63.10 The Role of the Etoposide Induced 2.4 Gene Ei24 in Regulating Pancreatic β Cell Function

Posted on January 26, 2017

S. R. Hamarneh1, J. M. Ramirez Decrescenzo1, F. M. Kuehn1, A. R. Munoz1, S. Morrison1, F. Adiliaghdam1, R. A. Hodin1  1Massachusetts General Hospital,Department Of Surgery, General & Gastrointestinal Surgery,Boston, MA, USA

Introduction:  Type 2 diabetes is characterized by insulin resistance, inadequate insulin secretion and declined pancreatic β-cell mass. EI24 is a tumor suppressor gene and has emerged as a regulator of autophagy and inflammatory pathways. We sought to explore the role that EI24 plays in insulin production and β-cell proliferation.

Methods:  We performed expression analysis of the EI24 gene using Gene-ontology data from patients with or without diabetes. To study the role of Ei24 in β-cell function, rat insulinoma cell line INS-1 was incubated with different inflammatory mediators and under nutrient deprivation conditions to study the effect of these stressors on Ei24 levels. Additionally, we studied the effect of Ei24 deletion or activation on inflammation levels, autophagy influx and insulin production in β-cells in vitro. Furthermore, functional analysis was performed after immunoprecipitation of Ei24 protein complexes to elucidate the plausible cellular pathways affected by Ei24 in pancreatic β-cells.

Results: Ei24 expression levels were lower in human β-cells from diabetic compared to non-diabetic patients (-1.27 vs. 1, p=0.0004). In INS-1 cells, inflammatory mediators such as TNF-α, LPS and bacterial contents suppressed Ei24 levels. Furthermore, the Ei24 expression was induced by nutrient availability. Overexpression of Ei24 increased insulin levels (Ei24 WT vs. Ei24-overexpressing Cells, 1.0 ± 0.3 Vs. 2.8 ± 0.45 Relative Expression, p< 0.01) in INS-1 cells in vitro.  Ei24 deletion in β-cells altered the expression of genes involved in β-cell activity such as Ppar-α (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.24 vs. 0.4 ± 0.056 Relative Expression, p< 0.05), Ppar-γ (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.16 Vs. 7.2 ± 1.0 Relative Expression, p< 0.01) and Pgc1-α (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.18 Vs. 3.7 ± 0.68 Relative Expression, p< 0.01). Also, EI24 deletion increased inflammatory cytokine levels: TNF-α (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.08 vs. 63 ± 7.8 Relative Expression, p< 0.001), IL-1β (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.23 Vs. 57 ± 5.5 Relative Expression, p< 0.001) and IL-6 (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.1 Vs. 42 ± 3.8 Relative Expression, p< 0.001) and impaired autophagic flux in β cells in vitro. Functional analysis demonstrated an extended role for Ei24 in β-cell function.

Conclusion: EI24 plays a major role in β-cell function and homeostasis. The EI24 pathway in pancreatic β-cells may represent an important therapeutic target to prevent or treat diabetes in humans.

 

63.09 Blockade of Canonical Notch Signaling in Helper T-Cells Impairs Wound Healing

Posted on January 26, 2017

A. S. Kimball1, A. Boniakowski1, A. Joshi1, M. Schaller1, R. Allen1, P. Henke1, I. Maillard1, S. Kunkel1, K. Gallagher1  1University Of Michigan,Ann Arbor, MI, USA

Introduction: For the past century, the study of wound healing has focused mainly on the role of the innate immune system in maintaining tissue homeostasis. Up to this point, there have been few studies looking directly at adaptive immune cells and their role in promoting tissue repair. In this study, we sought to quantify the presence of T-cells in wounds over time and to evaluate their effects on wound healing by blocking a well-described T-cell signaling pathway.  

Methods: C57BL/6 mice were obtained for general wound healing studies. DNMAMLf/f CD4-Cre+/- mice were obtained to evaluate wound healing in the setting of canonical Notch signaling blockade in CD4+ cells. 4mm punch biopsies were created on the mid-backs of the mice and wound healing was monitored daily using NIH ImageJ software. Wounds were harvested at various time-points for cell isolation and analysis. CD3+ cell volumes were calculated over-time post-injury using analytical flow cytometry.

