chi Minh City in Vietnam for further characterization of their antimicrobial resistance, ESBL genotype and its location, phylogenetic groups and virulence gene profiles. A total of 103 E. coli were successfully isolated, and most of the strains showed resistance to one or more antimicrobials. The results of antimicrobial susceptibility in this study are in accordance with a previous study of healthy children in FilaBavi, near Hanoi, Vietnam, except for CIP [14] in which only 6 antimicrobials, such as AMP, CIP, CHL, NAL, TET, and SXT were examined and the isolated E. coli strains were resistant to AMP (65%), TET (74%), CHL (40%), NAL (27%) and SXT (68%). However, <1% of the E. coli strains isolated in FilaBavi were resistant to CIP [14], although 15.5% strains were resistant to CIP in this study. Surprisingly, 72% of the E. coli strains were MDR, and furthermore, 42% of the E. coli strains were extensive MDR. This high rate of MDR was observed in the E. coli strains isolated from healthy adults in both urban and suburban districts in Ho Chi Minh City. These percentages are quite high in comparison to those observed in a French study [32], but consistent with those reported from Korea and Bolivia [2,8]. These results revealed a high risk of fecal carriage of antimicrobial resistant E. coli strains in healthy adults in Ho Chi Minh City, Vietnam. One reason could be due to improper use of antimicrobials for human in Vietnam where antimicrobials can be purchased over the counter without a prescription and there is no sanction for non-compliance of drug law regulations [28]. Alternatively, it could be due to improper use of antimicrobials for food-animals [7,48] and veterinary medicine. The phenomenon of over usage of antimicrobials and thus emergence of MDR is an emerging problem worldwide, especially in developing countries. It could be also possible that antimicrobial resistant E. coli might be transmitted from food-animals and raw foods to human. The previous studies from southern Vietnam suggested that food-animals and raw foods could be a potential reservoir of antimicrobial resistance bacteria that disseminated to human [31,49]. It was found that commensal E. coli isolates on household and small-scale chicken farms in the Mekong Delta of Vietnam have high prevalence of resistance to TET, AMP, SXT, and CHL ( >60%) and MDR (81.3%), indicating the high risk of transmission of antimicrobial-resistant E. coli to community [31]. It was also reported that 61.6% of E. coli isolates from raw foods in the marketplace in Ho Chi Minh City were MDR and some of the tested strains contained a pool of mobile genetic elements for dissemination of antimicrobial resistant genes to human [49]. The occurrence rate of ESBL-producing E. coli in this study was lower than that of community from Hanoi, Vietnam (46.7%) [46], Thailand (65.7%) [23] and China (50.5%) [21], but consistent with that of community in Libya (13.4%) [1], Tunisia (7.3%) [4], Japan (6.4%) [22], Germany (6.3%) [47], France (6.0%) [32], Netherlands (7.8%) [42] and Switzerland (5.8%) [15]. Higher doi: 10.1292/jvms.  prevalence of ESBL-producing E. coli in Hanoi, Vietnam, and other countries could be due to differences in the isolation protocols. In those studies, β-lactam supplemented agar media were used for the isolation of the E. coli. ESBLs can be broadly divided into three groups, such as TEM, SHV and CTX-M, and CTX-M β-lactamases are the most widespread and frequently detected in E. coli [39]. In addition, genotype of CTX-Ms was found to be different and specific between regions in the world [50]. In this study, bla CTX-M-9 was found to be the most predominant group in healthy adults in Ho Chi Minh City, which is similar in northern Vietnam [5], Thailand [23] and China [21]. Whereas bla CTX-M-1 group was the most predominant β-lactamase gene in ESBLproducing E. coli in healthy carriage in European countries, such as Germany, France, Netherland and Switzerland [15,32,42,47]. In Ho Chi Minh City, it has been reported that bla CTX-M-9 and bla CTX-M-1 groups were the two most predominant types among ESBL-producing E. coli strains from foods [29] and patients with nosocomial infections in hospitals [45]. These data suggest that bla CTX-M-9 and bla CTX-M-1 groups might be circulating among foods, healthy adults and clinical settings in Ho Chi Minh City, Vietnam.
