29.01 Treatment strategy for hepatocellular carcinoma with portal vein tumor thrombosis

Posted on December 22, 2014

T. Ochiai1, T. Sato1, Y. Ohata1, H. Ueda1, A. Oba1, K. Akahoshi1, K. Nakao1, T. Furuyama1, E. Katsuta1, H. Ito1, S. Matsumura1, A. Aihara1, D. Ban1, T. Irie1, A. Kudo1, S. Tanaka1, M. Tanabe1  1Tokyo Medical And Dental University,Hepato-Biliary-Pancreatic Surgery,Bunkyo-ku, Tokyo, Japan

Introduction: Hepatocellular carcinoma (HCC) is the most common type of primary liver tumor and is the fifth most common malignancy worldwide. The prognosis of patients with HCC accompanied by portal vein tumor thrombus (PVTT) is generally poor, therefore, the role of surgical resection for HCC with PVTT is controversial. This study aimed to evaluate the efficacy of initial surgery for HCC with PVTT and secondary treatments; hepatectomy, radiofrequency ablation (RFA), transcatheter arterial chemoembolization (TACE), chemotherapy, radiation, for recurrence after curative resection.

Methods: From April 2000 to December 2013, initial hepatic resection for 617 patients with HCC was performed at our hospital. Among these patients, a retrospective study was carried out on 79 patients (12.8%) with PVTT.

Results:The 1, 3- and 5-year overall survival rates were 65%, 41% and 38% in 66 patients who underwent curative resection and 18%, 0% and 0% in 13 patients who underwent non-curative surgery, respectively. Forty six patients with PVTT located in the segmental or sectoral portal vein showed significantly better survival than 20 with PVTT extended to right and/or left portal veins, the main portal vein or the superior mesenteric vein (Fig. 1). After initial curative resection, tumor recurrences were observed in 46 patients; 11 patients met Milan Criteria and 35 patients exceeded the criteria at first diagnosis of recurrence. Among 46 patients, 43 patients were treated by heaptectomy in 3 patients, RFA in 6, TACE in 16, or chemotherapy in 18. The overall survival was significantly better in hepatectomy and RFA than in TACE, chemotherapy and radiation.

Conclusion:Liver resection is justified in selected patients with PVTT located in the segmental or sectoral branches of the portal vein. Liver resection and RFA are suitable treatments for recurrence, however, 53% (35 of 66) of patients who underwent initial curative hepatectomy showed HCC recurrence that exceeded Milan criteria in this study. Effective adjuvant treatments need to be developed to counteract the high incidence of recurrence.

 

29.02 Mortality Following Pancreatoduodenectomy: The Influence of Fistula Risk

Posted on December 22, 2014

M. T. McMillan1, M. H. Sprys1, J. A. Drebin1, M. K. Lee1, R. E. Roses1, D. L. Fraker1, .. The Pancreatic Fistula Study Group1, C. M. Vollmer1  1University Of Pennsylvania Perelman School Of Medicine,Surgery,Philadelphia, PA, USA

Introduction:  Postoperative pancreatic fistula (POPF) is the most common and morbid complication following pancreatoduodenectomy (PD). The previously validated Fistula Risk Score (FRS) considers the presence of endogenous (gland texture, duct size, pathology) and operative (blood loss) risk factors to predict the occurrence of clinically relevant fistula (CR-POPF; ISGPF Grade B/C). These CR-POPF risk factors may also influence mortality; however, this has not been proven.

Methods:  This multinational study of 4,307 PDs involved 55 pancreatic surgical specialists at 15 high-volume institutions. Patients were stratified for 90-day mortality risk using the FRS (0-10 points) and assigned to one of four risk zones: negligible (0 points), low (1-2), moderate (3-6), or high (7-10). A Cox regression identified predictors for mortality while adjusting for FRS risk, as well as surgeon, institutional, and operative factors.   

Results: The overall mortality rate was 2.1% (N=89), with institutional rates ranging from 1.0 -8.6%. Individual surgeon rates—for those who contributed ≥ 25 cases—ranged from 0 – 11.1%. Clinically relevant fistulas accounted for 36% of the overall mortalities and their presence strongly correlated with higher rates of mortality (6.6 vs. 1.5%; P<0.001). Nearly 70% of deaths occurred in the setting of soft pancreatic parenchyma and intraoperative blood loss > 700 mL was associated with a greater than two-fold increase in mortality risk. The mean Fistula Risk Score was significantly greater in patients who suffered mortality (4.6 vs. 3.7; P<0.001). In fact, patients with high CR-POPF risk (FRS 7-10) had over a fivefold increase in mortality risk compared to patients at negligible risk (P=0.010; Figure). There was no significant difference in mean FRS between fistulous and non-fistulous mortalities (4.6 vs. 4.6; p=0.899); however, the median POD of mortality was two times greater in cases of mortality due to a CR-POPF (28 [IQR: 40] vs. 14 [IQR: 26] days; P=0.010). While surgeon years of experience and career PD volume did not significantly influence overall mortality, institutional PD volume > 75 cases per year correlated with reduced rates (1.9 vs. 4.9%; P=0.006).

Conclusion: Procedure-specific risk influences mortality after pancreatoduodenectomy. Improvements in pancreatic fistula outcomes will likely lead to improved survival following PD. 

 

29.03 Minimally Invasive Esophagectomy has Lower Severity of Complications than Open/Hybrid Esophagectomy

Posted on December 22, 2014

A. Chaudhary1, M. J. Pucci1, A. C. Berger1, E. L. Rosato1, N. R. Evans1, K. Chojnacki1, F. Palazzo1  1Thomas Jefferson University,Philadelphia, PA, USA

Introduction:  Minimally Invasive Esophagectomy (MIE) is increasingly utilized for the treatment of esophageal and gastroesophageal junction (GEJ) malignancies; however, perioperative morbidity remains significant. We sought to compare the severity of complications between patients undergoing MIE and Open/Hybrid Esophagectomy (OHE).

Methods:  Our single institution IRB-approved prospectively maintained database was retrospectively queried and a contemporary series of patients who underwent MIE (2008-2013) was compared to a cohort undergoing OHE – Mckeown approach (39), Ivor Lewis approach (16), Hybrid (13) (2000-2013). Hybrid esophagectomy was defined as having one component of the surgery performed minimally invasively—either thoracoscopic or laparoscopic. Perioperative complications were graded using the Clavien-Dindo classification.  Statistical analysis was performed using two-tailed t-test and Fisher exact test to assess the impact of operative approach (MIE vs. OHE) on operative blood loss (EBL), length of stay (LOS), rates of respiratory failure, anastomotic leaks, arrhythmias, and complication grades 3-5.

Results: MIE (n = 104) and OHE (n = 68) cohorts were similar with respect to age (p=0.288) and gender (p=0.322). The MIE cohort tended to have higher BMI (p=0.125), earlier stage disease (p=0.025), and was less likely to receive neoadjuvant CRT (p=0.001). The MIE cohort had significantly lower EBL (171 vs. 454 mL, p<0.001), decreased LOS (11 vs. 22 days, p<0.001), and lower grade 3 (1.9% vs. 10.3%, p=0.029) and grade 4 (13.5% vs. 27.9%, p=0.028) complications. No differences were noted in the rates of respiratory failure (21.2% vs. 36.2%, p=0.110), anastomotic leaks (20.2% vs. 14.7%, p=0.421), or arrhythmias (21.2% vs. 13.2%, p=0.308).