Results: CD3+ T-Cells are present and dynamic in wounds over-time post-injury, representing ~4% of live cells at day 2 and ~6% of the live cells at day 6. Given the two peaks of T-cell presence in the inflammatory and proliferative phases of wound healing, we sought to probe the relevance of these cells by blocking the well described Notch signaling pathway in CD4+ cells. Wound healing was evaluated in DNMAMLf/f CD4-Cre+  vs. Cre- mice and this demonstrated significantly delayed wound healing in those mice with impeded Notch signaling. 

Conclusion: Contrary to popular dogma, the adaptive immune system plays a not-insignificant role in wound healing and further studies are needed to evaluate the role of T-cells and Notch signaling. These findings are consistent with the impaired wound healing seen in immunocompromised patients and represents an exciting new area of future research.

 

63.08 Menin/MicroRNA-24 Regulatory Axis Drives Hepatic Fibrosis in Mdr2-/- Mice

Posted on January 26, 2017

C. Hall1, L. Ehrlich2, T. Shepperd2, A. O’Brien2, G. Alpini2, S. Glaser2, T. C. Lairmore1  1Scott & White Healthcare,Surgery,Temple, Texas, USA 2Texas A & M Health Science Center College Of Medicine,Bryan, TX, USA

Introduction:
Liver transplantation remains the primary treatment for Primary Sclerosing Cholangitis (PSC), one of several cholangiopathies that result in cholestatic liver disease. Mdr2-/- mice provide an in vivo model of PSC with characteristic biliary inflammation and fibrosis that subsequently develop cirrhosis and hepatic malignancies. Since cholangiocytes express a neuroendocrine phenotype within the liver, we tested the hypothesis that the tumor suppressor protein menin is implicated in the progression of liver fibrosis and that menin expression can be regulated in the liver via MicroRNA-24. 

Methods:
Menin expression was measured in human PSC and Mdr2-/- mice. Twelve-week old Mdr2-/- mice were treated with MicroRNA-24 Vivo Morpholino (miR-24 VM) to knockdown microRNA-24 expression levels. Liver fibrosis was evaluated using sirius red staining, hydroxyproline assay, and qPCR for genes associated with liver fibrosis (fibronectin 1 (FN1), collagen type 1 alpha1 (Col1α1), transforming growth factor-Beta1 (TGF-β1), and alpha-smooth muscle actin (α-SMA)). Intrahepatic bile duct mass was visualized using immunohistochemistry for CK-19, a cholangiocyte specific protein. Studies were replicated in vitro using mouse cholangiocytes and human hepatic stellate cells treated with microRNA-24 hairpin inhibitor and mimic. Menin and microRNA-24 gene expression measured via qPCR.

Results:
Menin gene expression was increased in Mdr2-/- mice and advanced stage human PSC samples. Treatment of Mdr2-/- mice with miR-24 VM increased menin expression, which correlated with increased expression of fibrotic genes (Figure 1). Mice treated with miR-24 VM showed significant increase in peri-ductular fibrosis and bile duct mass. In vitro, inhibition of microRNA-24 significantly increased cholangiocyte expression of FN1, Col1α1, TGF-β1, and α-SMA. 

Conclusion:
The menin/miR-24 regulatory system is implicated in cholestatic liver fibrosis. Inhibition of microRNA-24 increases menin and TGF-β1 expression, subsequently driving hepatic fibrosis and bile duct mass in Mdr2-/- mice, a murine model of PSC. Previous studies in other models of cholestatic liver disease have shown that TGF-β1 drives hepatic fibrosis through increased expression of phosopho-Smads. Modulation of the menin/microRNA-24 axis may provide novel targeted therapies to slow the progression of hepatic fibrosis into cirrhosis by altering TGF-β1 and phospho-Smad expression. 
 