It is well known that plasmid plays an important role in the horizontal transfer of antimicrobial resistant genes in microbes including ESBL [12,25]. In this study, bla CTX-M genes were detected on plasmids, whose sizes are ranged from about 50 to 200 kb, in 7 out of 10 ESBL-producing E. coli strains. Our data indicate that ESBL-producing E. coli in healthy carriage could be a potential reservoir and source of dissemination of plasmid-mediated ESBL genes.
Phylogenetic group of E. coli strains isolated from healthy adults was investigated. It has been reported that the intestinal pathogenic E. coli are mostly classified into phylogenetic groups A, B1 and D [41], whereas strains belonging to phylogenetic group B2 are highly virulent and associated with extra-intestinal infections [38]. Most of the E. coli strains isolated in this study were identified to be phylogenetic group A followed by groups B1, B2 and D. This result is consistent with many of the previous reports that E. coli from community mainly belonged to phylogenetic group A [9,47]. Notably, the majority of MDR E. coli strains in this study belonged to phylogenetic group A (34/74 strains). Furthermore, 13 of the MDR E. coli strains belonged to phylogenetic group B2, suggesting that these strains might have a potential virulence capacity and could pose a risk to public health.
Furthermore, virulence gene profile analysis indicated that indeed 18 E. coli strains carried one or two virulence genes associated with DEC (Table 2). Among 11 virulence genes tested, astA was most prevalent, and some of the strains carried eae, EAF, elt and eagg in addition to astA. astA encoding EAST1 was initially detected in enteroaggregative E. coli (EAEC) [26] and thought to be a specific virulence factor of EAEC. However, astA has been detected in E. coli strains belonging to various pathotypes [41], and significance of EAST1 in diarrhea is not still clear. On the other hand, there are several reports regarding association of E. coli containing only astA among various virulence genes for DEC with a few outbreaks all over the world [16,33,34,51], indicating  MDR E. coli having DEC-associated virulence genes, in particular astA-positive MDR E. coli strains, might be harmful to human.
In conclusion, most of the E. coli strains isolated from healthy adults in Ho Chi Minh City, Vietnam, are MDR including extensive MDR. Some of them have virulence genes associated with DEC. Together with phylogenetic analysis, these data suggest that E. coli strains in healthy adults in southern Vietnam could be reservoir of antimicrobial resistance genes including ESBL and might be harmful to human.


extfieldsofstudy: Medicine Chemistry

text:
Pyrintegrin Induces Soft Tissue Formation by Transplanted or Endogenous Cells

Focal adipose deficiency, such as lipoatrophy, lumpectomy or facial trauma, is a formidable challenge in reconstructive medicine, and yet scarcely investigated in experimental studies. Here, we report that Pyrintegrin (Ptn), a 2,4-disubstituted pyrimidine known to promote embryonic stem cells survival, is robustly adipogenic and induces postnatal adipose tissue formation in vivo of transplanted adipose stem/progenitor cells (ASCs) and recruited endogenous cells. In vitro, Ptn stimulated human adipose tissue derived ASCs to differentiate into lipid-laden adipocytes by upregulating peroxisome proliferator-activated receptor (PPARγ) and CCAAT/enhancer-binding protein-α (C/EBPα), with differentiated cells increasingly secreting adiponectin, leptin, glycerol and total triglycerides. Ptn-primed human ASCs seeded in 3D-bioprinted biomaterial scaffolds yielded newly formed adipose tissue that expressed human PPARγ, when transplanted into the dorsum of athymic mice. Remarkably, Ptn-adsorbed 3D scaffolds implanted in the inguinal fat pad had enhanced adipose tissue formation, suggesting Ptn’s ability to induce in situ adipogenesis of endogenous cells. Ptn promoted adipogenesis by upregulating PPARγ and C/EBPα not only in adipogenesis induction medium, but also in chemically defined medium specifically for osteogenesis, and concurrently attenuated Runx2 and Osx via BMP-mediated SMAD1/5 phosphorylation. These findings suggest Ptn’s novel role as an adipogenesis inducer with a therapeutic potential in soft tissue reconstruction and augmentation.

