80.20 Alterations in Biomarkers of Coagulation and Fibrinolysis Following Burn Injury

Posted on January 26, 2017

M. Vigiola Cruz2,3, K. E. Brummel-Ziedins4, T. Orfeo4, L. Moffatt2, J. W. Shupp2,3  2MedStar Health Research Institute,Firefighters’ Burn And Surgical Research Laboratory,Washington, DC, USA 3MedStar Washington Hospital Center,The Burn Center,Washington, DC, USA 4University Of Vermont,Department Of Biochemistry,Colchester, VT, USA

Introduction: There is limited understanding of the alterations in the coagulation cascade caused specifically by burn injury. Considering the fine balance between clot formation and degradation that is necessary for normal coagulation, further insight into the dynamics of pertinent biomolecules following thermal injury is crucial. Our aim is to analyze specific factors involved in clot formation and fibrinolysis over time to improve the current understanding of altered coagulation pathways in burn patients.

Methods: Blood samples were serially collected from 29  burn patients to quantify various biomarkers implicated in coagulation and fibrinolysis. Patients were grouped by injury severity, <10% (n=19) or 10-30% TBSA (n=10). Sampling began within four hours of burn injury, occurring every 2-4 hours in the initial 12 hours and subsequently twice daily for 7 days or until hospital discharge. Concentrations of fibrinogen, D-dimer, Plasminogen (PLG), Tissue Factor Pathway Inhibitor (TFPI), and Thrombin Activatable Fibrinolysis Inhibitor (TAFI) were quantified using ELISA.

Results:Fibrinogen levels on all patients were within normal limits on admission (mean 314mg/dL), and increased until they reached plateau at approximately 96 hours, a 2.3-fold elevation from initial evaluation (p=0.0007). Compared to the 10-30% TBSA group, the <10% cohort trended to have higher levels of fibrinogen measured at all timepoints; the differences were not statistically significant. D-dimer levels increased from presentation (mean 0.4µg/dL), and in the first 24 hours elevated to nearly 3-fold (p=0.0003), with markedly higher levels in the more severe burns as early as hour 12. Conversely, plasminogen levels initially decreased to the lower end of normal range in the initial 36 hours. Modest elevations were seen in TFPI, with fluctuations within a narrow range compared to early values. Mean TAFI concentrations peaked at hour 12 in the 10-30% TBSA group and returned to initial values after 36 hours. All patients survived.

Conclusion:The patterns in factor dynamics (e.g. fibrinogen) demonstrated by our analyses are not consistent with current concepts of coagulopathy, showing aberrancies that necessitate additional exploration. This observation emphasizes the suboptimally understood variation between hyper and hypocoagulability that follows burn injury, pointing to the complexity and multifactorial nature of the coagulation and fibrinolysis processes.  We have previously established that alterations in these cascades may not be detected by lab assays routinely used in the clinical setting, and increases in various acute phase reactants have been associated with higher risk of mortality in trauma and infection. Therefore, further work should continue to integrate specific factor data with clinical observations and measurable outcomes to improve resuscitative interventions in burn patients.

 

80.19 Angiogenin Regulates COX-2 Expression by TNF-α and Bradykinin in the Human Colonic Myofibroblast

Posted on January 26, 2017

E. Chu1, T. Liu1, N. Vanli1, G. F. Hu1, J. Yoo1  1Tufts Medical Center,Colon And Rectal Surgery,Boston, MA, USA

Introduction: The myofibroblast is an important stromal cell of the gastrointestinal (GI) tract that is a target of tumor necrosis factor-alpha (TNF-α ), a potent pro-inflammatory cytokine that has been strongly implicated in the pathophysiology of colitis-associated cancer.  Crosstalk mechanisms are known to exist between TNF-α and other pro-inflammatory mediators, including multiple G protein-coupled receptor (GPCR)-mediated agonists, that amplify inflammatory signaling.  However, the mechanism has not been previously determined.  Angiogenin (ANG) is a 14-kDa member of the ribonuclease superfamily that was the first tumor-derived angiogenesis protein. Like TNF-α , ANG levels are elevated in patients with inflammatory bowel disease (IBD) and colorectal cancer.  However, the role of ANG on inflammatory mediator crosstalk in the myofibroblast is unknown.

Methods: The human colonic myofibroblast cell line 18Co was grown to confluence on 35×10 mm cell culture dishes and was used from passages 8-14. 18Co cells were exposed to TNF-α  (10 ng/ml) and bradykinin (100nM) for varying times. ANG was quantified from the supernatant of serum-starved 18Co cells by ELISA.  The monoclonal antibody 26-2F was used to block the activity of ANG. The expression of cyclo-oxygenase-2 (COX-2) was assessed by Western Blot. 

Results:We have previously reported that 18Co cells exposed to both TNF-α  and the pro-inflammatory GPCR bradykinin (BK) lead to the synergistic expression of COX-2, evident after 4 h (P<0.05).  To determine whether ANG was involved in this process, we first measured ANG levels in the cell culture supernatant of 18Co cells by ELISA.  18Co cells secrete high levels of ANG (265.5 ± 4.7 pg/ml in serum-free media over 24 h).  Exposure of 18Co cells to TNF-α  (10ng/ml) led to a rapid (4 h, 127.8 ± 9.7 pg/ml, P<0.05) and sustained (24 h, 124.6 ± 25.1 pg/ml, P<0.05) reduction in the concentration of ANG in the supernatant, corresponding to an uptake of ANG by these cells.  The anti-ANG monoclonal antibody 26-2F, which neutralizes the activity of ANG, inhibited the synergistic expression of COX-2 induced by TNF-α  and BK at 4 h (P<0.05). 

Conclusion:TNF-α  stimulates ANG uptake by the myofibroblast, and inhibition of ANG blocks synergistic COX-2 expression induced by TNF-α  and BK.  Crosstalk signaling between TNF-α  and BK appears to be mediated by ANG.  Angiogenin may play an important role in the regulation of COX-2 expression in the setting of inflammation, and may be a novel therapeutic target for the management of colitis-associated cancer. 

 

80.18 Circulating Plasma MicroRNAs in Colorectal Neoplasia: A New Role in Assessing Response to Therapy

Posted on January 26, 2017

J. Carter1, U. Netz1, K. Feagins1, V. States1, M. R. Eichenberger1, S. Galandiuk1  1University Of Louisville,Department Of Surgery,Louisville, KY, USA

Introduction:

Recurrence following treatment for colorectal cancer is common. Current blood-based tests, such as serum carcinoembryonic antigen (CEA), are used both for post-operative surveillance and for monitoring response to therapy. CEA; however, lacks sufficient sensitivity and specificity to accurately detect recurrence of CRC or its precursor lesion, advanced adenoma (CAA). microRNAs (miRNAs) have been associated with both the diagnosis and regulation of different disease processes. They are short, non-coding RNAs that play an important role in gene expression. We believe miRNAs may have a potential role in monitoring therapy following removal of an adenoma or cancer. We have previously identified longitudinal changes in plasma miRNA in patients with CAA and CRC. Our aim is to confirm significantly dysregulated miRNAs identified from screening studies in plasma samples obtained from patients with CAA and CRC prior to and after endoscopic or surgical removal.