63.07 EP2 Receptor Blockade Decreases Intestinal Barrier Breakdown Following Cecal Ligation and Puncture

Posted on January 26, 2017

J. Golden1, P. Kavarian1, L. Illingworth1, J. Uppuluri1, O. Escobar1, M. Isani1, C. Gayer1, A. Grishin1, H. Ford1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA

Introduction:  Cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) are inflammatory mediators that have been implicated in sepsis, inflammation, and intestinal barrier breakdown. Previous work in our lab has shown that COX-2 can be upregulated by its end-product PGE2 in enterocytes. We have identified pro-inflammatory prostanoid receptor, EP2, as a key mediator in this feedback loop leading to upregulation of COX-2 during inflammation. Therefore, we hypothesized that EP2 inhibition would protect against induction of COX-2 and intestinal barrier breakdown in experimental peritonitis.

Methods: Following IACUC approval, C57/Bl6 mice underwent sham operation as a control or cecal ligation and puncture (CLP) to induce experimental peritonitis. Mice were injected intraperitoneally with or without 10mg/kg EP2 receptor antagonist PF-04418948 at time of operation and were orally gavaged with fluorescein isothiocyanate (FITC)-dextran 8 hours later. All mice were sacrificed 12 hours following sham or CLP. Blood samples were analyzed for FITC-dextran to determine intestinal barrier breakdown and terminal ileum was analyzed for COX-2 expression.

Results: CLP led to increased serum FITC-dextran levels and higher intestinal COX-2 mRNA and protein expression compared with sham. PF-04418948 attenuated serum FITC-dextran levels and intestinal barrier breakdown from 4.2±0.8 to 2.0±0.5 fold change from sham (p<0.05) in mice who underwent CLP. PF-04418948 decreased COX-2 mRNA levels in terminal ileal samples from 25±19 to 9±3.3 fold compared with sham mice. Additionally, COX-2 protein levels in terminal ileal samples decreased from 1.8±0.1 to 1.2±0.1 fold sham levels (p<0.05) in CLP mice who received PF-04418948. 

Conclusion: EP2 receptor inhibition protects against intestinal barrier breakdown following cecal ligation and puncture and may inhibit the positive feedback induction of COX-2 via PGE2 activation of EP2. This suggests that EP2 receptor specific inhibition may have important therapeutic implications in the treatment of inflammation and gut-origin sepsis.
 

63.06 Intestinal Alkaline Phosphatase Regulates Gut Barrier Function And Enhances Life Span In Drosophila

Posted on January 26, 2017

F. Adiliaghdam1, A. Tsurumi1,2,3, Y. Dhole1, J. Ramirez1, F. Kuehn1, A. Munoz1, S. Hamarneh1, L. Rahme1,2,3, R. Hodin1  1Massachusetts General Hospital,General Surgery,Boston, MA, USA 2Shriners Hospitals For Children,Boston, MA, USA 3Harvard School Of Medicine,Department Of Microbiology And Immunobiology,Brookline, MA, USA

Introduction: Intestinal barrier dysfunction is considered to be an underlying factor in the pathophysiology of aging.We have previously shown that the gut enzyme intestinal alkaline phosphatase(IAP) maintains a healthy gut mucosal barrier.We therefore hypothesized that IAP could play an important role in aging process.

Methods: The wild-type(WT) Oregon-R Drosophilia Melanogaster was used to study the changes of gut alkaline phosphatase(AP)activity with aging and the effect of IAP supplementation on lifespan.Strain y/w67C23 was used to develop double-cross mutants for the midgut-specific AP genes in D.melanogaster, CG5150 and CG10827, for lifespan analysis. Furthermore,Gut barrier function was assessed using feeding with a blue food dye. Junction protein gene expression (DECad: Drosophila E-Cadherin and dlg1:discs large 1genes) assayed by qPCR from dissected mid-guts of 35 day-old WT and AP double-cross mutants. Three independent trials were done for all experiments.