Ptn alone failed to induce adipogenesis (Fig. 1B), suggesting that Ptn only augments adipogenic differentiation in the presence of adipogenic supplements in AIM. Quantitatively, Ptn upregulated PPARγ and C/EBPα in AIM, significantly more than AIM alone and DMEM (Fig. 1C). Again, Ptn alone failed to upregulate PPARγ or C/EBPα (Fig. 1C). Human ASCs isolated from different donors were tested similarly, and were shown to exhibit similar lipid accumulation differences (data not shown).
Upon exposure to 2 μ M Ptn-supplemented AIM, PPARγ expression began to elevate by Day 4 and peaked between 7 and 14 days, followed by a decline at 21 days (Fig. 1D); C/EBPα expression began to increase by Day 4,and continued to escalate up to the tested 21 days (Fig. 1E), suggesting Ptn's concerted and sequential effects on PPARγ and C/EBPα expression during adipogenic differentiation in ASCs. Quantitatively, ASCs secreted significantly more levels of adiponectin, leptin, total triglycerides and glycerol ( Fig. 1F-I, respectively) upon 2-or 4-wk exposure to 2-μ M Ptn-supplemented AIM than cells cultured in DMEM or AIM.

Pyrintegrin primed human adipose stem/progenitor cells yielded adipose tissue formation in vivo.
A microporous scaffold was fabricated by 3D layer-by-layer apposition using 100 wt% poly-ε -caprolactone (PCL) with ~65 kDa molecular weight per our prior approach 43,44 . Cylindrical PCL scaffolds were fabricated with an overall dimension of 5 × 3 mm 2 (diameter × height) ( Fig. 2A) and with interconnecting microchannels as conduits to promote tissue regeneration. Interlaid strand diameters of 250-300 μ m and interconnecting microchannel diameters of 400-500 μ m were selected to facilitate host cell infiltration and angiogenesis per our previous data 43,44 (Fig. 2A). Human ASCs were culture-expanded in DMEM, AIM, or 2-μ M Pyrintegrin supplemented AIM for 2 wks. A total of 10 × 10 6 cells/mL were homogenously seeded in 3 mg/mL type I collagen solution, and infused into the scaffold's microchannels (Fig. 2B), followed by incubation at 37 °C for 1 h to allow gelation. Subcutaneous pockets were surgically created in 8-9-wk-old athymic mice (Fig. 2B). Cell-infused or cell-free PCL scaffolds were implanted subcutaneously in the dorsum. Following 4-wk implantation, tissue ingrowth was apparent in all groups (N = 6/group) as exemplified in Fig. 2B. Cell infiltration occurred in all groups: scaffold alone, ASC-infused scaffolds, AIM-primed ASC scaffolds and Ptn-and AIM-treated ASC scaffolds (Fig. 2C). A moderate amount of adipose tissue was found in Ptn-and AIM-treated ASCs as revealed by Oil-red O staining (Fig. 2C). Human PPARγ expression was most pronounced in Ptn-and AIM-treated ASC scaffolds, significantly more than AIM-primed ASC scaffolds, ASC-infused scaffolds or scaffold only groups (Fig. 2D).

Pyrintegrin promotes adipogenesis from host endogenous cells without cell transplantation.