Methods:

Plasma was isolated from 24 patients, 12 with CAA (>0.6cm diameter) and 12 with stage II-III CRC prior to treatment and 4-6 weeks following endoscopic removal or surgical resection. Total RNA was extracted and RNA quality and quantity were assessed. Reverse transcription and quantitative real-time polymerase chain reaction was performed using specific primers and probes for the miRNAs of interest. A total of 11 miRNAs were included for assessment: 4 miRNAs identified from our screening cohort to be significantly dysregulated between pre-treatment and post-removal samples and 7 miRNAs that were significantly different between CAA and controls and CRC and controls in a prior study. Data was analyzed comparing pre-treatment samples to post-removal samples using paired t-tests after normalizing raw cycle threshold data to endogenous RNU6 and miR-16.

Results:

Of the 11 analyzed miRNAs, miR-29c (sensitivity 50% 95% CI:25-75, specificity 83% 95% CI:54-97%, AUC 0.67 95% CI:0.45-0.90) was found to be downregulated in pre-treatment plasma samples compared to post-removal samples in CRC (p<0.05 at α=0.05). miR-374a (sensitivity 50% 95% CI:25-75, specificity 83% 95% CI:54-97%, AUC 0.66 95% CI:0.43-0.89) was found to be downregulated in pre-treatment plasma samples compared to post-removal samples in CAA (p<0.05  at α=0.05).

Conclusion:

The expression levels of miR-29c and miR-374a, were different in pre-treatment as compared to post-removal plasma samples in patients with CRC and CAA, respectively. These findings may help provide for a relatively non-invasive method of monitoring therapy or assessing response to treatment. Future considerations should include standardized protocols for assessing miRNA pre- and post-neoadjuvant therapy in patients with rectal cancer in order to determine the effect of such treatment on tumor regression.

80.12 Periosteal Cells are Skeletal Progenitor Cells

Posted on January 26, 2017

T. V. Boyko1,2, Z. Wang1, 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:  We have identified the mouse skeletal progenitor cells consisting of 8 distinct subpopulations capable of self-renewal and giving rise to all three components of the skeleton: bone, cartilage and stroma. The periosteum consists of a thin layer of cells that have been shown to expand and contribute to bone fracture healing. Separately, we recently reported the strong expression of Del1 in cartilage and its potential role in osteoarthritis. Del1 was also strongly expressed in skeletal progenitors, and histology had shown strong expression in the periosteum. We hypothesized that the expression of Del1 in the periosteum served as a marker for the presence of skeletal progenitors and that periosteal cells would be found to consist primarily of skeletal progenitor cells.

Methods:  Del1-LacZ mice were anesthetized, the left femur exposed, and the periosteum was injured by scraping with the side of a pair of scissors. The right femur was untouched and used as a control. On post-operative day 7, mice were harvested and processed for histology. To obtain cells for Fluorescent Activated Cell Sorting (FACS) analysis uninjured femurs were stripped of periosteum, which was digested to obtain a cell suspension. Cells were then stained for surface markers of skeletal progenitor cells and FACS was performed to analyze for presence of skeletal progenitor cells. 

Results: Strong expression of Del1 could be seen in uninjured periosteum. Using LacZ staining, we demonstrated that these cells underwent expansion with differentiation into hypertrophic cartilage and bone at the injury site. To identify skeletal progenitors within the periosteum, we harvested cells and subjected them to FACS analysis. FACS showed that the largest subpopulation of skeletal progenitors in the periosteum were Bone Cartilage Stroma Progenitor cell population (BCSPs), the skeletal progenitors found to contribute most to fracture healing.

Conclusion: We have previously shown that mouse skeletal progenitor cells play an integral role in bone fracture healing. It has long been known that the periosteum contributes cells to bone repair. We show here that the periosteum contains a large number of skeletal progenitor cells and that the largest population were BCSPs, the cells most involved in fracture repair. Furthermore, we have identified Del1 expression as a strong marker of skeletal progenitor cells. Separately, we have shown that Del1 deletion leads to fracture healing with a decreased bony callus. We conclude that Del1 represents a novel regulator of skeletal progenitor cell biology. 

 

80.10 Regulation of Cell Proliferation by the MicroRNA-200 Family in Colorectal Cancer Cell Lines

Posted on January 26, 2017

J. Burton1, J. Carter1, K. Ramos2, B. G. Oxford3, M. R. Eichenberger1, S. Galandiuk1  1University Of Louisville,Department Of Surgery,Louisville, KY, USA 2University Of Arizona,College Of Medicine,Tuscon, AZ, USA 3University Of Louisville,School Of Medicine,Louisville, KY, USA

Introduction:
Chromosomal instability (CIN) is the major molecular pathway associated with development of sporadic colorectal cancer (CRC). With increasing CIN, mutations in the KRAS gene contribute to the progression from normal mucosa to invasive CRC. K-Ras protein is a product of the KRAS gene and acts within the mitogen-activated protein kinase (MAPK) pathway, a major regulator of cell proliferation.The miR-200 family has been linked to several cancers, including CRC, and is known to target a negative regulator of K-Ras, RASSF2. We have previously identified increased expression of all members of the miR-200 family and decreased expression of RASSF2 in CRC cell lines as compared to a normal colon epithelial cell line. Therefore, we hypothesize that the miR-200 family regulates cell proliferation through targeting of this negative regulator and subsequent K-Ras activity in the MAPK pathway.

Methods:
A K-Ras wild type CRC cell line (HT29) and normal colon epithelial cell line (CCD841) were acquired (ATCC®, Manassas, VA). Cells were grown in appropriate culture medium until confluent. Once grown, cells were harvested and plated into separate wells on a 12-well plate at a concentration of 100,000 cells/mL culture media and then allowed a 24-hour period to adhere. At 24 hours, cells were serum starved for 2 hours and transfected with miR-200 family mimics or antagomirs, and their respective negative controls, using the Lipofectamine®RNAiMAX Transfection Reagent protocol (ThermoFisher Scientific, Waltham, MA). Transfection was stopped after 24 hours and cell number was measured each day for 5 days using an automated cell counter (TC20™ Bio-Rad, Hercules, CA).