Results: We found that AP activity in WT D.melanogaster’s midgut decreased with aging (P< 0.0001). In WT flies, oral IAP supplementation caused major lifespan extension compared to vehicle controls (median survival: 53d vs. 47d, P<0.0001). Climbing assay showed that IAP-treated flies climbed better than the vehicle-treated controls (%24 increase at week3, P< 0.0001 and 10% increase at week5, P<0.01). We also found that the double-cross mutants lacking the two AP genes died earlier than the WT-flies (median survival:42d vs. 47d, P<0.0001) and also performed worse in the climbing assay (21% decrease P<0.001). WT-flies showed a significant age-dependent breakdown in the gut barrier and this impairment was much worse in the AP double-cross mutants.(Figure1) Furthermore, AP double-cross mutants had significantly reduced mRNA levels of Junction proteins in the midgut compared to WT flies: DECad (Mutants vs. WT, 0.491± 0.070 Vs.1.0± 0.14 Relative Expression ,P=0.001) and dlg1 (Mutants vs. WT, 0.607± 0.07 Vs.1.0± 0.26 Relative Expression, P<0.01)

Conclusion: Decreasing endogenous levels of IAP is an important factor in age-dependent loss of intestinal integrity.We believe that IAP supplementation could represent a novel therapy to delay aging process.

 

63.05 Endogenous Intestinal Alkaline Phosphatase Modulates Inflammatory Pathways and Homeostasis in the Gut

Posted on January 26, 2017

F. M. Kuehn1, S. R. Hamarneh1, J. M. Ramirez1, A. R. Munoz1, F. Adiliaghdam1, S. A. Morrison1, R. A. Hodin1  1Massachusetts General Hospital,Department Of Surgery,Boston, MA, USA

Introduction:  The inability of epithelial cells to cope with various stresses and cellular damage plays a crucial role in the development of many inflammatory diseases in the gut. The exact functions of the brush border enzyme intestinal alkaline phosphatases (IAP) in various cellular pathways are not well understood. We propose that IAP functions as an important modulator of intracellular homeostasis and stress responses in the gut.

Methods: Functional analysis and identification of IAP-binding proteins were performed using Liquid chromatography-tandem mass spectrometry (LC-MS) after immunoprecipitation of IAP complexes from Caco-2 cells in vitro. Furthermore, IAP knockout and overexpressing Caco-2 cells were developed using CRISPR/Cas9 gene editing technique and cell transfection, respectively. Subsequently, the effect of IAP activation or inhibition on inflammatory cytokine levels was measured using qPCR in the IAP-knockout and overexpressing Caco-2 cells. Additionally, the role of IAP in Caco-2 cell survival was assayed after incubation with high doses of TNF-α.

Results: Analysis of IAP protein complexes showed that IAP binds to key modulators of the NFκB, TNF-α and TLR-4 pathways. Overexpression of human IAP significantly reduced mRNA-levels of inflammatory cytokines in Caco-2 cells: TNF-α (IAP WT vs. IAP-overexpressing Cells, 1.0 ± 0.08 Vs. 0.19 ± 0.1 Relative Expression, p= 0.01), IL-1β (IAP WT vs. IAP-overexpressing Cells, 1.0 ± 0.4 Vs. 0.47 ± 0.06 Relative Expression, p= 0.001) and IL-8 (IAP WT vs. IAP-overexpressing Cells, 1.0 ± 0.26 vs. 0.46 ± 0.16 Relative Expression, p= 0.046). IAP deletion increased the expression of TNF-α (IAP WT vs. IAP-KO Cells, 1.0 ± 0.2 Vs. 7.9 ± 0.7 Relative Expression, p= 0.007) and IL-1β (IAP WT vs. IAP-KO Cells, 1.0 ± 0.6 Vs. 11.4 ± 0.1.1 Relative Expression, p= 0.004) and IL-8 (IAP WT vs. IAP-KO Cells, 1.0 ± 0.12 Vs. 15.9  ± 0.8 Relative Expression, p= 0.0013). Furthermore, overexpression of IAP resulted in significantly less inflammation and cytokine production in Caco-2 cells when incubated with inflammatory mediators such as TNF-α (IAP WT vs. IAP-overexpressing Cells, 9.7 ± 1.8 Vs. 1.2 ± 0.46 Relative Expression, p= 0.004), LPS and bacterial contents. Additionally, higher IAP levels significantly increased Caco-2 survival after incubation with TNF-α 20ng/mL for 24 hours (IAP WT vs. IAP-overexpressing Cells, 30.5 ± 7.2 Vs. 90.3 ± 11.1 % Survival, p= 0.018). 