Conclusion: These data support the use of MIE over OHE for the surgical treatment of esophageal and GEJ malignancies. These findings need to be confirmed in future prospective studies.

 

29.04 Combined Surgery and Chemotherapy Correlates with Superior Survival in Hepatic Embryonal Sarcoma

Posted on December 22, 2014

Y. Shi1, W. Zhang4, E. Beierle5, J. Doski6, A. Goldin7, M. Goldfarb8, K. Gow7, M. Langer9, J. Nuchtern1,3, S. A. Vasudevan1,3  1Baylor College Of Medicine,Department Of Surgery,Houston, TX, USA 3Texas Children’s Hospital,Department Of General Pediatric Surgery,Houston, TX, USA 4Texas Children’s Hospital,Outcomes And Impact Service,Houston, TX, USA 5University Of Alabama,Department Of Surgery,Birmingham, AL, USA 6University Of Texas Health Science Center – San Antonio,Department Of Surgery,San Antonio, TX, USA 7University Of Washington,Department Of Surgery,Seattle, WA, USA 8University Of Southern California,Department Of Surgery,Los Angeles, CA, USA 9Tufts University,Department Of Surgery,Portland, ME, USA

Introduction: Embryonal sarcoma of the liver (ES) is a rare tumor that primarily afflicts children. With an incidence of 1 per million and less than 100 cases reported in the literature since 1955, few data exist to help guide therapy and counsel patients. The aim was to identify factors that correlate with survival in patients with ES.

Methods: The National Cancer Data Base was queried for primary cases of ES diagnosed between 1998 and 2011. Data were examined using log-rank comparisons of Kaplan-Meier survival curves. Multivariate analysis was performed using a Cox proportional hazards model. Chemotherapy and surgery together are referred to as combined therapy. Survival is reported as 5-year overall survival (OS).

Results: There were 156 total patients, 107 pediatric (<18y) and 49 adult (≥18y). The OS of the entire study group was 72%. On univariate analysis, the OS of pediatric patients was significantly better than that of adults (82% vs. 52%; p<0.01). Regarding treatment, the OS of patients who had combined therapy was greater than that of patients who had one of the other treatment modalities, which were surgery only, chemotherapy only, and no treatment (75% vs. 48%, 29%, 20%, respectively) (Figure A). On subgroup analysis based on age, the OS of children who had received combined therapy was greater than that of adults (88% vs. 32%; p<0.01) (Figure B). When other factors were examined, greater OS was associated with high vs. low income (79% vs. 18%; p=0.02) and private vs. public insurance (51% vs. 12%; p<0.01) in adults. This difference based on income (86% vs. 78%; p=0.22) and insurance status (84% vs. 80%; p=0.49) was not seen in children. Metastatic disease, tumor size, race, and ethnicity were not significant factors in either age group. For all patients, factors that improved OS in the multivariate analysis (shown as hazard ratio; 95% confidence interval) were: surgical resection (0.094; 0.015 – 0.62), combined therapy (0.083; 0.017 – 0.41), negative surgical margins (0.16; 0.37 – 0.68), and age <18y (0.14; 0.057 – 0.35). No factors significantly affected OS in the adult group. In children, combined therapy (0.044; 0.007 – 0.28) was associated with greater OS.

Conclusion: Superior OS was associated with age <18y, negative surgical margins, surgical resection, and combined therapy. The optimal treatment for ES appears to be combined therapy. Patients with ES seem to respond differently to treatment depending on age; survival benefit is greater in children than in adults. Further study with a larger patient population may identify additional prognostic factors related to survival in these patients.

29.05 Predictors of Postoperative Mortality Among Patients With Disseminated Cancer and Perforation

Posted on December 22, 2014

C. E. Cauley1,3, M. T. Panizales5, G. Reznor2,4, A. B. Haynes1,3, J. M. Havens2,4, Z. Cooper1,2,4  1Ariadne Labs,Boston, MA, USA 2Brigham And Women’s Hospital,Department Of Surgery,Boston, MA, USA 3Massachusetts General Hospital,Department Of Surgery,Boston, MA, USA 4Brigham And Women’s Hospital,Center For Surgery And Public Health,Boston, MA, USA 5Partners Healthcare International,Boston, MA, USA

Introduction:  Surgical intervention in patients with serious preexisting illness is an important area of inquiry due to a lack of clarity about the impact of such interventions on quality of life and overall disease course. Identifying patients with the highest mortality risk prior to surgery would help guide clinical decisions, increase family understanding, and avoid non-beneficial operations. The outcomes after emergency surgery for patients with disseminated cancer have not been well described. We sought to characterize the 30-day postoperative mortality of patients with disseminated cancer who underwent surgery for intestinal perforations, and to identify preoperative factors and patient characteristics associated with worse survival.

Methods:  We performed a retrospective cohort study using the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) database from 2005-2012 to identify patients with disseminated cancer who underwent emergency operations for intestinal perforation. Patient demographics and clinical data such as age, gender, race, comorbidities, American Society of Anesthesiology (ASA) Class, preoperative lab results, and survival were included. We conducted univariate analysis using Chi square and Wilcoxon Sum to determine differences between survivors and non-survivors. We used stepwise multivariate logistic regression including variables significant at p <0.10 level to determine patient factors predicting death at 30 days. We used the multiple imputation chained equations (MICE) method to account for missing data. Analysis was performed using SAS 9.3.

Results: Among 499 patients in our cohort, 30-day postoperative mortality was 34% (n=170). Patients who died were more likely to have ascites (43% vs. 41%, P<0.001), dyspnea at rest (25% vs. 8%, P<0.001), renal failure (11% vs. 3%, P<0.001), septic shock (39% vs. 12%, P<0.001), ASA Class >3 (69% vs. 51%, P<0.001), and preoperative do not resuscitate orders (10% vs. 2%, P<0.001), than survivors; they were less like to have body mass index (BMI) >25 (45% vs. 53%, P=0.067). Renal failure, septic shock, ascites, dyspnea at rest, and dependent functional status were independent predictors of death at 30 days (See table). BMI > 25 was protective with an odds ratio of 0.5 (0.28-0.75).

Conclusion: More than one in three patients with disseminated cancer who has surgery for gastrointestinal perforation dies within thirty days. Preoperative factors can help identify patients at highest risk of postoperative mortality and frame expectations for patients, clinicians and caregivers. Further studies are needed to examine longer-term survival and quality of life for advanced cancer patients undergoing these procedures.

29.06 Image Related Factors Important for AKI Risk Assessment After Colectomies

Posted on December 22, 2014

E. T. Chang1, A. Lussiez1, J. Li1, N. Wang1, S. C. Wang1  1University Of Michigan,General Surgery,Ann Arbor, MI, USA

Introduction:

Acute kidney injury (AKI) occurs in approximately 20% of hospitalized adults and costs the US $10 billion annually. Prolonged hospital stay, cost, and mortality have been shown to be increased in these patients and those requiring renal replacement therapy have an even higher mortality rate. In the setting of colon surgery, a recent large scale study found several associated factors with postoperative AKI including advanced age, chronic renal failure, and total colectomy. However, a surgical decision informed by standard clinical data alone may not be sufficient. We set out to study the relationship between AKI and quantitative kidney morphomic factors from cross-sectional preoperative imaging for patients undergoing colectomies and comparing the results with demographic and intra-operative data. We hypothesized that individualized kidney characteristics would correlate with the risk of postoperative AKI development.