Results:
Confirmation of successful transfection was performed. CCD841 cells had significantly increased proliferation when transfected with miRNA mimics of all members of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) both individually and combined, as compared to the negative control at day 5 (p<0.05). Conversely, we observed a significant decrease in cell proliferation at days 4 and 5 when HT29 cells were transfected with miR-200 antagomirs for the miR-200 family members both individually and combined, compared to the negative control (p<0.05). A representative growth curve is shown in Figure 1.

Conclusions:
These findings suggest that the gain and loss of function of the miR-200 family affects cell proliferation. Overexpression of the miR-200 family increases cell proliferation, an important process in tumor growth, whereas silencing of the miR-200 family decreases cell proliferation, reducing neoplastic progression. miR-200 should be investigated further as a potential therapeutic target in the treatment of colorectal cancer.

80.09 Effect of Biomechanical Stretch on Regulation of Extracellular Matrix Hyaluronan by Fibroblasts

Posted on January 26, 2017

M. Fahrenholtz1, X. Wang1, H. Li1, Y. Dhamija1, P. Duann1, M. Rae1, K. Grande-Allen2, S. Keswani1, S. Balaji1  1Baylor College Of Medicine,Surgery,Houston, TX, USA 2Rice University,Bioengineering,Houston, TX, USA

Introduction:  Mid gestation fetus heals cutaneous wounds without scar and its anti-fibrotic phenotype is defined by negligible resting tension and distinct extracellular matrix (ECM) with elevated levels of hyaluronan (HA) produced by fetal fibroblasts. In contrast, adult skin is characterized by relatively low levels of HA, and much higher resting tension. Biomechanical tension induces a pro-fibrotic phenotype in fibroblasts which is characterized by increased inflammatory cytokine and ECM production, which leads to the formation of scar tissue in vivo. The role of HA production by fibroblasts under biomechanical stretch has not been fully examined. We hypothesize that higher mechanical tension will alter HA metabolism of fibroblasts via differential regulation of HA synthases (HAS1-3) and hyaluronidases (Hyal1-2).

Methods:  Primary murine adult dermal fibroblasts (AFb) were cultured on collagen-coated silicone membranes +/-10% static strain. AFb expression of HA synthesis(HAS1-2), remodeling(HYAL1-2), and receptor(CD44) genes, as well as phenotype(ASMA) were assessed by qPCR. Total HA production was measured by ELISA. Data is presented as mean+/-SD, n=3/group, p values by ANOVA with post-hoc Tukey HSD.

Results: Mechanical tension induced differential HA gene regulation in AFb, with significantly increased HAS1 gene expression (1.67+/-1.03 vs 5.13+/-0.70, p < 0.01) and decreased HAS2 expression (0.92+/-0.10 vs 0.063+/-0.009, p < 0.01), but no change in HAS3 expression after 24h application of stretch, as compared to unstretched condition. Both Hyal1 and Hyal2 were down-regulated under tension (1.72+/-0.84 vs 0.17+/-0.10, p < 0.05, and 1.64+/-0.70 vs 0.13+/-0.05, p < 0.01, respectively). Total HA quantification at 24 h showed no significant influence of stretch on AFb production of HA, despite differential regulation of HA synthesis and HA remodeling genes, indicating a need to assess additional time points and hyaluronidase activity. ASMA gene expression was not influenced by tension at 24h. Tension downregulated CD44 gene expression (1.18+/-0.32 vs 0.33+/-0.08, p < 0.05), which may influence AFb ability to interact with HA produced under mechanical stretch.

Conclusion: Our data suggest that biomechanical forces have a significant role in influencing the dermal fibroblasts’ cell-matrix interaction and their regulation of the ECM-specifically HA. Understanding the time course of these effects on fibroblast responses to tension, especially under the influence of exogenous wound factors and chemokine imbalance, may yield novel therapeutic interventions to promote anti-fibrotic healing. Understanding the contribution of mechanical environment via morphological and phenotypic alterations may yield novel therapeutic targets in recapitulating fetal regenerative healing in postnatal tissues.

80.07 Sphingosine Demonstrates Effective Killing of E. coli in Models of Urinary Tract Infection

Posted on January 26, 2017

R. M. Boudreau1, G. E. Martin1, C. Couch1, A. E. Mahdy1, M. J. Edwards1, E. Gulbins1,2, A. P. Seitz1, P. L. Jernigan1  1University Of Cincinnati,Department Of Surgery,Cincinnati, OH, USA 2Universitat Duisburg-Essen,Division Of Molecular Biology,Essen, NORTH RHINE-WESTPHALIA, Germany

Introduction:  Catheter-associated bacteriuria is the most frequently diagnosed nosocomial infection globally. The conventional treatment of these infections with systemic antibiotics creates an opportunity for the selection of antimicrobial resistance, adds to burgeoning medical costs, and increases the risk for antibiotic-related side effects. Furthermore, this approach remains suboptimal, with approximately one-quarter of acutely infected patients developing recurrent infection. Sphingosine, a membrane sphingolipid with broad-spectrum anti-microbial properties, has been described as an important part of the innate immunity of the respiratory epithelium to bacterial invasion. Our group has recently discovered high amounts of sphingosine in the transitional epithelium of mouse bladders. We hypothesize that sphingosine is important in the bladder’s innate immunity and that exogenous sphingosine may serve as an effective anti-microbial in murine models of E. coli UTI.

Methods:  E. coli was incubated with normal saline control or varying concentrations of sphingosine in vitro for 2 hours at 37C and 125 rpm agitation.  Bacterial growth was quantified by the plate-dilution method. To evaluate sphingosine’s effectiveness in vivo, wild type mice were sterilely catheterized and inoculated with E. coli for one hour before undergoing bladder irrigation with normal saline control or varying concentrations of sphingosine. Bladders were then harvested and homogenized; and bacterial load was quantified by the plate-dilution method.

Results: Sphingosine demonstrated impressive killing of E. coli compared to saline control in our in vitro study (94% reduction, p<0.001). Similarly, in our murine model of UTI, the bladder irrigations showed maintenance of this antimicrobial effect (Table 1, 95% reduction vs. saline control, p=0.007). 

Conclusion: It was demonstrated previously that sphingosine both plays a crucial role in innate mucosal immunity and possesses antimicrobial activity against E. coli in solution. Here, we present the first study to demonstrate that exogenous sphingosine causes effective bacterial killing in a murine model of UTI. Our data support the need for further investigation toward a possible role for sphingosine-based bladder irrigation in the management of UTIs.