Conclusion: Endogenous IAP functions as a modulator of the stress response and inflammatory pathways in intestinal epithelial cells. Intracellular IAP pathways in the gut may represent an important therapeutic target to prevent or treat a variety of gut inflammatory conditions.

 

63.04 Hepatic Exosomes Regulated by Rab27a Promote Liver Ischemia/Reperfusion (I/R) Injury

Posted on January 26, 2017

M. Yang1,2, Q. DU1, P. R. Varley1, J. Goswami1, R. Wang1, B. Chen1, N. D. Anderson1, D. B. Stolz3, D. A. Geller1  1University Of Pittsburgh,Department OF Surgery,Pittsburgh, PA, USA 2The First Hospital Affiliated To Nanchang University,Department OF Surgery And Department Of Transplantation,Nanchang, JIANGXI, China 3University Of Pittsburgh,Center Of Biology Imaging,Pittsburgh, PA, USA

Introduction:  Exosomes play an important role in cell communication, and Rab27a is a GTPase that has been shown to promote exosome secretion. However, the role of Rab27a and exosomes in liver I/R injury is unknown. We hypothesized that exosome secretion regulated by Rab27a promotes liver I/R injury.

Methods:  70% liver I/R injury mouse model was used. Adenoviral Rab27a shRNA (Ad-Rab27a shRNA) was used to knock down hepatic Rab27a expression. Circulating serum exosomes (Exo) in vivo or murine hepatocyte-secreted exosomes in vitro were isolated with ultracentrifugation. Exosome pellets were verified by TEM and by exosome markers CD63, CD81 and HSP70. Serum Exo concentration was analyzed with CD81 exosome ELISA kit.  Liver damage during I/R was calculated with ALT and liver HE staining. Hypoxia or normoxia primed hepatocyte-secreted exosomes were injected to mice in vivo during warm I/R with/without Rab27a knockdown.

Results: Hepatic Rab27a protein was induced in vivo during liver I/R in a time-dependent manner with strong induction 6 hr after I/R (Fig. A).  Liver I/R induced increase in hepatic Rab27a protein expression was diminished by knockdown of Rab27a in vivo with Ad-Rab27a shRNA (Fig. B, last lane), but not scrambled shRNA. Knockdown of Rab27a resulted in a dramatic reduction in liver damage measured by ALT (Fig. C) and liver necrosis on HE staining (not shown).

During liver warm I/R injury, serum exosomes were increased in a time-dependent manner with maximal serum exosome pellet seen 6-12 hr after I/R (Fig. D). Knockdown of hepatic Rab27 in vivo with Ad-Rab27a shRNA decreased serum exosome concentration (Fig. E). Liver damage in vivo during warm I/R was reduced by Rab27a knockdown (Fig. F, third column), and this protective effect was abrogated by injecting hypoxia primed hepatocyte-secreted exosomes (Fig. F, last column). 

Conclusion: Hepatic Rab27a protein expression was markedly increased in vivo during liver warm I/R injury, and this resulted in increased serum exosome concentrations. Knockdown of Rab27a decreased exosome secretion and liver damage. Hypoxia primed exosomes recapitulated liver damage reversed by Rab27a knockdown. These findings increase our understanding of fundamental exosome cell signaling and regulation during liver I/R injury.
 

63.03 Inflammatory Cytokine Regulation of Extracellular Matrix Results in Attenuated Renal Fibrosis

Posted on January 26, 2017

X. Wang1, P. Duann1, C. Lu1, C. Moles1, H. Li1, M. Fahrenholtz1, M. Rae1, Y. Dhamija1, J. Cheng2, S. Balaji1, S. Keswani1  1Baylor College Of Medicine,Surgery,Houston, TX, USA 2Baylor College Of Medicine,Medicine,Houston, TX, USA

Introduction:

Renal fibrosis is a pathological characteristic of chronic kidney disease (CKD), which affects nearly 700 million patients globally, and is a product of aberrant inflammation and extracellular matrix (ECM) deposition. Patients with CKD are associated with a three-fold or higher mortality rate compared to the general population. We have previously shown a novel role for interleukin-10 (IL-10) in dermal fibrosis, beyond its accepted anti-inflammatory role. In this role, IL-10 regulates the ECM, specifically hyaluronan (HA), and TGFβ isoforms, which are crucial for regenerative tissue repair. However, the roles of IL-10 and HA in renal fibrosis are not completely elucidated. We hypothesize that IL-10 might regulate HA and TGFβ expression in the kidney, and attenuate renal fibrosis in murine unilateral urethral obstruction (UUO) model. 