Next, we performed another in vivo experiment to determine whether Ptn promotes endogenous adipogenesis without cell transplantation. A total of 10-μ g/mL Ptn was homogenously seeded in 3 mg/mL neutralized type I collagen solution (20 μ L). Ptn-adsorbed or Ptn-free collagen solution was infused into PCL scaffold's microchannels (Fig. 3A), followed by gelation for 1 h at 37 °C. Ptn-adsorbed or Ptn-free PCL scaffolds were implanted in the inguinal fat pad in vivo in C57BL/6 mice (N = 6/group) (Fig. 3A). Following 4-wk in vivo implantation, little adipose tissue was formed in the representative Ptn-free scaffolds (Fig. 3B). Remarkably, Ptn-adsorbed collagen gel induced formation of adipose tissue that was positive to Oil Red O (Fig. 3B). Mouse PPARγ expression with RNA extracted from in vivo harvested tissue within the scaffold's microchannels was significantly higher in Ptn-adsorbed collagen gel scaffolds than Ptn-free collagen gel scaffolds (Fig. 3C). Given that no cells were transplanted, Ptn promotes endogenous adipogenesis in the native adipose environment.
Pyrintegrin attenuates SMAD-dependent BMP signaling. To explore Ptn signaling mechanisms, we first performed a human PPARγ luciferase receptor assay. PPARγ activity was unresponsive to Ptn stimulation at 0.02, 0.2 or 2 μ M, but was promoted by Rosiglitazone, a known potent PPARγ agonists (Fig. 4A,B). Accordingly, we screened several known adipogenesis signaling pathways and found that Ptn down-regulated the phosphorylation of Smad1/5, a downstream BMP target (Fig. 4C). However, Ptn did not inhibit the phosphorylation of p42/44 MAPK (Fig. 4C), a downstream target of MEK1/2 pathway. To further explore whether Ptn acts through SMAD-dependent BMP signaling, hASCs were treated with no BMP4, or BMP4 + Ptn (0 to 10 μ M) media. Both western blot and densitometry analysis confirm our finding that Ptn inhibited BMP4-mediated phosphorylation of BMP responsive SMAD1/5 in a dose-dependent manner (half maximal inhibitory concentration (IC 50 ) = 1.14 μ M), (Fig. 4D), but failed to attenuate SMAD2 activation by TGFβ 1 at concentrations equal to or greater than those that were inhibitory to BMP signaling (Fig. 4E). Ptn activated phosphorylation of p38 MAPK at higher doses (5 and 10 μ M), but not at lower concentrations ( Fig. 4F) in the presence of BMP4. p38 MAPK is responsive to both cellular non-stress and stress stimuli signals 45 and our results suggest that at higher doses of Ptn, the cells might be experiencing cytotoxic stress.
Pyrintegrin attenuates osteogenesis and promotes adipogenesis. At 80-90% confluence, hASCs were treated with osteogenesis induction medium (OIM), and OIM containing 2-μ M Ptn (Fig. 5A). In comparison with expected robust Alizarin Red staining with ASCs in OIM for 21 days, Ptn drastically attenuated Alizarin Red staining (Fig. 5A). By 21 days, Ptn significantly attenuated Runx2 and Osx (Fig. 5B,C). Ptn further upregulated PPARγ (Fig. 5D) and C/EBPα (Fig. 5E) even in chemically defined medium for osteogenesis. Ptn further stimulated lipid droplets in OIM at 7, 14 and 21 days (Fig. 5F). Importantly, Ptn alone failed to induce lipid accumulation (Fig. 5F). Given that Ptn induced adipogenesis in OIM but not in DMEM, we compared the known ingredients between osteogenesis induction medium and adipogenesis induction medium based on common protocols including the ones used in the present study, and found that the only common component is dexamethasone (Dex). With a postulation that Dex may have synergistic effect with Ptn in promoting adipogenesis, we found that indeed, 2-μ M Ptn and Dex at 0.1-, 1-and 10-μ M concentrations conjunctively promoted lipid accumulation for the tested 21 days (Fig. 5G), suggesting that Dex is likely the key ingredient in both osteogenesis and adipogenesis induction media that enables Ptn's ability to promote adipogenesis.