80.06 Characterization of the Abdominal Adhesion Fibroblast

Posted on January 26, 2017

C. D. Marshall1, M. S. Hu2, R. C. Ransom1, L. A. Barnes1, A. A. Moore1,3, T. D. Leavitt1, H. P. Lorenz1, M. T. Longaker1  1Stanford University School Of Medicine,Department Of Surgery,Stanford, CA, USA 2University Of Hawai’i John A. Burns School Of Medicine,Department Of Surgery,Honolulu, HI, USA 3Brigham And Women’s Hospital,Department Of Surgery,Boston, MA, USA

Introduction:
Abdominal adhesions resulting from surgery are the most common cause of small bowel obstruction. Their presence complicates subsequent operations and contributes to infertility. Adhesion formation depends on fibroblast collagen production. The precise cell populations and molecular signals that induce adhesion formation are not known. As a result, no effective pharmaceutical anti-adhesion therapies exist. An improved understanding of the cellular and molecular basis of adhesions would allow for the development of improved treatments.

Methods:
Laparotomy was performed on wild type mice and the cecum and abdominal sidewall were abraded using sandpaper. The resulting adhesion tissue was examined with immunohistochemistry and was digested with collagenase, allowing adhesion fibroblasts to be subjected to immunocytochemical analysis. In an abdominal wall transplant model, the abdominal muscular wall of a pan-RFP mouse was sutured onto the inner surface of the abdominal wall of a pan-GFP mouse before adhesions were created. Fluorescent imaging of adhesion tissue forming within the red-green interface was used to determine the origin of adhesion cells. Finally, adhesions were created in transgenic inducible multi-color Rainbow mice, allowing for the assessment of adhesion cell clonality.

Results:
Adhesion fibroblasts expressed known fibroblast markers: vimentin, fibronectin, FSP, PDGFRα , and collagen. Additionally, many fibroblasts expressed the myofibroblast and smooth muscle marker α SMA, the mechanical transduction mediator FAK, and CD26, a surface marker implicated in fibrosis. After two weeks, smooth muscle cells migrated out of the intestinal wall into the adhesion space. Abdominal wall transplantation revealed that >80% of cells in the adhesion tissue were green and hence derived from the bowel surface, rather than red and derived from the abdominal wall (Figure 1). Assessment of cell proliferation in the adhesion using rainbow mice showed that individual intestine surface cells multiply after injury and expand clonally into the adhesion space.

Conclusion:
Adhesion fibroblasts express several cell markers associated with fibrosis that may provide molecular targets for future anti-adhesion therapies. Smooth muscle cells of the intestinal wall migrate into the adhesion and may contribute to inflammation and the formation of permanent adhesions. There is likely significant overlap between cells traditionally defined as fibroblasts and smooth muscle cells in the adhesion. Cells of the intestinal surface are substantially more active in adhesion formation than cells of the abdominal wall surface. Individual progenitor cells of the intestinal surface produce progeny that proliferate clonally and populate the adhesion. 
 

80.03 Hydrogen peroxide promotes polarization of macrophages to the M1 phenotype

Posted on January 26, 2017

J. Xu1, J. Hu1, C. Zgheib1, M. Hodges1, K. W. Liechty1  1Laboratory For Fetal And Regenerative Biology, Children’s Hospital Colorado And The University Of Colorado Anschutz Medical Campus,Department Of Surgery,Aurora, CO, USA

Introduction: Macrophages play an essential role during wound healing and have the ability to dynamically transition between M1 and M2 phenotypes in response to signals from the surrounding microenvironment. Reactive oxygen species (ROS), including hydrogen peroxide, are one of many stimuli that have the potential to polarize macrophages. However, the role of hydrogen peroxide in macrophage polarization remains poorly defined.   We hypothesize that treatment of macrophages with hydrogen peroxide will polarize the macrophage into M1 phenotype with induction of M1 gene expression and reduction of M2 gene expression.

Methods: To test our hypothesis, Raw cells from the murine macrophage cell line RAW264.7 were treated with 0, 10 uM and 100 uM hydrogen peroxide for 1 hour.  Additional macrophages were treated with100 uM hydrogen peroxide for 5, 20, or 60 minutes.  M1 and M2 gene expression was analyzed using real-time PCR. 

Results: Macrophages treated with hydrogen peroxide showed significantly increased gene expression of the M1 macrophage markers (iNOS, and IL1-beta), while demonstrating decreased gene expression of M2 macrophage markers (CD206) in a dose and time dependent manner.

Conclusion: These findings provide evidence that hydrogen peroxide can polarize macrophages to the M1 phenotype. Furthermore, our results demonstrate that increased reactive oxygen species can perpetuate chronic inflammation through persistent M1 macrophage polarization and decreased M2 macrophage polarization. The use of anti-ROS therapies may help to create a microenvironment that decreases M1 polarization and promote M2 polarization and resolution of the inflammatory response.

 

80.02 Direct Peritoneal Resuscitation Decreases Lung ICAM and MPO After Resuscitated Hemorrhagic Shock

Posted on January 26, 2017

M. A. Eid1, P. J. Matheson1,2, V. S. Graham1, C. D. Downard1,2, R. N. Garrison1,2, J. W. Smith1,2  1University Of Louisville,Department Of Surgery,Louisville, KY, USA 2Robley Rex Veterans Affairs Medical Center,Research,Louisville, KY, USA

Introduction:  Potential complications of hemorrhagic shock (HS) include gut and liver hypoperfusion, splanchnic hypoxia, gut cytokine storm, acute lung injury (ALI), and/or acute respiratory distress syndrome (ARDS).  While ALI/ARDS pathophysiology is multifactorial, lung polymorphonuclear neutrophil (PMN) infiltration occurs early in the cascade via increased intracellular adhesion molecule-1 (ICAM-1).  We hypothesized that DPR treatment improves gut and liver perfusion to prevent these sequelae to decreased ALI/ARDS, and might mitigate ALI/ARDS following HS/CR.

Methods:  Anesthetized male Sprague-Dawley rats (225-250g) were randomized to groups (n=8/group): 1) Sham, 2) HS/CR, 3) HS/CR+DPR (0), or 4) HS/CR+DPR (120).  HS was 40% of baseline MAP for 60 minutes.  CR was shed blood plus two volumes of normal saline over 30 minutes.  DPR was intraperitoneal injection of 30mL pre-warmed 2.5% dextrose peritoneal dialysis solution.  Serum and tissue were collected at 4 hours post-CR.  Lung ICAM-1 ELISA and MPO activity assay were performed.  Lung H&E and IHC for ICAM-1, VCAM-1, and MPO were blindly graded.  