Methods:
Primary renal fibroblasts (FB) were isolated from 8-10 week-old male C57BL/6J (WT) mice. IL-10 (200 ng/ml) with or without hyaluronidase (HYAL, 1.5 unit/ml) was added to cultures. HA matrices were analyzed by particle-exclusion assay at 24h. Gene expression of HA synthases 1, 2, and 3 (HAS1-3), hyaluronidases 1 and 2 (HYAL1-2) and TGFβ-1 were assessed by qPCR at 1, 2, 3 and 6 h. 8 weeks C57BL/6J (WT) and IL-10 KO male mice were injected with lenti-IL-10/ lenti-GFP (1×1010 IU) under the kidney capsule. Three days after the injection, unilateral ureteral obstruction (UUO) was performed. UUO/sham kidneys and serum were collected at 14 days after UUO for RNA, ELISA, and immunohistochemical (IHC) analysis. n=3/treatment group; p-values by ANOVA.

Results:
In vitro, IL-10 resulted in an upregulation of HAS-1,2, and 3 expression at 2h after treatment, and a significant downregulation of HYAL 1, 2 and TGFβ-1. IL-10 resulted in a 1.88-fold increase in HA-rich matrix formation at 24h, and the effect was abolished by HYAL treatment (p<0.05). In vivo, IL-10 KO mice demonstrated more fibrosis than WT mice. Lenti-IL-10 treatment resulted in less dilated tubules and decreased kidney fibrosis, as well as reduced α-SMA expression as compared to lenti-GFP treated kidneys in both WT and IL-10 KO mice. The HA level in serum was 1.7-fold higher in lenti-IL-10 treated mice as compared to lenti-GFP treated (p<0.05) (Fig.1).

Conclusion:
Our data demonstrates that IL-10 regulates HA metabolism and TGFβ expression of renal FB in vitro, and is effect of IL-10 is validated in the UUO model. The endogenous IL-10 is essential for normal kidney integrity against excessive fibrosis with UUO injury. This previously unreported mechanism for IL-10 regulation of ECM in the kidney may have a significant impact for future therapies to ameliorate kidney fibrosis.

63.02 Del1 Knockout Affects Bone Cartilage Stroma Progenitor Cells Following Femur Fracture in Mice

Posted on January 26, 2017

T. V. Boyko1,2, O. Marecic1, E. Y. Seo1, C. K. Chan1, T. Leavitt1, M. T. Longaker1, G. P. Yang1,3  1Stanford University,Surgery,Palo Alto, CA, USA 2State University Of New York At Buffalo,Surgery,Buffalo, NY, USA 3VA Palo Alto Healthcare Systems,Surgery,Palo Alto, CA, USA

Introduction:  DEL1 is a secreted protein, which has been shown to be involved in bone fracture healing. In previous experiments we have demonstrated that Del1 gene knockout (KO) mice healed fractures with 15% less bone when compared to wildtype (WT) mice. Increased apoptosis was also seen in fracture calluses of KO mice. Separately, we identified the mouse skeletal stem cell (mSSC) and 7 other unique subpopulations of skeletal progenitor cells that are capable of self-renewal and giving rise to all three components of the skeleton: bone, cartilage and stroma. One subpopulation, the Bone Cartilage Stroma Progenitor Cells (BCSPs), are the primary skeletal stem cell population involved in fracture repair. Following fracture, BCSPs transition to another sub-type, f-BCSPs, that has greater osteogenic potential. We hypothesized that Del1 deletion leads to decreased fracture callus due to an effect on BCSP biology. 