Methods:
A retrospective review of prospectively gathered data from adult patients undergoing colectomies from 2006-2012 at the University of Michigan was conducted. 390 initial subjects without elevated baseline creatinine values, ESRD, a prior nephrectomy, or renal injury were included. Polycystic kidneys or other kidney abnormalities identified on imaging were excluded resulting in 326 subjects. AKI was defined by the KDIGO criteria within 30 days of injury. Demographic and intraoperative factors were evaluated along with measurements of pertinent kidney morphomic data including volume, surface area, and average Hounsfield Units (HU) which were subjected to univariate analyses. 

Results:
Of the 326 patients, 60 developed AKI (18.4%). Of demographic factors, advanced age (p=0.0002) and a higher BMI (p=0.02) were associated with AKI. Comorbidities associated with AKI included hypertension (p=0.008) and diabetes (p=0.013). Interestingly, intraoperative factors including surgical time (p=0.13), blood transfusions (p=0.14), blood loss (p=0.42), and hypotensive episodes (p=0.17) were not significantly associated with AKI. Table 1 shows the results of the univariate analysis of the morphomic factors.

Conclusion:
AKI is a serious complication and the prevalence after colorectal surgery at our institution was 18.4%. Our analysis shows that a lower average parenchymal HU was the most significant morphomic factor associated with postoperative AKI. This study is the first to provide information that morphomic analysis can be useful in clinical practice to determine which patients are at risk for developing AKI after a colectomy.
 

29.07 Outcomes in Cirrhotic Patients After Major Laparoscopic vs Open Gastrointestinal Non-Hepatic Surgery

Posted on December 22, 2014

D. Hsu1, S. Tohme1, D. Chalhoub2, A. Tsung1  1University Of Pittsburgh,General Surgery,Pittsburgh, PA, USA 2University Of Pittsburgh,Department Of Epidemiology,Pittsburgh, PA, USA

Introduction:  Patients with liver cirrhosis are considered to be at higher risks of developing peri-operative morbidity and mortality. Laparoscopic surgery is considered a safe and feasible alternative to open surgery in cirrhotic patients undergoing cholecystectomy or hepatic resections. However, outcomes after major nonhepatic laparoscopic surgery in cirrhotic patients remain unclear. The aim of this retrospective study was to compare patient characteristics, short term morbidity and mortality between major laparoscopic and open nonhepatic gastrointestinal surgery at a single center in patients with histologically proven cirrhosis.

Methods: Electronic medical records of 4758 patients who were diagnosed with cirrhosis and underwent open or laparoscopic surgery from 1990 to 2014 at University of Pittsburgh Medical Center were reviewed. Out of those patients, 186 patients underwent open and 63 underwent laparoscopic major non-hepatic abdominal surgeries. Demographic information (age, gender, etiology of cirrhosis, MELD score, pre-operative hemoglobin, albumin, and platelets) and peri-operative data (re-admission rate, 90 day morbidity and mortality) were reviewed and analyzed. Adjusted and nonadjusted Cox proportionate hazard ratio was used to compare outcomes. 

Results: 

With the laparoscopic group compared with the open surgery group, mean age was 55 vs. 59 (p=0.03), MELD score was 5.3 vs. 16.1 (p<0.001), emergent surgery was performed in 11.1% vs. 68.3% (p<0.001). With regards to short term outcomes, operative time was 189 minutes vs. 210 minutes (p = 0.452), hospital stay was 6 days vs. 16 days (P < 0.001), 90 day morbidity grade was 0.3 vs. 3.1 (p<0.001), 90 day readmission rate was 9.5% vs. 21.5% (p=0.05), 90 day mortality rate was 3.2% vs. 45.2% (p<0.001).

We next did subgroup analysis of patients who underwent elective surgery with adjustment of MELD score since the open surgery group were more likely to have more advanced cirrhosis and have emergent surgery performed which likely is behind the significantly worse outcomes. In non-emergent cases and after adjusted for MELD score and age, the hazard ratio for 90 day mortality was 6.2 (95%CI 0.7-52) times greater in open compared to laparoscopic surgery. The hazard ratio for 90 day readmission after adjusting for MELD score was 4 (95%CI 1.2-12.9) times greater in open compared to laparoscopic surgery. In addition, 90 day complication rate after adjusting for MELD score was 16.8 (95%CI 4.1-69.2) times greater in open compared to laparoscopic surgery. 

Conclusion: Laparoscopic surgery is more likely to be offered to patients with early stage cirrhosis on an elective basis. Open approach is more likely to be performed in the emergent setting. Laparoscopic surgery has fewer overall postoperative complications, a shorter hospital stay, decreased morbidity and mortality than open surgery even after adjusting for emergency of surgery and degree of cirrhosis. 
 

29.08 The Diagnostic Accuracy of Abdominal Ultrasound and HIDA Scan for Acute Cholecystitis

Posted on December 22, 2014

C. Kaoutzanis1, E. Davies1, S. W. Leichtle3, K. B. Welch2, S. Winter1, M. G. Franz1, W. Arneson1  1St. Joseph’s Mercy Hospital,Department Of Surgery,Ann Arbor, MI, USA 2University Of Michigan,Center For Statistical Consultation And Research,Ann Arbor, MI, USA 3University Of Southern California,Department of Surgery,Los Angeles, CA, USA

Introduction: Acute cholecystitis is one of the most common surgical problems, yet accurate diagnosis of these patients can be difficult given the variable presentation and nonspecific findings that may be present.  A substantial debate remains over the utility of physical examination, abdominal ultrasound (AUS), or advanced imaging such as hepato-imino diacetic acid (HIDA) scan to support the diagnosis, alone or in combination.  The aim of this study was to establish the diagnostic value of AUS and HIDA scan, which are the two of the most commonly used imaging modalities used for patients with suspected acute cholecystitis.

Methods: The diagnostic imaging workup of patients who presented to the Emergency Department with acute abdominal pain and suspected diagnosis of acute cholecystitis was reviewed to calculate the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of AUS and HIDA scan. The diagnostic value of each AUS finding (gallstones, sludge, sonographic Murphy's sign, gallbladder distension, pericholecystic fluid, gallbladder wall thickening) was also assessed by comparing the percent of patients with and without each finding who were later confirmed histologically to have acute cholecystitis, using a Pearson chi-square or Fisher's exact test.