Discussion
The present data provides the first experimental evidence that a small molecule, Ptn is potent in concurrently capable of promoting adipogenic differentiation and attenuating osteogenic capability of human ASCs by mediating SMAD dependent BMP signaling attenuation in vitro (Fig. 6). It further demonstrates that this small molecule, Ptn promotes adipose tissue formation from either transplanted human adipose stem/progenitor cells or host endogenous cells. Ptn's ability to induce in vivo adipogenesis is supported not only by its promotion of crucial adipogenic genes including PPARγ and C/EBPα , but also by adipocytokines such as adiponectin and leptin. PPARγ , known as a key adipogenesis transcription factor, is activated by prostaglandins and anti-inflammatory agents including indomethacin and thiazolidinedione [46][47][48] . Ptn appears to signal differently than PPARγ agonists. By itself, Ptn fails to promote PPARγ gene expression and thereby does not directly promote adipogenesis. Nonetheless, Ptn robustly promotes adipogenesis in the presence of known adipogenic supplements including insulin, dexamethasone, and cAMP activators. Ptn's ability to stimulate adipogenesis only in the presence of known adipogenic supplements is supported by the outcome of our two in vivo experiments in which adipogenesis is induced not only by Ptn primed adipose stem/ progenitor cells, but also adipogenesis by host endogenous cells. Either way, adipogenesis within the microchannels of 3D printed scaffold suggests that transplanted cells or recruited host endogenous cells are the cell sources for adipose tissue formation. In this regard, Pyrintegrin acts similarly to IGF1 28 that has been shown to orchestrate adipose tissue formation from endogenous cells in an existing adipogenic environment.
In multiple bone disorders, stem/progenitor cells differentiate towards adipogenic lineage at the expense of osteogenesis. PPARγ activation with TZDs promotes the differentiation of precursor cells into adipocytes and inhibits osteoblast differentiation 49 . By comparing Ptn with known PPARγ promoters such as Rosiglitazone by performing human PPARγ luciferase assay, we concluded that Ptn does not appear to be a classic PPARγ agonist. These data suggest that Pyrintegrin promotes adipocyte differentiation through a mechanism that is distinctively different from that of PPARγ agonists. The functional importance of BMP signaling in promoting adipogenesis is supported by Ptn's ability to inhibit osteogenic differentiation and promote lipid accumulation in vitro. BMP responsive SMAD1/5/8 promotes osteogenesis by interacting with Runx2 to activate target gene transcription, leading to osterix induction 50 . Since the present data show that Ptn attenuates BMP-mediated SMAD1/5 activity and promotes adipogenesis even in osteogenic differentiation medium, we suggest that Ptn mediated BMP signaling might be adjunctive to Ptn-induced adipogenesis. The ability of Pyrintegrin to block SMAD1/5/8 phosphorylation in the absence and presence of BMP ligand suggests that Ptn attenuates BMP receptor kinase activity. Additional work will test Ptn effects in cells lacking BMP type II receptors and understand Pyrintegrin functions in the absence or presence of BMP receptors. Inhibition of BMP signaling is typically achieved by sequestering its ligands by administering soluble receptor extracellular domains or ligand-specific neutralizing antibodies [51][52][53] , or endogenous antagonists such as noggin or follistatin [54][55][56] . Hence, additional studies utilizing gain-of-function or loss-of function of BMP signaling in the presence of Ptn will likely elucidate BMP signaling in Ptn mediated adipogenesis. Pyrintegrin induces lipid accumulation and adipogenic differentiation of adipose stem/progenitor cells in the presence of dexamethasone alone and in the absence of insulin and cAMP activators. Dexamethasone is a glucocorticoid and known as a potent stimulator for 3T3-L1 and TA-1 preadipocytes 57-60 . The glucocorticoid receptor is a member of the nuclear hormone receptor family and upon ligand binding can directly activate C/EBPδ transcription. The present findings demonstrate that the simultaneous activation of dexamethasone's canonical signaling pathways, through the glucocorticoid receptor and Ptn through PPARγ and C/EBPα promotes adipogenic differentiation. In the absence of Dex alone treatment group, we wish to highlight the role of Dex based on the current literature. Dexamethasone (Dex) is known to alter bone marrow 61,62 and muscle derived mesenchymal stem cells (MSCs) proliferation rate 61 , and enhances their osteogenic differentiation capability 61,63 . Dex alone does not induce adipogenic differentiation in MSCs over a 16-day culture period 64 or in human adipose derived stem cells 65 . Dex and Rosiglitazone together are necessary and sufficient to induce adipogenic differentiation in MSCs 35  and therefore we tested the effect of Pyrintegrin in the presence of Dex. Remarkably, lipid droplet formation and mineralization occur concurrently in response to Ptn treatment during osteogenesis.
