Sofia Karkampouna

BMP7 activates brown adipose tissue and reduces diet-induced obesity only at subthermoneutrality

Background/Aims Brown adipose tissue (BAT) dissipates energy stored in triglycerides as heat via the uncoupling protein UCP-1 and is a promising target to combat hyperlipidemia and obesity. BAT is densely innervated by the sympathetic nervous system, which increases BAT differentiation and activity upon cold exposure. Recently, Bone Morphogenetic Protein 7 (BMP7) was identified as an inducer of BAT differentiation. We aimed to elucidate the role of sympathetic activation in the effect of BMP7 on BAT by treating mice with BMP7 at varying ambient temperature, and assessed the therapeutic potential of BMP7 in combating obesity. Methods and Results High-fat diet fed lean C57Bl6/J mice were treated with BMP7 via subcutaneous osmotic minipumps for 4 weeks at 21°C or 28°C, the latter being a thermoneutral temperature in which sympathetic activation of BAT is largely diminished. At 21°C, BMP7 increased BAT weight, increased the expression of Ucp1, Cd36 and hormone-sensitive lipase in BAT, and increased total energy expenditure. BMP7 treatment markedly increased food intake without affecting physical activity. Despite that, BMP7 diminished white adipose tissue (WAT) mass, accompanied by increased expression of genes related to intracellular lipolysis in WAT. All these effects were blunted at 28°C. Additionally, BMP7 resulted in extensive ‘browning’ of WAT, as evidenced by increased expression of BAT markers and the appearance of whole clusters of brown adipocytes via immunohistochemistry, independent of environmental temperature. Treatment of diet-induced obese C57Bl6/J mice with BMP7 led to an improved metabolic phenotype, consisting of a decreased fat mass and liver lipids as well as attenuated dyslipidemia and hyperglycemia. Conclusion Together, these data show that BMP7-mediated recruitment and activation of BAT only occurs at subthermoneutral temperature, and is thus likely dependent on sympathetic activation of BAT, and that BMP7 may be a promising tool to combat obesity and associated disorders.

TGFβ Signaling in Liver Regeneration

Adult organ regeneration occurs in many systems such as in liver, skin, intestine and heart, indicating that postnatal life is not a static or quiescent state but a dynamic and complex process. The liver is a spectacular organ, exhibiting high regenerative capacity crucial for homeostasis and tissue repair: injuries induced mechanically or chemically, can be completely restored. Regeneration involves extensive cell division, inflammation and extracellular matrix remodeling processes. At the molecular level, one of the key mediators of regeneration response is the secreted cytokine transforming growth factor-β (TGFβ). TGFβ is a profibrogenic and anti-proliferative protein with pleiotropic functions depending on the cellular context. In this review, we discuss the role of TGFβ in the development of the liver and in adult liver regeneration, with particular emphasis on its role in regulation of hepatocyte regeneration and in hepatic progenitor cell-induced regeneration. Finally, we give an overview of the current direction of liver research towards cell replacement therapies.

Novel ex vivo culture method for the study of dupuytren's disease:Effects of TGFβ Type 1 receptor modulation by antisense oligonucleotides

Dupuytrens disease (DD) is a benign fibroproliferative disease of the hand. It is characterized by the excessive production of extracellular matrix (ECM) proteins, which form a strong fibrous tissue between the handpalm and fingers, permanently disrupting the fine movement ability. The major contractile element in DD is the myofibroblast (MFB). This cell has both fibroblast and smooth muscle cell-type characteristics and causes pathological collagen deposition. MFBs generate contractile forces that are transmitted to the surrounding collagen matrix. Μajor profibrotic factors are members of the transforming growth factor-β (TGFβ) pathway which directly regulate the expression levels of several fibrous proteins such as collagen type 1, type 3, and α-smooth muscle actin. Molecular modulation of this signaling pathway could serve as a therapeutic approach. We, therefore, have developed an ex vivo “clinical trial” system to study the properties of intact, patient-derived resection specimens. In these culture conditions, Dupuytrens tissue retains its three-dimensional (3D) structure and viability. As a novel antifibrotic therapeutic approach, we targeted TGFβ type 1 receptor (also termed activin receptor-like kinase 5) expression in cultured Dupuytrens specimens by antisense oligonucleotide-mediated exon skipping. Antisense oligonucleotides targeting activin receptor-like kinase 5 showed specific reduction of ECM and potential for clinical application.