Results: HS/CR increased ICAM-1 levels and MPO activity compared to Sham, while DPR diminished these effects (see Table).   In HS/CR, ICAM-1, VCAM-1, and MPO IHC staining increased compared to Sham, which was decreased with DPR.  MPO IHC revealed increased PMN extravasation and increased absolute number per high-powered field in HS/CR groups compared to Sham, which both decreased below HS/CR levels with DPR. 

Conclusion: Lung ICAM-1, VCAM-1, and MPO expression following hemorrhagic shock are modulated by peritoneal resuscitation using hypertonic peritoneal dialysis solution.  These data suggest that resuscitation applied to the peritoneal space has a remote effect on lung pathophysiology associated with hemorrhagic shock.  This study supports the finding of resuscitation with DPR in prior human trauma patient studies.

 

80.01 Damage-Associated Molecular Patterns Are Mitigated by DPR in Resuscitated Hemorrhagic Shock

Posted on January 26, 2017

M. A. Wilson1,2, P. J. Matheson1,2, J. L. Weaver1, C. D. Downard1,2, R. N. Garrison1,2, J. W. Smith1,2  1University Of Louisville,Department Of Surgery,Louisville, KY, USA 2Robley Rex Veterans Affairs Medical Center,Research,Louisville, KY, USA

Introduction:  Hemorrhagic shock (HS), a significant cause of mortality in trauma patients, has traditionally been resuscitated with intravenous blood and fluid infusion (CR).  While central hemodynamic variables can be restored with CR, vital organ blood flow can often drop causing intestinal hypoperfusion, hypoxia, gut inflammation, and remote organ dysfunction.  The addition of Direct Peritoneal Resuscitation (DPR) can prevent intestinal and hepatic hypoperfusion and inflammation.   We hypothesized that DPR would improve lung function in resuscitated HS (HS/CR) by altering levels of serum and lung inflammatory mediators (DAMPs).

Methods:  Anesthetized Sprague-Dawley rats were randomly assigned to groups (n=8/group):  1) Sham (matching timeline but no HS, CR, or DPR) 2) HS/CR (HS=40% MAP for 60min, CR=shed blood + volumes NS); and 3) HS/CR+DPR.  All groups were followed for 4hr post-RES.  ELISA was used to measure serum and/or lung lipopolysaccharide (LPS), cytokines, hyaluronic acid (HA), high mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), MYD88, TRIF.  Statistics were by analysis of variance and Tukey-Kramer test a priori P value of 0.05.

Results: HS/CR increased serum levels of LPS, HA, pro-inflammatory cytokines (IL-1a, IL-1b, IL-6, and interferon-g), and HMGB1, and lung levels of TLR4 and MYD88 were increased but not TRIF compared to Shams.  HS/CR+DPR decreased LPS, HA, cytokines, HMGB1, TLR4 and MYD88 levels but did not alter TRIF levels compared to HS/CR alone.  

Conclusion: Gut-derived mediators of systemic inflammation can be modulated by peritoneal application of hypertonic DPR to prevent activation of lung inflammatory processes.  DPR after hemorrhagic shock improved visceral blood flow, reduced tissue injury, reduced DAMP formation and serum levels of multiple inflammatory cytokines and chemokines.  Direct peritoneal resuscitation has the potential to significantly improve morbidity and mortality by downregulating the systemic inflammatory response following hemorrhagic shock

 

79.19 Long non-coding RNA Lethe regulates NOX2 expression through inhibiting ERK pathway in macrophages

Posted on January 26, 2017

J. Xu1, C. Zgheib1, J. Hu1, M. Hodges1, K. W. Liechty1  1Laboratory For Fetal And Regenerative Biology, Children’s Hospital Colorado And The University Of Colorado Anschutz Medical Campus,Department Of Surgery,Aurora, CO, USA

Introduction: Recent studies reveal that long non-coding RNAs (lncRNAs) play important regulatory roles in many biological processes. We have previously shown that lncRNA Lethe is down-regulated in diabetic wounds and is involved in the regulation of Reactive oxygen species (ROS) production through modulation of NOX2 gene expression. We hypothesize that Lethe regulates NOX2 expression through the ERK pathway.

Methods: To test our hypothesis, we incubated the murine macrophage cell line RAW264.7 with media containing 5 mM glucose (low glucose), or 25 mM glucose (high glucose) for 24 hours. Overexpression of Lethe was achieved by plasmid transfection. Western blot analysis was used to measure ERK protein level and Real-time PCR used to quantify relative gene expression.

Results: NOX2 was significantly upregulated in high glucose conditions and was associated with significantly decreased Lethe gene expression. Overexpression of Lethe significantly reduced NOX2 gene expression but did not affect the levels of SOD2, SOD3, or Catalase. Western blot analysis showed significantly increased levels of phosphorylated ERK1/2 in high glucose conditions, and that the overexpression of Lethe significantly reduced the levels of phosphorylated ERK1/2.

Conclusion: These findings demonstrate that the lncRNA Lethe regulates NOX2 gene expression and ROS production in macrophages via the ERK pathway.  Furthermore, these results suggest a potential role of lncRNA Lethe in the pathogenesis of the diabetic wound healing impairment and may represent a potential novel therapeutic target to correct the impaired diabetic wound healing response.

 

79.15 Long non-coding RNA GAS5 Regulates Proinflammatory Macrophage Polarization

Posted on January 26, 2017

C. E. Liechty1, J. Hu1, C. Zgheib1, K. W. Liechty1, J. Xu1  1Laboratory For Fetal And Regenerative Biology, Children’s Hospital Colorado And The University Of Colorado Anschutz Medical Campus,Department Of Surgery,Aurora, CO, USA

Introduction:

Macrophages have the ability to dynamically transition between M1 (pro-

inflammatory) and M2 (anti-inflammatory) phenotypes in response to signals from the

surrounding microenvironment. Prolonged M1 macrophage polarization is thought to play a

central role in the chronic inflammation and pathogenesis of diabetic wounds. Recent studies

reveal that long non-coding RNAs (lncRNAs) play important regulatory roles in many biological

processes. However, the role of lncRNAs in macrophage polarization or in diabetic wounds

remains undefined. We have previously shown that lncRNA GAS5 (Growth Arrest-Specific 5) is

up-regulated in diabetic wounds. We hypothesize that increased GAS5 expression contributes

to prolonged M1 macrophage polarization in diabetic wounds.

Methods:

To test our hypothesis, we incubated the murine macrophage cell line RAW264.7

with media containing 5 mM glucose (low glucose), or 25 mM glucose (high glucose) for 4, 8,

and 24 hours and examined GAS5 gene expression by Real-time PCR. In addition, we

examined macrophage polarization and STAT1 gene expression in response to GAS5

overexpression using plasmid transfection.