Results: A total of 3969 emergency room visit charts from November 2009 through May 2011 were reviewed, with 1217 patients meeting inclusion criteria. 1176 patients underwent AUS, 301 underwent HIDA scan, and 260 had both studies performed.  Sensitivity, specificity, PPV and NPV of AUS and HIDA scan for all cases with each imaging modality and for cases with both imaging modalities, based on ultimate histological diagnosis, are shown in Table 1. When both imaging modalities were used, the sensitivity, specificity, PPV, and NPV were better for HIDA scan than AUS for diagnosing acute cholecystitis. In addition, the odds of having histologically proven acute cholecystitis were higher when each AUS finding was present versus absent. As the number of AUS findings increased, the odds of having histologically proven acute cholecystitis also increased – 1 AUS finding Odds Ratio (OR) 41.4 (CI 10.8-371.4), 2 AUS findings OR 113.8 (CI 29.7-1020.6), and 3 or more AUS findings OR 261.8 (CI 68.3-2349.4).

Conclusion: In adults with acute abdominal pain presenting to the Emergency Department, the effectiveness of AUS in diagnosing acute cholecystitis increases with the number of AUS findings. However, HIDA scan has superior diagnostic value for identifying patients with acute cholecystitis.  An improved understanding of the real-world diagnostic ability of imaging modalities may help develop algorithms for optimum surgical resource utilization.

 

29.09 The Management of Cholecystectomy in Patients Receiving Anticoagulant and/or Antiplatelet Therapy.

Posted on December 22, 2014

K. Akahoshi1, T. Ochiai1, S. Matsumura1, A. Aihara1, D. Ban1, T. Irie1, A. Kudo1, S. Tanaka1, M. Tanabe1  1Tokyo Medical And Dental University,Department Of Hepato-Biliary-Pancreatic Surgery,Bunkyo-ku, Tokyo, Japan

Introduction: Patients receiving anticoagulant and/or antiplatelet therapy are increasing with aging of the society. In case of emergent surgery, if they continue taking anticoagulant and/or antiplatelet therapy up to the time of surgery, they face an increased risk of bleeding. Therefore, most patients need to bridge with heparin or to discontinue it before surgery. However, some patients don’t have adequate time to weaken the antiplatelet effect, or need to continue it to avoid thrombosis. We retrospectively studied the acute cholecystitis patients on anticoagulant and/or antiplatelet therapy and report the introduction of hemostatic techniques for liver surgery into the acute cholecystitis patients on anticoagulant and/or antiplatelet therapy.

Methods: Between 2008 and 2013, 85 patients were performed cholecystectomy for acute cholecystitis in our hospital and 18 patients were on anticoagulant and/or antiplatelet therapy. The medical records of 85 patients were retrospectively reviewed.

Results:Of the 18 patients, 9 were on antiplatelet, 5 were on anticoagulant and 4 were on both anticoagulant and antiplatelet. Prior to emergent surgery, anticoagulant and/or antiplatelet were discontinued in 2 patients, bridged with heparin in 8 and continued in 7. The average amounts of blood loss of those who discontinued the anticoagulant and/or antiplatelet, bridged with heparin and continued were 289ml, 283ml and 528ml, respectively. Among all 85 patients of acute cholecystectomy, intraoperative bleeding more than 1000ml occurred in 29% (2 of 7) of the patients who continued antiplatelet therapy, 13% (1 of 8) of heparinization group and 2.9% (2 of 68) of control group. In addition, hemostatic agents such as Tachocomb and hemostatic devices such as TissueLink dissecting sealer were effective to control the bleeding from liver bed. No postoperative hemorrhage was confirmed in this study.

Conclusion:The risk of intraoperative bleeding increased when anticoagulant and/or antiplatelet therapy was continued. In our experiences, hemostatic techniques for liver surgery, such as hemostatic agents and TissueLink dissecting sealer, are effective to control intraoperative and postoperative bleeding.

29.10 The Impact of Smoking on Ventral Hernia Repair: An Analysis of NSQIP Data

Posted on December 22, 2014

S. Ross1, C. R. Huntington1, T. Cox1, L. Blair1, B. Oommen1, A. Walters1, A. Lincourt1, R. Sing1, B. T. Heniford1, V. Augenstein1  1Carolinas Medical Center,Charlotte, NC, USA

Introduction:
Several studies have shown that smoking increases rates of complications following surgery.  This study quantifies the effect of smoking on ventral hernia repair (VHR) using national outcomes data.

Methods:
The NSQIP database was queried from 2005-2011 for all elective laparoscopic VHR (LVHR) and open VHR (OVHR).  Patients were stratified by surgical approach, and outcomes were compared by current smoking status using standard statistical tests.  Multivariate regression (MVR) was performed for outcomes controlling for age, sex, BMI, diabetes, Charlson Comorbidity Index (CCI), recurrent and incarcerated hernia.

Results:

There were 75,332 VHRs identified: 9,153 LVHR and 66,179 OVHR.  In their respective strata, LVHR and OVHR were similar (means or percentage): smokers (19.5% vs 21.4%), age (56.8±13.4 vs 56.4±14.2), male (40.4% vs 42.6%), BMI (33.6±8.1 vs 32.5±8.4), CCI (0.4±0.8 vs 0.5±1.1), diabetic (16.6% vs 16.7%), tobacco use in pack years (10.3±21.0 vs 11.0±21.4), recurrent hernia (23.6% vs 22.8%), and incarcerated hernia (28.2% vs 22.1%).  In LVHR, smokers were similar to nonsmokers by rates of recurrent hernia, gender, and mean CCI (p>0.05); however, they had decreased age, BMI, and diabetes rates and increased rate of incarceration (p<0.05).  In OVHR, smokers had equivalent CCI (p>0.05) but were more often male with increased rates of recurrent and incarcerated hernias.  For non smokers, OVHR had decreased mean age, BMI, and CCI(p<0.05).  Results of MVR are displayed in the Table and show a higher odds ratio for all complications examined for smokers undergoing OVHR, including reoperation, readmission and mortality (p<0.05).  Additionally, major complications and readmission odds were increased in the smokers undergoing LVHR (p<0.05).  Paradoxically, length of stay was actually shorter in the smoker group for both repair types (p<0.05). 

Conclusion:
Smoking has a substantial negative impact on operative morbidity in LVHR, but has a markedly significant effect on patient outcomes in OVHR.  Smoking can be a prognostic indicator of wound and major complications, reoperations, readmission, and mortality.  If a repair must be performed, a LVHR is less morbid than an OVHR in a current smoker.  Based on these data, smoking may be an absolute contraindication in elective OVHR. 

26.08 Transdermal Deferoxamine Prevents Pressure-Induced Diabetic Ulcers

Posted on December 22, 2014

D. Duscher1, Z. N. Maan1, A. J. Whittam1, V. W. Wong1, M. S. Hu1, G. G. Walmsley1, R. C. Rennert1, M. Rodrigues1, A. J. Whitmore1, D. Atashroo1, E. R. Zielins1, R. Tevlin1, J. Barrera1, J. Rajadas2, G. C. Gurtner1  1Stanford University,Division Of Plastic Surgery,Palo Alto, CA, USA 2Stanford University,Biomaterials And Advanced Drug Delivery Center,Stanford, CA, USA

Introduction:
The primary reason for compromised diabetic wound healing is impaired neovascularization in response to tissue ischemia. Hypoxia inducible factor-1 alpha (HIF-1α) is the governing transcriptional factor in response to hypoxia and has been shown to be impaired in diabetes. We examined whether activation of HIF-1α via a transdermal drug delivery system (TDDS) of deferoxamine (DFO) was effective to treat and prevent diabetic wounds. 