Taken together, the present data show that Ptn promotes adipogenesis in an adipogenic environment either by Ptn-primed ASCs or host endogenous cells in Ptn-adsorbed scaffolds implanted in the inguinal fat pad. For translational studies, Ptn's efficacy to promote endogenous adipogenesis without cell transplantation clearly offers advantages, similar to our previous work on tissue regeneration by cell homing, without the need for cell transplantation 43,44,66,67 . Pyrintegrin may offer potential advantages of tunable doses as a potential treatment for lipoatrophy, and an alternative to autologous fat transfer that has an intrinsic disadvantage of unpredictable post-operative volume reduction. Ptn attenuation of osteogenesis may have potential usage in promoting soft tissue healing and avoiding ectopic mineralization. Nevertheless, when using a small molecule the possibility of confounding effects from secondary targets should be considered, which we did not see in our in vivo studies due to the lowest effective concentration used. This study further warrants a more detailed and different dose analysis to maximize selectivity for effective adipogenic signaling.

Human adipose stem/progenitor cells. All experimental protocols were approved by Columbia
University's IRB approval and were carried out in accordance with their outlined guidelines and regulations. Human adipose stem/progenitor cells (hASCs) were isolated from lipectomy samples (anonymous female donors; age: 37 to 52 years old) per our prior methods 2,11,16,17 . We also obtained lipoaspirate-extracted ASCs from different donors from Dr. Farshid Guilak's laboratory at Duke University Medical Center as a generous gift. An informed consent was obtained from all the human subjects whose tissue samples were utilized for this study design. Growth medium (DMEM) was defined as Dulbecco's modified Eagle's medium:Nutrient mixture per manufacturer's instructions. RNA samples were eluted in 30-μ l DEPC-water. Total RNA was quantified using NanoDrop 1000 Spectrophotometer (Invitrogen, Carlsbad, CA) and stored at − 80 °C prior to reverse transcription. All RNA samples were reverse-transcribed using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). The cDNA was synthesized using 0.1-0.2 μ g RNA to obtain 20-μ L cDNA. All cDNA samples were stored at -20 °C until further use. For qPCR reactions, cDNA samples were mixed with TaqMan ® Universal PCR Master Mix (Applied Biosystems, Foster City, CA), primers, and nuclease-free water per manufacturer's protocol. PCR primers included peroxisome proliferator-activated receptor γ (PPARγ , Hs01115513_m1), and CCAT-enhanced binding protein α (C/EBPα , Hs00269972_s1) gene-specific FAM dye tagged primers. Osteogenic markers included Runt-related transcription factor 2 (RunX2, Hs00231692_m1), and Osterix (Sp7, Hs01866874_s1) gene-specific FAM dye tagged primers (Applied Biosystems, Carlsbad, CA). All mRNA was measured using ViiA ™ 7 Real-Time PCR System (Applied Biosystems) in 25-μ l reaction volume, with GAPDH as a house-keeping gene.