Results:

High glucose conditions significantly induced macrophage GAS5 gene expression in a

time dependent manner. Plasmid mediated over-expression of GAS5 resulted in significantly

increased gene expression of M1 macrophage markers (iNOS, TNFa, and IL1-Beta), while

demonstrating no change M2 macrophage markers (Arg1, and Mrc1). Mechanistically, GAS5

overexpression promoted M1 macrophage through significantly induced STAT1 expression, the

crucial transcriptional factor for M1 activation.

Conclusion:

These findings provide the first evidence that the lncRNA GAS5 is involved in the

regulation of macrophage polarization. Furthermore, these results suggest a potential role of

lncRNA GAS5 in the pathogenesis of the diabetic wound healing impairment and may represent

a novel therapeutic target to correct the diabetic wound healing impairment.

79.14 Phenotypic and Genotypic Changes in Metastatic Neuroblastoma

Posted on January 26, 2017

W. E. Barry1, J. R. Jackson1, G. Asuelime1, Z. Wan2, R. C. Seeger2, E. S. Kim1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA 2Children’s Hospital Los Angeles,Pediatrics,Los Angeles, CA, USA

Introduction:  Neuroblastoma is the most common solid tumor in children and despite current multimodal therapies, survival is poor.  While massive primary tumors are a clinical challenge for these children, the main cause of death is recurrent, incurable, metastatic disease following surgical resection.  It is critical to further characterize the biologic differences between the primary tumor and the metastatic cells which appear to respond differently to treatment and are the major cause of mortality in these patients. We hypothesize that cells derived from liver and bone marrow metastases in a murine model will be phenotypically and genotypically different when compared to their parental cell of origin. 

Methods:  Multiple metastatic cell lines were created de novo from the bone marrow and liver of NSG mice utilizing two human neuroblastoma cell lines (CHLA 255, SH-SY5Y). A metastatic neuroblastoma mouse model of minimal residual disease was used whereby the parental cell line was injected into the renal capsule, followed by primary tumor resection 7 days later. The metastases were allowed to grow until euthanasia criteria were met. Metastatic neuroblastoma cells from the liver and bone marrow were harvested and cultured. In vitro studies comparing the parental cell line to the metastatic cell lines were performed. Response to increasing doses of chemotherapy (etoposide) was analyzed using a DIMSCAN cytotoxicity assay. Gene expression was characterized using a low-density metastatic pathway PCR microarray. The PCR-array was performed using the parental and metastatic cell lines from the SH-SY5Y cell line. 

Results: Metastatic cell lines derived from the liver and bone marrow had a significantly lower response to increasing doses of etoposide (p<0.05) compared to the parental cell line, which followed a dose response curve. PCR array revealed numerous genes which were upregulated in the liver and bone marrow cell lines compared to the parental (Figure). Numerous neuroblastoma associated genes appeared to be upregulated including APC, CDH1, CXCL12, FGFR4, MET and NR4A3. These genes play critical roles in cell adhesion, migration, proliferation and survival. Many of the other upregulated genes were also vital to cell migration and adherence including CDH6 and MMP10. 

Conclusion: Metastatic neuroblastoma cells appear to have significantly different gene expression as well as response to standard chemotherapy when compared to the parental cell line. These preliminary data provides the background for further in vitro and in vivo studies to elucidate the effects of treatments particularly on genes specific to metastatic disease in our novel murine model of minimal residual disease.

79.09 Necrotizing Enterocolitis Is Associated With Changes In Intestinal Epithelial Claudin-2 Expression

Posted on January 26, 2017

G. J. Ares1,2, D. R. Wood1, C. Y. Yuan1, C. J. Hunter1  1Northwestern University,Pediatrics,Chicago, IL, USA 2University Of Illinois At Chicago,General Surgery,Chicago, IL, USA

Introduction:
Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in neonates, affecting 5-10% of patients in the neonatal intensive care unit (NICU). NEC is mostly seen in premature infants, but its pathophysiology remains unknown. Tight junction (TJ) proteins are paracellular complexes in intestinal villi important in maintenance of protective intestinal barrier. Claudin-2 (C2) is an intercellular pore forming TJ protein that regulates permeability. We hypothesize that Claudin-2 expression levels are upregulated in human NEC, as well as in an experimental model of bacteria-induced NEC.

Methods:
Caco-2 cells were exposed to LPS in an in vitro model of NEC in a 10 day time course experiment with samples taken on days 0, 1, 3, 5, 7, and 10 along with untreated controls per day. An in vivo model of rat pups subjected to hypoxia twice per day and fed clean formula (control) vs bacteria + formula (NEC). Cells were fragmented into cytosol, membrane, nucleus, and cytoskeleton compartments. C2 was analyzed by immunofluorescence mean fluorescent intensity (MFI), western blot, and quantitative PCR in Caco-2 cells, rat intestinal segments, and human intestines from patients with NEC vs humans without NEC requiring bowel resection. Western blots were standardized to loading controls.  Data was analyzed with student’s T-test.

Results:
Staining showed increased expression of C2 in humans with NEC vs control (MFI=710±24 vs 1530±98 (p<0.0005), respectively) and in rats with experimental NEC vs control (MFI= 514±22 vs 706±21 (p<0.0005), respectively). There appears to be a change in localization of C2 from cytosol to membrane in IF for Caco-2 cells. Western blot and qPCR confirmed 2-fold increase in C2 expression in the cellular NEC model vs control over time, with an increased proportion of C2 in the membrane compartment. In rats, western blot showed increased C2 expression in NEC vs control, and a greater than 2-fold increase on qPCR (p<0.001). Figure 1 shows western blot for C2 in Caco-2 cells exposed to LPS (L) vs control group (C) and the measured immunoblot density of LPS group relative to Control per day.

Conclusion:
In conclusion, human cells and experimental cellular and rat models both showed increased expression of Claudin-2 TJ protein in the intestinal epithelium with NEC. The change in expression of this pore forming protein may play an important role in the breakdown of intestinal barrier integrity in NEC. Changes seen in localization may elucidate the mechanism by which this breakdown occurs. Further research in TJ proteins could help define the pathophysiology of NEC and provide targets for therapy.