Methods:
A TDDS containing the FDA approved small molecule DFO, known to increase HIF-1α activation, was developed. The TDDS was assessed for its physicochemical characteristics and its effects on acute and chronic diabetic wound healing. DFO TDDS application was compared to vehicle patch control in chronic and to 1mM, 100mM drip-on application and vehicle patch control in acute diabetic wounds. Upon closure tensile testing of the wound was performed and histological samples were collected.

Results:
The TDDS displayed satisfactory physicochemical characteristics and significantly accelerated excisional wound closure (12 days) vs. 1mM (15.25 days) and 100mM drip-on (15.6 days), and vehicle control patch (19.4 days) (*P < 0.05). No significant differences were observed between the 1mM and 100mM drip concentration. DFO TDDS therapy also reduced chronic diabetic wound healing time by 50% (*P < 0.05). Prophylactic DFO TDDS application was further able to prevent diabetic pressure ulcer formation (*P < 0.05). Histological examination revealed an increase in dermal thickness, collagen density, and vascularity in the DFO patch group (*P < 0.05). Uniaxial skin tensile testing demonstrated increased wound strength in the treatment group according to Young’s modulus (*P < 0.01) and ultimate tensile strength (*P < 0.05).

Conclusion:
TDDS application outperforms direct application of DFO solution in diabetic wound healing. DFO TDDS treatment further results in significantly accelerated healing of chronic diabetic ulcers and can be used prophylactically to massively reduce the incidence of ulcer formation in diabetic mice. The developed TDDS improves overall wound quality and can be rapidly translated into clinical practice.

 

Figure Legend

(A) An established model of chronic ulcer formation by applying 2 magnets on a dorsal skinfold of diabetic mice was employed. (B,C) Significantly improved ulcer healing was achieved by DFO TDDS treatment. (D) The closed wounds were more vascularized represented by CD31 immunohistochemistry. (E) Picrosirius red staining revealed increased collagen density in the DFO TDDS treatment group.

 

26.09 Delivery of Monocyte Lineage Cells in a Biomimetic Scaffold Enhances Tissue Repair

Posted on December 22, 2014

G. G. Walmsley1,2, M. S. Hu1, K. Weiskopf2, R. C. Rennert1, M. Januszyk1, Z. N. Maan1, D. Duscher1, K. Senarath-Yapa1, A. J. Whittam1, R. Tevlin1, D. A. Atashroo1, I. L. Weissman2, H. P. Lorenz1, G. C. Gurtner1, M. T. Longaker1,2  1Stanford University School Of Medicine,Department Of Surgery, Division Of Plastic & Reconstructive Surgery,Stanford, CA, USA 2Stanford University School Of Medicine,Institute For Stem Cell Biology And Regenerative Medicine,Stanford, CA, USA

Introduction:
Macrophages are thought to play a critical regulatory role in many stages of wound healing, including angiogenesis, reepithelialization, and remodeling. Evidence for the importance of macrophages in these processes comes from experiments demonstrating impaired wound healing in mice following DTR-based ablation of macrophages, genetic knockout of G/M-CSF, or administration of anti-macrophage antiserum. We have previously shown that transplantation of macrophages into excisional wounds on wild type (FVB/NJ) mice significantly increases the rate of wound healing. Here, we expand the analysis to include diabetic wound healing and monocyte transplantation.

Methods:
Macrophages derived from the bone marrow of L2G (FVB-Tg(CAG-luc,-GFP)L2G85Chco/J) were seeded on pullulan-collagen hydrogels and transplanted onto splinted excisional wounds on the dorsum of diabetic (FVB.BKS(D)-Leprdb/ChuaJ) mice. Human monocytes isolated from drawn blood were similarly transplanted on pullulan-collagen hydrogels onto splinted excisional wounds on the backs of immunodeficient nude (Foxn1nu) mice. Histologic analysis allowed for in vivo tracking of the survival, localization, and phenotype of transplanted macrophages and monocytes. Microfluidic single-cell gene expression analysis of transplanted L2G macrophages (GFP+Luc+) FACS-isolated on the basis of GFP expression from cutaneous wounds provided further insight into macrophage phenotype and behavior during wound healing.

Results:
L2G macrophage-seeded hydrogels improved wound healing compared to un-seeded hydrogel controls on days 4-20 (*p<0.01) in diabetic mice. The average time for complete wound healing was 17.2 days in the macrophage group versus 20.3 days in the control group (*p<0.001). IVIS imaging revealed survival of transplanted macrophages in diabetic wounds through day 20 of wound healing. Microfluidic single-cell gene expression analysis revealed that macrophages transplanted into wounds displayed a predominantly M2 phenotype after being in the wound environment for 24 hours. Human monocyte-seeded hydrogels significantly improved healing compared to un-seeded control hydrogels. The average time to complete healing was 17.8 days in the monocyte group versus 21 days in the control group (*p<0.005). Histologic analysis of monocyte treated wounds showed that transplanted monocytes differentiate in vivo to a predominantly M2 phenotype after 48 hours in the wound environment. Importantly, scar size and quality was not affected in wounds receiving either monocyte or macrophage transplant as compared to controls.

Conclusion:
Here we demonstrate that by increasing the number of monocyte lineage cells in the wound site above physiologic levels in diabetic and nude mice the rate of wound healing can be significantly accelerated with no adverse impact on the quality of repair. These findings hold promise for translational medicine aimed at accelerating wound healing across a broad spectrum of diseases.

26.10 Heterotopic Ossification is Inhibited in Leptin-deficient (ob/ob) Mice Despite Robust Vessel Growth

Posted on December 22, 2014

S. Agarwal1, J. Peterson1, O. Eboda1, S. Loder1, C. Brownley1, A. Donneys1, D. Fine1, K. Stettnichs1, K. Ranganathan1, S. Wang1, S. Buchman1, P. Cederna1, B. Levi1  1University Of Michigan,Surgery,Ann Arbor, MI, USA

Introduction:  Clinical and laboratory experience has shown that diabetes contributes to delayed wound and bony healing. However, the relationship between diabetes and the generation of heterotopic ossification (HO) following trauma has not been previously studied. By understanding how diabetes potentiates ectopic bone formation, we may be able to identify treatment strategies which target similar local or systemic factors to prevent HO in patients following trauma, large surface-area burns, and surgical procedures.

Methods:  Male leptin-deficient (ob/ob) or wild type mice (C57BL/6 background) underwent 30% total body surface area burn injury with left hind limb Achille’s tenotomy.  At 7 weeks, mice were injected with Microfil contrast and hind limbs were imaged with micro-CT to quantify aggregate vessel volume.  Vessel volumes were normalized to hind limb mass to account for differences in limb size between diabetic and wild type mice.  A second set of mice underwent micro-CT every 2 weeks up to 9 weeks to quantify HO volume (Hounsfield unit threshold 1000) after adjusting images to remove normal tibia and fibula bone.  