Methods:  Femurs were fractured in KO and WT mice. Fracture calluses were harvested on post-operative day 7. BCSPs, mSSCs and f-BCSPs were isolated by Fluorescent Activated Cell Sorting (FACS) following staining for signature cell surface markers. Antibody staining for Annexin V was used to determine extent of apoptosis. KO and WT BCSPs were cultured in vitro and colony-forming units (CFUs) were counted 14 days after plating. 

Results: FACS analysis revealed that the BCSP populations in KO and WT femurs were equivalent prior to fracture (p=0.498, n=3 each). There is an increase of skeletal progenitors in the callus following fracture, but fewer BCSPs were found in KO mice compared to WT (11,700 cells/1 million events vs 77,706 cells/1 million events, p<0.01, n=4 each). Additionally, mSSCs showed a similar pattern in the KO mice with an attenuated increase after fracture (12,452 cells/1 million events in KO vs 57,451 cells /1 million events in WT, p<0.02, n=4 each). The percentage of apoptotic cells was found to be higher in both BCSPs (23.98% KO vs. 6.56% WT, p<0.001, n=4 each) as well as for mSSCs (14.83% KO vs. 4.74% WT, p<0.002, n=4 each). Following culture in vitro, KO BCSPs showed no difference in proliferation compared to WT, but did form significantly fewer CFUs (16.3 vs. 24 CFUs, p<0.05, n=6 each).  Examining f-BCSPs showed an equivalent percentage in KO compared to WT.

Conclusion: Following fracture, skeletal progenitors expand to create bone. In KO mice, this expansion is attenuated leading to decreased bone formation. BCSPs still transition to a more osteogenic phenotype, but there are just fewer of them. These data suggest DEL1 may have a therapeutic role in promoting fracture healing or regenerating bone.

 

63.01 Obeticholic acid accelerates liver regeneration following portal vein embolization in a rabbit model

Posted on January 26, 2017

P. B. Olthof1, F. Huisman1, K. Van Lienden2, R. F. Van Golen1, M. Heger1, J. Verheij3, F. F. Schaap4, P. L. Jansen4, S. W. Olde Damink4, T. Van Gulik1  1Academic Medical Center,Surgery,Amsterdam, ZUID-HOLLAND, Netherlands 2Academic Medical Center,Radiology,Amsterdam, ZUID-HOLLAND, Netherlands 3Academic Medical Center,Pathology,Amsterdam, ZUID-HOLLAND, Netherlands 4NUTRIM School Of Nutrition And Translational Research In Metabolism, Maastricht University,Surgery,Maastricht, LIMBURG, Netherlands

Introduction: Portal vein embolization (PVE) is used to increase future remnant liver volume in patients scheduled for major liver surgery. The bile salt-activated transcription factor farnesoid X-receptor (FXR) is a key mediator of bile salt signaling, an event implicated in the early phase of liver regeneration following partial hepatectomy. The aim of this study was to evaluate the effect of a potent FXR agonist (obeticholic acid, OCA) on PVE-induced liver hypertrophy.

Methods: Twenty-four rabbits (female, 2.9±0.4kg) were given a daily oral gavage with OCA (10mg/kg) or vehicle starting 7 days pre-PVE until 7 days post-PVE of the cranial liver lobes.  Effectiveness of the embolization procedure (coils, PVA particles) was confirmed by portography. Caudal liver volume (CLV) was analyzed by CT-volumetric analysis at days -7, -1, +3 and +7. Rabbits were sacrificed at day +3 and +7.

Results:Three days after PVE the increase in CLV was 2.0-fold (59.3 ± 19.2% vs. 29.7 ± 16.1, p=0.0013) greater in the OCA group compared to controls. No differences in CLV increase were measured after 7 days. OCA had no effect on volume of the atrophic cranial lobes at the respective time points. Likewise, OCA did not cause spontaneous liver growth, as liver volume before PVE was proportional to body weight increase over the days before PVE.

Conclusion:Obeticholic acid accelerated liver regeneration in a rabbit model of PVE by 2.0-fold over the first 3 days. The ultimate increase in CLV is the same in both groups. OCA treatment has potential in extending resectability as well as the prevention of postoperative liver failure.