79.08 Localization of muscarinic receptors to the crypt stem cell compartment of the murine small intestine

Posted on January 26, 2017

C. J. Greig1, S. J. Armenia1,2, V. R. Klump3, R. A. Cowles1  1Yale School Of Medicine,Department Of Surgery,New Haven, CT, USA 2New Jersey Medical School,Newark, NJ, USA 3Yale School Of Medicine,Department Of Dermatology,New Haven, CT, USA

Introduction:

Maintenance of the highly plastic intestinal epithelium relies upon a small population of stem cells localized to intestinal crypts. These specialized stem cells are responsible for both self-renewal and generation of all mature intestinal epithelial cell types, but the mechanisms by which these processes are controlled remain incompletely understood. Recent evidence suggests that muscarinic acetylcholine (ACh) signaling is involved in epithelial barrier function, epithelial proliferation and inhibition of apoptosis. Furthermore, all muscarinic ACh receptor (mAChR) subtypes have been documented throughout the gastrointestinal tract but the precise location of these receptors within the functional crypt-villus unit is unclear. Thus identification of ACh receptors present in the region of the stem cell niche would allow for design of therapies targeting the muscarinic cholinergic signaling system. We hypothesized that the intestinal crypt base would express only specific mAChRs that drive proliferation in this critical area of the mucosa.

Methods:

With institutional approval, proximal and distal intestinal segments from the jejunum and ileum were procured from wild type C57Bl/6 mice aged 12-14 weeks. Collected segments from each region of the bowel were divided and either formalin fixed and used to create paraffin sections or used for RNA extraction in an RNase-free environment per standard protocols. Paraffin sections on specialized membrane slides were used in capturing cells at the base of the intestinal crypt, performed on a LCM microscope at 100X magnification into RNAlater solution to allow for subsequent RNA extraction without significant degradation. Care was taken to avoid any subepithelial tissue and include only the bottom half of the crypt, where the intestinal stem cells are thought to reside. Using primers targeting the five subtypes of mAChRs (M1-M5), RT-PCR was performed using RNA from full-thickness intestinal and LCM crypt cell (LCM-CC) samples to determine the presence or absence of each subtype.

Results:

In full-thickness intestinal samples, mAChR subtypes M1-M4 were found in all regions of the jejunum and ileum examined. The M5 mAChR subtype was present in the proximal jejunum, but was absent from all other regions of the bowel. For LCM-CC samples, only the M1 mAChR subtype was present and was found in crypt cells from all regions of the intestine examined.

Conclusion:

All subtypes of muscarinic acetylcholine receptors were present throughout the small intestine with the exception of the M5 mAChR, which appears to be localized to the proximal jejunum. However, when cells from the crypt base were collected by laser capture microdissection and analyzed, only the M1 subtype was present by RT-PCR. Given the known location of the stem cell niche to this area, the cholinergic system, possibly via the M1 muscarinic acetylcholine receptor, is likely to be a key mediator of intestinal homeostasis.

79.04 Decreased Angiogenesis in Slit3Knockout Mice with CDH Results in Reduced Pulmonary Arterial Branching

Posted on January 26, 2017

M. Shah1, M. R. Phillips1, T. Rao1, L. J. Edwards1, Y. Z. Lee1, S. E. McLean1  1University Of North Carolina At Chapel Hill,Chapel Hill, NC, USA

Introduction:
Congenital diaphragmatic hernia (CDH) is a structural birth defect associated with pulmonary hypoplasia and pulmonary arterial hypertension (PAH). Impaired angiogenesis is thought to play a role in the pathophysiology of PAH in CDH. We hypothesize that decreased angiogenesis in Slit3 knockout (KO) mice with CDH and PAH results in attenuated pulmonary arterial (PA) branching patterns.

Methods:
Slit3 wild-type (WT) and KO mice were harvested at 2-weeks, 4-weeks, and 2-3 months (adult) of age. The pulmonary arterial system was perfused with Microfil casting agent and scanned with MicroCT (Scanco). Vascular tree analysis was performed using AMIRA software, based on size and degree of vessel branching (rank) from the main pulmonary artery. Lungs were harvested for RNA isolation. Real-time PCR was performed for angiogenesis markers. Lungs were also lysed in RIPA buffer for protein isolation for use in Western Blot analysis.

Results:
There was a 2-fold decrease in FGF1 expression in KO vs. WT mice at 4-weeks, and a 2-fold decrease in HIF1A and eNOS expression in adult KO vs. WT mice. There was a 28% decrease in eNOS protein levels in KO vs. WT mice based on Western Blot. There was a trend towards fewer number of vessels in 2-week, 4-week, and adult KO vs. WT mice, with a more pronounced difference in adult mice (5404 vs. 7247, p=0.32, 8989 vs. 14229, p=0.12, 11293 vs. 21292 vessels, p=0.06, respectively). Number of ranks were similar in KO vs. WT mice at 2-weeks (61-70 vs. 51-60 ranks), with a trend towards decreased number of ranks in 4-week and adult KO vs. WT mice (61-70 vs. 81-90, 61-70 vs. 91-100 ranks, respectively). There were also fewer vessels per rank in KO vs. WT mice at 2-weeks, 4-weeks, and adult (1185 vs. 1902, 2186 vs. 3675, 2660 vs. 5311, respectively for ranks 31-40, p<0.05). 

Conclusion:
Slit3 KO mice with CDH and PAH have decreased angiogenesis over time with decreased pulmonary arterial branching patterns. Further elucidation of the mechanisms of decreased angiogenesis is needed in order to further understand the mechanisms leading to PAH in CDH.

79.03 Mitochondrial DNA Promotes a Pro-Inflammatory Profile in Intestinal Epithelial Cells

Posted on January 26, 2017

V. Siow1,2, E. A. Novak1, G. J. Vincent1, K. Cunningham1,2, K. P. Mollen1  1Children’s Hospital Of Pittsburgh,Pediatric General And Thoracic Surgery,Pittsburgh, PA, USA 2University Of Pittsburgh,General Surgery,Pittsburgh, PA, USA

Introduction:  The role of mitochondrial dysfunction in the pathogenesis of inflammatory bowel disease (IBD) is just beginning to be understood. Our lab and others have shown evidence of mitochondrial stress and a disruption in mitochondrial function within the intestinal epithelium of humans with IBD as well as mice undergoing experimental colitis. We now hypothesize that mitochondrial stress leads to a release of the damage-associated molecular pattern (DAMP) mitochondrial DNA (mtDNA), which in turn contributes to immune activation and the potentiation of inflammation.

Methods: Serum samples were obtained from patients with severe active IBD and from control subjects according to University and IRB protocol. MtDNA content was determined using qRT-PCR. Mice were subjected to 3% Dextran Sodium Sulfate (DSS) for 7 days. The mitochondria within the intestinal epithelium of control mice vs. DSS-subjected mice were analyzed via electron microscopy. Total DNA was isolated from murine intestinal crypts in control and DSS-subjected mice. This DNA was then utilized to determine the levels of mtDNA relative to nuclear DNA via qPCR and the ΔΔCt method. Intestinal epithelial HT29 cells were treated with mtDNA at 37°C. RNA was then isolated, cDNA synthesized, and qPCR setup to determine the expression of cytokines in control vs. mtDNA-treated cells.