Results: HO volume in diabetic mice was significantly lower than in wild type mice at 9 weeks following burn and tenotomy injury (3.99 mm3 v. 6.20 mm3, p<0.01). Diabetic mice exhibited a decreasing trend in HO volume from week 5 to week 9 based on micro-CT (r=-0.52, p =0.15), suggesting that HO had actually resorbed in these mice.  The mean vessel volume in the hind limbs of diabetic mice 7 weeks after burn and tenotomy was significantly greater than the mean vessel volume in wild type mice (20.6 mm3 v. 4.7 mm3, p <0.05).  When adjusting for limb mass, we found that diabetic legs continued to have more vessel volume (14.1 mm^3/g v. 7.2 mm3/g).  Further examination of vessel architecture showed that the diabetic hind limbs had small vessel growth with disorganized character, while the wild type hind limbs had fewer, but larger vessels.

Conclusion: Here we use Microfil with micro-CT imaging to compare vessel formation between diabetic (leptin-deficient) and wild-type mice in a model of ectopic bone formation. Our findings demonstrate that despite robust small vessel formation at the site of hind limb trauma, diabetic mice produce less ectopic bone than wild type mice.  We also found that diabetic mice actually exhibit resorption of ectopic, suggesting that these mice may be unable to sustain bone growth. This may be related to the disorganized pattern of vessel growth characteristic of the wounds in diabetic mice, as demonstrate by Microfil.  Our findings may elucidate additional targets, such as the local vascular milieu, to prevent initial ectopic bone growth or recurrence following surgical excision.

27.01 Electrophysiological Characterisation Of Human Visceral Afferent Nerves: First In Man

Posted on December 22, 2014

K. S. Ng1,2, N. A. Montes-Adrian2, D. A. Mahns3,M. A. Gladman1,2 1Academic Colorectal Unit, Sydney Medical School – Concord Hospital Campus, University of Sydney, NSW, Australia. 2Enteric Neuroscience & Gastrointestinal Research Group, ANZAC Research Institute, University of Sydney, Australia 3Department of Integrative Physiology, School of Medicine, University of Western Sydney, NSW, Australia

Introduction:  During the last decade, abnormal afferent activity gained recognition as being important in the development of functional gastrointestinal disorders. Since it is not possible to directly measure visceral afferent activity ‘in vivo’ in humans, in this study we aimed to make direct electrophysiological recordings (in vitro) from extrinsic afferents supplying the human colon and rectum. 

Methods:  SSections of normal rectum and colon were procured from anterior resection and right hemicolectomy specimens, respectively. Sections were pinned flat and mesenteric nerves dissected. Extracellular visceral afferent nerve activity was recorded. Neuronal responses to chemical (capsaicin and ‘inflammatory solution’ [IS]1) and mechanical (Von Frey probing) stimulation were recorded and quantified by determining peak firing rates [range] in onesecond intervals. 

Results: 21 nerves were studied from six rectums. Of these, spontaneous afferent activity was recorded in 18 nerves. Peak discharge rates increased significantly following capsaicin (7 [4-25] spikes/sec vs. 3 [2-6], P=0.001) and IS (5 [3-18] spikes/sec vs. 4 [3-12], P=0.003) application. Punctate mechanosensitive ‘hot-spots’ were identified in 11 nerves (threshold 2.0g [1.4–4.0g]). In six of these, the threshold decreased following IS (1.0g [0.4–1.4g]). By comparison, no ‘hot spots’ were identified and spontaneous activity in only one of 18 nerves studied from five
colons. 

Conclusion:  This is the first study to record from extrinsic rectal afferent nerves and to confirm their chemoand mechanosensitivity. Colonic afferents appear less responsive to mechanical stimulation, suggesting differences in electrophysiological characteristics. This technique offers the opportunity to measure electrophysiological properties of extrinsic nerves in disease states. 

References:  

  1. 10μM each of histamine, serotonin, bradykinin, and prostaglandin E2, as in Feng B, Gebhart GF. Characterization of silent afferents in the pelvic and splanchnic innervations of the mouse colorectum. Am J Physiol Gastrointest Liver Physiol. 2011; 300(1): G170-80.

 

 

27.02 BET Inhibitor Blocks Neurosphere Formation And Promotes Differentiation In Neuroblastomas

Posted on December 22, 2014

E. J. Rellinger1, S. Lee1, J. Qiao1, B. T. Craig1, K. Kim1, C. V. Romain1, D. H. Chung1  1Vanderbilt University Medical Center,Pediatric Surgery,Nashville, TN, USA

Introduction:  High-risk group neuroblastoma (NB) patients have a poor prognosis with ~50% overall survival. Nearly half of high-risk NBs demonstrate MYCN amplification; however, conventional drug discovery strategies have had limited success in targeting this transcription factor. The bromodomain and extraterminal (BET) family of proteins coactivates transcription by interacting with histones. BET inhibitors, such as JQ1, have recently been shown to block NB proliferation and induce apoptosis in vitro and halt tumor formation in in vivo murine models by inhibiting MYCN signaling. Our laboratory has characterized the role of gastrin-releasing peptide (GRP) signaling in NB tumor initiation, progression, and metastasis. Its cognate receptor, GRP-R, modulates expression of N-myc, along with PI3K/AKT and ERK. As such, we set out to delineate the effects of JQ1 treatment on GRPR signaling and assess the phenotypic effects of this epigenetic regulator in NBs.

Methods:  Cell viability was determined using CCK-8 assay. Immunoblotting was performed to assess for N-myc, GRP-R, p-AKT, p-ERK expression after JQ1 treatment in NB cells. Tumor sphere formation in serum-free media was used to select for stem-like cancer cells. Tumor sphere formation and expression of neural and stem cell markers served as measures of JQ1 efficacy. We used a subcutaneous murine xenograft model to determine the effects of intraperitoneal JQ1 administration on tumor progression. At harvest, tumor size and immunohistochemistry were our primary measures of tumor progression and differentiation.

Results: JQ1 inhibited cell growth in both MYCN-amplified and MYCN-nonamplified NBs; n-myc expression was decreased in MYCN-amplified cell lines (Fig. A; *p < 0.05 vs. control). Notably, JQ1 blocked the expression of GRP-R and its downstream pathways, PI3K/AKT and ERK, highlighting that BET inhibitors act on both MYCN-dependent and MYCN-independent signaling pathways (Fig. B). We next selected for purported tumor-initiating cells utilizing an in vitro sphere assay and found that JQ1 diminished sphere formation, decreased expression of stem cell markers, and induced neural differentiation. JQ1 was shown to decrease tumor size and induced neural differentiation in our xenograft model.

Conclusion: BET inhibitor downregulates NB cell proliferation, induces apoptosis, and promotes neural differentiation irrespective of MYCN copies, further implicating its therapeutic potential in the treatment of high-risk group of NBs.

 

27.03 Circulating Tumors Cells as a Preoperative Biomarker of Metastatic Disease in Pancreatic Cancer

Posted on December 22, 2014

J. Ankeny1, C. Court1, H. Tseng2, J. S. Tomlinson1  1University Of California – Los Angeles,Surgical Oncology,Los Angeles, CA, USA 2University Of California – Los Angeles,Molecular & Medical Pharmacology,Los Angeles, CA, USA

Introduction: The majority of pancreatic ductal adenocarcinoma (PDAC) patients who undergo pancreatic resection ultimately succumb to metastatic disease. This fact strongly points to our inability to accurately stage patients at the time of disease presentation. We are in serious need of a biomarker which predicts systemic disease in order to better select our patient’s for surgery versus systemic therapy. Our goal was to investigate the presence of circulating tumor cells (CTCs) in early stage PDAC as a biomarker of metastatic disease.