Lipid staining. Human ASC cultures were washed with PBS (Sigma-Aldrich), fixed in warmed 4% paraformaldehyde solution (Fisher-Scientific, Pittsburgh, PA) for 10-30 min at room temperature. Cells were gently washed twice with PBS and then stained with LipidTOX TM neutral lipid stain (Invitrogen) for 10-15 min per our prior methods.
Adipogenesis assays. Human ASCs were plated at 5 × 10 3 /cm 2 and differentiated in chemically defined medium for 28 days. Culture medium and lysed cell supernatant were individually collected on 14 and 28 days. Cells were lysed using 1% Triton-X solution (Sigma-Aldrich, St. Louis, MO). Cell culture lysate was collected by scrapping with a cell scraper (Fisher-Scientific) and briefly sonicated for 10 s using an ultrasonic water bath Scientific RepoRts | 7:36402 | DOI: 10.1038/srep36402 (Fisher-Scientific) on ice. Total secreted human adiponectin and leptin were measured using ELISA (R&D, Minneapolis, MN) per our prior methods 2,10,11,16,17 . Absorbance was read using a microplate reader (Biorad, Hercules, CA). DNA content was measured using Quant-iT ™ PicoGreen ® dsDNA Assay Kit (Life Technologies) per our prior methods. A high-range standard curve of 1 μ g/mL was used for measuring the DNA concentration of the samples per our prior methods. The samples were prepared in a 96 well black bottom plate (Fisher-Scientific) with absorbance read using microplate reader and standard fluorescein wavelengths (excitation ~480 nm, emission ~520 nm; Molecular Devices, Sunnyvale, CA). Total triglycerides and glycerol contents were measured by processing the sample lysate with Free Glycerol Determination kit (Sigma-Aldrich) per our prior methods 2,10,11,16,17 . Scaffold design and synthesis. Scaffold design and synthesis followed our previous methods to create 3D printed scaffolds with interconnecting microchannels 43,44,66,67 . Using 100 wt% poly-ε -caprolactone, PCL (Sigma-Aldrich Corp.,) with molecular weight ~65 KDa was 3D-printed using a bioplotter (EnvisionTec, Gladbeck, Germany). PCL pellets were first melt at 120 °C and then printed layer-by-layer as a cylinder (5 × 3 mm: diameter × height) using CAD software. Interlaid strands had diameters of 250-300 μ m and interconnecting microchannels had a diameter of 400-500 μ m. Scaffolds were sterilized in Ethylene Oxide (ETO) gas (Anprolene AN74i, Fisher, Pittsburgh, PA).
Cell seeding in scaffolds. Cells were washed, trypsinized, counted and seeded at a density of 10 × 10 6 cells/ mL by mixing the cells with 3 mg/mL Collagen Type I solution (BD Bioscience, Sparks, MD). Collagen solution was allowed to crosslink for 1 h at 37 °C before in vivo implantation. Sample size per group was 6.
In vivo implantation of cell-seeded scaffolds. All experimental protocols were approved by Columbia University's Institutional Animal Care and Use Committee (IACUC) and were performed in accordance with their guidelines and regulations. Athymic nude male mice (7-8 wks old; Harlan, Indianapolis, IN) were anesthetized with intraperitoneal ketamine (100 mg/kg) and xylazine (5 mg/kg), and maintained at 1-3% isoflurane (Fisher-Scientific). The skin was disinfected with 70% Ethanol and wiped with Betadine and Povidone-Iodine (Fisher-Scientific). Following a linear midsaggital incision, subcutaneous pockets were created using fine scissors and housed all in vivo implanted scaffolds, followed by skin closure with a 4-0 P-3 vicryl coated undyed braided suture (Ethicon, Somerville, NJ). All mice received post-surgical Rimadyl IP injections for pain control (5 mg/ kg, Pfizer, New York, NY). All scaffolds were retrieved after 4 wks following euthanasia with CO 2 asphyxiation.