MP44-17 PATIENT DERIVED XENOGRAFTS AS PRECLINICAL MODELS OF UROLOGICAL MALIGNANCIES

INTRODUCTION AND OBJECTIVES: The cross-talk of mammalian target of rapamycin (mTOR) pathway is clinical limitation of mTOR inhibitor for the treatment of urothelial carcinoma (URCa). This study is to search mTOR pathway downstream genes to overcome cross-talk at non muscle invasive low grade (LG)-URCa of the bladder. METHODS: Gene expression patterns, gene ontology, and gene clustering by dual (p70S6K and S6K) siRNAs or rapamycin in 253J and TR4 cell lines were investigated by microarray analysis and we selected mTOR/S6K pathway downstream genes which were suppressed to siRNAs, and rapamycin up-regulated or rapamycin down-regulated. We validated mTOR downstream genes with immunohistochemistry using tissue microarray of 90 non-muscle invasive LG-URCa patients whether genes can predict clinical outcomes, and knockout study to evaluate the synergistic effect with rapamycin. RESULTS: In the microarray analysis, we selected mTOR pathway downstream genes which consisted of 4 rapamycin downregulated (FOXM1, KIF14, MYBL2, and UHRF1), and 4 rapamycin upregulated (GPR87, NBR1, VASH1 and PRIMA1). In tissue microarray, FOXM1, KIF14, and NBR1 were more expressed at T1, and MYBL2 and PRIMA1 were more expressed at tumor size more than 3 cm. In multivariate Cox regression model, KIF14 and NBR1 were significant predictors of recurrence in non-muscle invasive LG-URCa of the bladder. In NBR1 knock out model, rapamycin treatment showed synergistic effect to inhibit cell viability and colony forming ability compared to rapamycin only. CONCLUSIONS: KIF14 and NBR1 were mTOR/S6K pathway downstream genes to predict recurrence in non-muscle invasive LGURCa of the bladder and NBR1 knockout overcome the rapamycin coss-talk.

MP41-10 MODELLING PROSTATE CANCER USING PRIMARY AND METASTATIC CANCEROIDS

INTRODUCTION AND OBJECTIVES: A recent study showed that adipose-derived stem cells are able to counteract urethral stricture formation in rats. The aim of this study was to evaluate the feasibility of autologous adipose derived stromal vascular fraction (SVF) transplantation into male urethra stricture walls after direct vision incision of urethra (DVIU). METHODS: A prospective clinical study was undertaken after ethics approval and appropriate patient consent. The inclusion criteria were: male patients older than 18 years, with single short recurrent not-obliterating urethral stricture (<2 cm). The exclusion criteria were: patients not willing to consent, multiple strictures and those not deemed suitable for endoscopic management. Failure was defined as need for further interventions. Preoperative workup included history, examination, retrograde urethrogram (RGU), voiding cystourethrogram (VCUG), urine culture, renal function tests, AUA score, IIEF, PROM. Plastic surgery team performs liposuction to extract 50 mls of fat from the patient’s abdominal wall. 50ml fat-SVF was obtained using a Goog Manufacturing Practice collagenase (Celase , Cytori Therapeutics, San Diego, USA) according to a standard protocol. SVF was diluted in 5 ml saline solution for the injection. A cystoscopy was performed and the stricture evaluated, a glide wire was placed and an urethrotomy performed at 6 o’clock position in bulbar urethra. Gide wire was left in situ. The SVF solution was injected at the site of the stricture and on either side of the stricture. A 12 Fr urinary catheter was placed. The urinary catheter was removed after 24 hrs. Follow up was of 3.5 months. RESULTS: Two patients were included in the study. The main characteristics are reported in table 1. No local or systemic side effects or complications were recorded. No recurrence of urethral stricture was detected in both patients after 3.5 months. Table 1 CONCLUSIONS: This is the first study to demonstrate a successful autologous SVF transplantation in male urethral stricture after DVIU. Further studies are necessary to confirm the efficacy of SVF in preventing urethral stricture recurrence.