Methods: We obtained a venous blood (VB) in the preoperative setting from 21 consecutive PDAC patients deemed to be AJCC Stage I or II based on preoperative imaging. Four milliliters of VB was evaluated for the presence and number of CTCs. Capture and enumeration was carried out with a novel microfluidic NanoVelcro technology enhanced by anti-EpCAM enrichment. CTCs were defined by immunocytochemical staining (CK+ or CEA +, CD45-, DAPI+). CTC number was correlated with postoperative AJCC stage and the presence or absence of metastatic disease discovered at surgery.

Results:Of the 21 PDAC patients taken to the operating room, 7 were found to have visible macroscopic metastatic disease in the peritoneal cavity and thus the planned resection was aborted. CTC number correlated with stage and distinguished patients with local disease versus metastatic disease. Mean CTC counts for stage I, 2A, 2B and 4 were 0, 0.67, 0.78, 13.1 respectively.  AUROC utilizing a CTC cutoff of > 3 CTCs/4ml VB was 0.93 (95% CI (0.772-1.086) p-value = 002).

Conclusion:In this small prospective study, the presence of 3 or more CTCs in 4ml VB demonstrate a strong ability to predict macroscopic metastatic disease undetectable by preoperative cross sectional imaging. CTCs are a promising biomarker which may allow for strong preoperative prediction of metastatic disease in the PDAC patient and guide first line treatment decisions. Longer follow-up with the addition of outcomes data is needed to firmly establish CTCs as a predictive biomarker in PDAC.

 

27.04 A Novel Selective Retinoid X Receptor Agonist, 9-cis-UAB30, Inhibits Hepatoblastoma Cell Growth

Posted on December 22, 2014

A. M. Waters1, J. E. Stewart1, V. R. Atigadda1, D. D. Muccio1, C. J. Grubbs1, E. A. Beierle1  1University Of Alabama,Birmingham, Alabama, USA

Introduction:  Hepatoblastoma, a malignant tumor that arises from immature liver cells, is the most commonly diagnosed liver tumor in children.  The incidence of hepatoblastoma is increasing but survival for children with advanced, metastatic or recurrent disease remains dismal at less than 50%.  Therefore, innovative therapeutic strategies are needed to treat this disease.  A novel retinoid, 9-cis-UAB30 (UAB30), has been developed that has significantly less toxicity than other retinoids.  We hypothesized that treatment with UAB30 would induce cell cycle arrest, inhibit cellular migration and invasion, and lead to apoptosis and cell death in hepatoblastoma cells in vitro and impede tumor growth in vivo.  

Methods:  The human hepatoblastoma cell line HuH-6 was utilized.  Cell cycle analysis was completed with flow cytometry.  Migration and invasion assays were performed with scratch assays and Transwell plates. AlamarBlue®  and trypan blue exclusion was used to measure cell survival and proliferation, respectively.  Apoptosis was detected via Western blotting for PARP cleavage and caspase 3 activity was detected by a colorimetric assay kit.  HuH-6 hepatoblastoma cells (2.5 x 106) were injected into the right flank of athymic nude mice.  Mice were fed standard chow or UAB30-treated chow and tumor volumes were measured twice weekly with calipers.  Animals were euthanized when parameters mandated by IACUC policy were reached.  Student’s t-test was used to compare data between groups, with p ≤ 0.05 considered significant.  

Results: The following results were noted with increasing concentrations of UAB30: a) cell cycle arrest with a significant increase in the percentage of cells in G1 and a decrease in S phases; b) a significant decrease in cellular migration and invasion; c) a significant decrease in cellular proliferation; and d) a significant decrease in cell survival.  Further, increased PARP cleavage and caspase 3 activity confirmed that cell death was due to apoptosis.  Additionally, after four weeks of treatment in the in vivo study, tumor size of the UAB30 treated mice injected with HuH-6  cells were significantly smaller than the controls (275 ± 402 mm3 vs. 1035 ± 164 mm3, treated vs. control, p<0.0001). 

Conclusion: The synthetic retinoid, 9-cis-UAB30, had a significant impact upon the HuH-6 human hepatoblastoma cell line resulting in cell cycle arrest, decreased proliferation, migration and invasion, and increased apoptosis in vitro as well as inhibition of tumor growth in vivo.  These results suggest a potential therapeutic role for UAB30 in hepatoblastoma treatment.

 

27.05 ERK MAP Kinase Interacts with and Up-regulates Pancreatic Duodenal Homeobox-1

Posted on December 22, 2014

G. Zhou1, E. Rozengurt2, J. Sinnett-Smith2, S. Liu1, J. Yu1, J. Wu1, R. Sanchez1, F. C. Brunicardi1  1University Of California – Los Angeles,General Surgery/Surgery/David Geffen School Of Medicine,Los Angeles, CA, USA 2University Of California – Los Angeles,Digestive Diseases/Medicine/David Geffen School Of Medicine,Los Angeles, CA, USA

Introduction: Pancreatic and duodenal homeobox-1 (PDX-1) is a key pancreatic transcription factor known to be involved in tumorigenesis and markedly overexpressed in pancreatic ductal adenocarcinoma (PDAC). However, little is known about the related oncogenic signaling pathways causing the overexpression. The activation of the extracellular signal-regulated kinases (ERK) pathway driven by the K-RAS mutation plays a critical role in promoting survival, invasion and migration of PDAC cells. The purpose of this study is to determine the role of ERK in regulation of PDX-1 expression in PDAC.

..

Methods: Antibody array screen was performed in GFP-PDX-1 stable HEK293 cells using a membrane filter arrayed with 400 antibodies, which was preblocked in a buffer containing 5% nonfat milk. After overnight incubation at 4°C, the presence of the antibody-antigen-PDX-1 complex was detected by horseradish peroxidase-conjugated anti-GFP antibody, followed by chemiluminescence. The ERK-PDX-1 interaction was confirmed in PDAC cells by performing immunoprecipitation/Western blotting using anti-ERK and anti-PDX-1 antibodies. Protein expression levels were determined by performing Western blotting using appropriate antibodies. Lipofectamine 2000 was used for transient transfection of PDX-1, ERK1, c-Jun N-terminal kinase 1 (JNK1) and Ubiquitin into HEK293 cells. PDAC cells were treated with various concentrations of epidermal growth factor (EGF) in the presence of 2% serum. Serine 268 residues of human PDX-1 were mutated into alanine by performing site-directed mutagenesis as instructed by the manufacture’s manual. The mutations were confirmed by standard PCR sequencing. Ubiquitination of PDX-1 was monitored by immunoprecipitating PDX-1, followed by followed by Western blotting using an antibody against ubiquitin.

Results:1) ERK1 and ERK2 were identified as PDX-1-interacting proteins in an antibody array screen; 2) The ERK-PDX-1 interaction was confirmed by immunoprecipitation/Western blotting in PDAC cells in response to EGF; 3) PDX-1 expression was specifically up-regulated by ERK1, but not by JNK1; 4) EGF enhanced PDX-1 expression in PDAC cells; 5) ERK inhibitor blocked EGF-stimulated PDX-1 expression in PDAC cells; and 6) Phosphorylation of proline-directed Ser 268 suppressed PDX-1 ubiquitination and stabilized PDX-1, thus, Ser 268 is a potential ERK phosphorylation site within PDX-1, whose phosphorylation contributes to the stabilization of PDX-1.