In vivo implantation of Pyrintegrin adsorbed scaffolds. A total of 3 mg/mL neutralized collagen type I solution was prepared per our prior method 43,44,66,67 with 10 μ g/mL Ptn adsorbed. Ptn-adsorbed or Ptn-free collagen gel was infused into the scaffolds' microchannels by pipetting, followed by collagen crosslinking for 1 h at 37 °C before in vivo implantation. Following Columbia University IACUC approval, C57BL/6 male mice (age: 10-11 wks, weight 15-25 g; Harlan, Indianapolis, IN) were fed with adjusted irradiated calories diet (Harlan). Given the mouse susceptibility to diet-induced obesity, the mice gained ~10-15 g in 6 wks (data not shown), after which we surgically implanted Ptn-adsorbed and collagen gel alone scaffolds in separate mice (N = 6 per group). On the day the surgery, the mice were anesthetized with 3-5% isoflurane. The skin was disinfected with 70% Ethanol, Betadine and Povidone-Iodine. An 1-1.5 cm long incision was made in the upper segment of the hindlimbs on both sides to expose the inguinal fat pad. A small pocket was created using fine dissecting scissors in this fat pad followed by implantation of Ptn-adsorbed or collagen gel only scaffolds, and closure of fat pad and skin using a 4-0 P-3 vicryl coated undyed braided suture. All mice received post-surgical Rimadyl IP injections for pain control (5 mg/kg, Pfizer). All scaffolds were retrieved after 4 wks following euthanasia with CO 2 asphyxiation.
Histology and immunohistochemistry. The retrieved scaffolds were embedded in optimal cutting temperature medium (Fisher-Scientific) in cryomolds and frozen in isopentane (Fisher-Scientific) pre-cooled in liquid nitrogen and processed for cryosectioning (Histoserv, Germantown, MD) into 8-μ m sections. Frozen sections were fixated in 4% paraformaldehyde (Fisher-Scientific) at room temperature for 15 min, permeabilized with 0.25% Triton X-100 (Sigma-Aldrich, St. Louis, MO) and blocked with Licor Odyssey Blocking buffer (Licor) and stained with Oil Red O and hematoxylin.
Human PPARγ reporter assay. PPAR Reporter assay kit (Indigo Biosciences, State College, PA) utilizing non-human mammalian cells engineered to express constitutive, high-level expression of human PPARγ , a ligand-dependent transcription factor. These reporter cells include the luciferase reporter gene functionally linked to a PPARγ -responsive promoter. Thus, quantifying changes in luciferase expression in the treated reporter cells provides a sensitive surrogate measure of the changes in PPARγ activity. Rosiglitazone was used as a positive control due to its ability to bind to the nuclear PPARγ receptor. As per manufacturer's instructions, the reporter cells were plated and immediately treated with Rosiglitazone or different Ptn doses. No adipogenic inductive factors were used in this assay. Following overnight incubation at 37 °C, the treated medium was discarded followed by addition of 100-μ l luciferase detection reagent (LDR), and incubation at room temperature for 15 min. Light intensity emitted was quantified using a plate-reading luminometer.
Alizarin Red staining. ASCs were seeded onto a 24 well plates and treated with osteogenesis induction medium (OIM) with or without 2-μ M Ptn for 3 wks. Cells were fixed in warmed 4% paraformaldehyde and stained with Alizarin Red S stain (Sigma-Aldrich) for 5 min as per manufacturer's protocol.
Dexamethasone treatment. ASCs were seeded at 5 × 10 3 cells/cm 2 in 24 well plates. After cells were exposed to 2-μ M Ptn in DMEM with different dexamethasone doses (0.1 to 10 μ M) for 3 wks, culture medium was removed with cells stained for lipid visualization.
Statistical analysis. Upon confirmation of normal distribution, all quantitative data were analyzed using a one-way analysis of variance (ANOVA) with post-hoc Bonferroni tests to determine significant differences between or within groups, using a statistical package (SPSS, Chicago, IL). Results were considered significant for p < 0.05.