Conclusion:These data show that 1) ERK MAP kinase is a positive regulator of PDX-1; and 2) ERK up-regulates PDX-1 expression via a mechanism involving promoting phosphorylation of Ser 268, leading to decreased ubiquitination and enhanced stabilization of PDX-1. Thus, these data suggest that ERK MAP kinase plays an important role in PDX-1 overexpression in PDAC.

 

27.06 Focal Adhesion Kinase as a Mechanism of Distraction-Mediated Enterogenesis

Posted on December 22, 2014

F. R. Demehri1, Y. Feng1, R. Sueyoshi1, H. K. Yoon1, J. Guan2, D. H. Teitelbaum1  1University Of Michigan,Surgery,Ann Arbor, MI, USA 2University Of Michigan,Internal Medicine,Ann Arbor, MI, USA

Introduction:   Distraction-mediated enterogenesis (DME) is a novel therapeutic strategy for short bowel syndrome (SBS), wherein distractive force leads to substantial lengthening of functional bowel.  The mechanism(s) by which this occurs is incompletely understood.  Using a mouse model of DME, we recently identified increased expression of phosphorylated focal adhesion kinase (p-FAK) – a mediator of mechanotransduction in other tissues.  We hypothesized that DME is dependent upon a FAK phosphorylation pathway.

Methods:   C57BL/6 mice underwent creation of an isolated 3-cm jejunal segment with instillation of 300 µl of high molecular weight polyethylene glycol (PEG, 3350 kD) to induce osmotic stretch (and growth) of the segment.  PF-562,271, a selective FAK inhibitor, was administered via oral gavage.  Control mice received vehicle alone.  A second group of tamoxifen-induced intestine-specific FAK knockout(KO) mice (Villin-Cre-ER-FAKf/f) underwent the same procedure, and were compared to non-induced and wild-type controls. After 5 days, gross morphology of the distracted segment was evaluated, volume measured via CT-imaging, and intestinal epithelial cell (IEC) proliferation measured via PCNA immunostaining.  Results (mean±SEM) were analyzed for significance by t-test.

Results:  Chemical inhibition of FAK significantly prevented DME, resulting in a decrease in length of 23.1±9.2%, vs an increase in length of 11.7±1.4% in control DME mice (p<0.05; Fig A, C).  Volume gain was limited to 37.5±2.4% with FAK-inhibition, vs 154.0±8.3% in control mice (p<0.01).  FAK inhibition with PF-562,271 also prevented the increase in IEC proliferation seen with DME (26.9±2.2% vs 52.9±3.3% PCNA positive cells; p<0.0001; Fig B, D).  The proliferative index with FAK inhibition was similar to that of non-distracted controls (30.3±1.2% PCNA positive; p=0.34).  Inhibition of FAK phosphorylation was confirmed via Western blot, with a blunted increase in p-FAK expression in treated mice vs controls.  We next examined enterogenesis in tamoxifen-induced FAK-KO mice.  A similar prevention in DME was noted, with a decrease in length of 9.0±7.1%, versus an increase in length of 19.5±3.5% in controls (p<0.05).  Interestingly, FAK-KO led to a reduction in IEC proliferation during distraction (14.5±1.3% PCNA positive) relative to non-distracted FAK-KO (25.2±1.9% PCNA positive; p<0.001) and FAK-competent bowel (24.2±1.5% PCNA positive; p<0.001).     

Conclusion:  FAK activation is required for DME, with p-FAK inhibition restricting overall volumetric growth and IEC proliferation.  FAK may represent a potential target to enhance DME as a novel treatment for SBS.

27.07 ”Construction of the subcutaneous liver tissue by transplantation of of hepatic NPCs sheets.”

Posted on December 22, 2014

M. Fujii1, K. Yamanouchi1, Y. Sakai1, A. Kinoshita1, M. Hidaka1, A. Soyama1, K. Kobayashi1, M. Takatsuki1, K. Kanetaka1, F. Fujita1, T. Kuroki1, S. Eguchi1  1Department Of Surgery, Nagasaki University Graduate School Of Biomedical Sciences,Nagasaki, NAGASAKI, Japan

Introduction:  

Subcutaneous hepatocyte transplantation using cell sheet technology could be one of the attractive therapeutic options for inherited liver metabolic disease. However, subcutaneous hepatocytes reportedly do not survive for even 2 weeks without pre-vascularization. On the other hand, adipose-derived mesenchymal stem cells (ADSCs) are known to produce various humoral factors, such as VEGF and HGF, which should induce vascularization. Liver tissue is comprised of 70% hepatocytes, 30% non-parenchymal cells (NPCs), including endothelial cells, biliary epithelial cells, and small hepatocytes, etc. The small hepatocytes have the capacity to proliferate more effectively than hepatocytes, and can differentiate into mature hepatocytes. To construct liver tissue by transplanting co-cultured cell sheets consisting of NPCs/ADSCs subcutaneously without pre-vascularization.

Methods:  

Male 5- to 7-week-old Fischer rats were used. ADSCs were separated from the inguinal adipose tissue of the donor by collagenase-digestion and ADSCs sheets were created by culturing them on temperature-responsive culture-dishes (TRCDs). The donor liver was digested by the collagenase perfusion method and we thereby separated hepatic cells into NPCs and hepatocytes employing centrifugations.

Ⅰ) In vitro evaluation of NPCs and hepatocytes: We spread 1.0 × 106 NPCs or hepatocytes each on 35-mm collagen-coated dishes and evaluated their structures and functions. 

Ⅱ) Transplantation of co-cultured cell sheets and the corresponding evaluations: We spread NPCs or hepatocytes onto ADSCs cultured beforehand on TRCDs for 2 or 3 days to create co-cultured cell sheets. We then transplanted these sheets into subcutaneous tissues of the recipient rats 2 days after dissemination of the NPCs or hepatocytes. After 2 and 4 weeks, animals were sacrificed and samples were examined histologically.

Results:

Ⅰ) Cultured NPCs showed hepatocyte-like structures. They formed functioning bile canaliculi and produced glycogen. In addition, their albumin secretion capacity was equal to that of hepatocytes (3-day: 98.0 ± 8.4, 90.2 ± 5.8; 7-day: 67.4 ± 10.3, 59.5 ± 3.4μg/mL/dish/2-day). 

II) The 1 to 3 layered cell sheets of NPCs/ADSCs survived subcutaneously for 4 weeks while producing glycogen and albumin and expressing CD26, the marker of bile canaliculi. Moreover, vascularization around and in the cell sheets was confirmed by CD31 immunohistochemistry.

Conclusion:

The co-cultured sheets of NPCs/ADSCs had liver-specific functions and survived subcutaneously without pre-vascularization. These results suggest NPCs to be a potential cell source for liver disease therapy and that the technique of creating co-cultured cell sheets might be useful for regenerative medicine by promotion of vascularization.

 

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