 

Results: For the first time, we show evidence of mtDNA release in the bloodstream of humans with severe active IBD. Mice undergoing experimental colitis exhibit dramatically altered mitochondrial structure as well as a decrease in overall mtDNA within the intestinal epithelium as compared to control mice.  Although total mtDNA content was decreased, there was evidence of free mtDNA within the cytosol of intestinal epithelial cells of mice subjected to experimental colitis. Additionally, HT29 cells treated with mtDNA exhibited an increase in the expression of pro-inflammatory cytokines, including TNF-α as well as TLR9 expression.

Conclusion: Here we investigate the role of mtDNA in intestinal inflammation. Our previous studies demonstrate a role for mitochondrial dysfunction in the pathogenesis of colitis.  However, it remains unclear how this dysfunction leads to inflammatory change. We now demonstrate that the amount of circulating mtDNA in the blood of patients with IBD is increased as compared to healthy control patients, suggesting that a release of mtDNA from dysfunctional mitochondria occurs during disease. We also show that intestinal epithelial cells treated with mtDNA demonstrate an increase in expression of pro-inflammatory cytokines. Our studies suggest that mtDNA may contribute to local and systemic inflammation in patients with IBD. Strategies aimed at limiting mitochondrial dysfunction or sequestering cell-free mtDNA may lead to new therapeutic approaches to combat disease.

 

79.02 The IEC-6 Cell Line as a Model of Muscarinic Signaling Pathways in the Murine Intestinal Epithelium

Posted on January 26, 2017

S. J. Armenia1,2, C. J. Greig1, R. A. Cowles1  1Yale University School Of Medicine,Department Of Surgery,New Haven, CT, USA 2New Jersey Medical School,Newark, NJ, USA

Introduction:  Growth of the intestinal epithelium is critical during intestinal adaptation and after intestinal mucosal injury. While several peptide hormones have been shown to stimulate intestinal mucosal growth, the role of enteric neurotransmitters, such as acetylcholine (ACh), in mucosal homeostasis remains poorly defined. Previous work documenting serotonergic regulation of enterocyte proliferation implicated muscarinic ACh receptors (mAChRs) in the signaling mechanism. The distribution of the five mAChR subtypes on dividing enterocytes has not been reported but may assist in the development of targeted therapies aimed at stimulating intestinal mucosal growth. In order to establish an in vitro model for study of cholinergic signaling in the intestinal mucosa, we aimed to document the presence of mAChRs in cultured enterocytes. We hypothesized that while mAChRs would be widely distributed in the small bowel, the IEC-6 cell line would express enterocyte-specific muscarinic receptors allowing these cells to respond to muscarinic receptor agonists in a well-controlled model system. 

Methods:  The IEC-6 small intestine epithelial cell line was cultured under standard conditions and, with institutional approval, small intestinal segments were obtained from male wild type C57Bl/6 mice between the ages of 12-14 weeks. RNA was extracted from IEC-6 cells and whole intestinal segments in an RNase-free environment per standard protocols. RT-PCR was performed using targeted primers to document the presence or absence of each of the five mAChRs. PCR products were separated by gel electrophoresis and visualized using ethidium bromide staining.  To assess the responses of epithelial cells to agonists, cultured IEC-6 cells were treated with serial dilutions of bethanechol, a muscarinic receptor agonist, for 48 hours. Cellular viability and proliferation were assessed using an MTT assay. Statistical analysis was performed using Student’s t-test, with significance assumed when p<0.05.

Results: In whole intestinal segments, the presence of RNA from all five mAChRs was demonstrated by RT-PCR. In cultured IEC-6 cells, RNA for two of the five mAChR subtypes (M1 and M4) was amplified by RT-PCR. Treatment of IEC-6 cells with bethanechol stimulated cellular proliferation in a dose-dependent manner. 

Conclusion: Muscarinic ACh receptors are widely distributed in the small intestine. While all mAChR subtypes can be documented in whole small intestinal segments, only M1 and M4 mAChRs appear to be expressed in IEC-6 cells. The ability of bethanechol to stimulate IEC-6 cell proliferation suggests that ACh may play an important role in enterocyte proliferation in-vivo. The specific mAChR responsible for this action and the subsequent intracellular signaling pathways warrant further study of this model. 

 

79.01 FAK Inhibition Abrogates Neuroblastoma Cancer Stem Cell Phenotype in Patient-Derived Xenografts

Posted on January 26, 2017

L. Stafman1, E. Garner1, A. Hjelmeland1, J. Stewart1, S. Mruthyrunjayappa1, K. Yoon1, S. Cramer1, E. Beierle1  1University Of Alabama,Birmingham, Alabama, USA

Introduction:

Neuroblastoma (NB) is the most common extra-cranial solid tumor of childhood and is responsible for over 15% of pediatric cancer deaths. Focal adhesion kinase (FAK) is a tyrosine kinase affecting proliferation, adhesion, and migration, and is prominent in aggressive forms of NB. FAK inhibition in immortalized human NB cell lines has been shown to decrease NB tumorigenicity. Immortalized cell lines do not always recapitulate the human condition, so we have adopted the use of patient-derived xenografts (PDXs), which are tumors implanted into immunosuppressed mice from human patients.  Cancer stem cells are a subpopulation of cells with stem cell-like properties. NB cancer stem cells can be identified by expression of cell surface proteins, including CD133, and the ability to grow as spheres in vitro. We hypothesized that FAK inhibition would decrease tumorigenicity and cancer stem cell maintenance in human NB PDXs.

Methods:
Cells from two human NB PDXs (COA3, COA6) were treated with the FAK inhibitors 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15) or PF-573,228 (PF). FAK expression and phosphorylation was determined with immunoblotting. Viability and proliferation were assessed with alamarBlue and CellTiter 96 assays, respectively. Expression of the cell surface protein and stem cell marker, CD133, was determined using flow cytometry after 24 hours of treatment with PF or Y15. The ability of the NB PDXs to grow as spheres was assessed using an in vitro limiting dilution analysis. Student’s t-test, extreme limiting dilution analysis, and χ2 statistics were used. Data reported as mean ± SEM with p<0.05 significant.

Results:
FAK expression and phosphorylation was inhibited in both COA3 and COA6 cells following treatment with PF or Y15.  FAK inhibition with the small molecules (PF or Y15) significantly decreased viability and proliferation in a dose dependent fashion in both NB PDXs. Additionally, PF or Y15 treatment yielded a significant decrease in the expression of the cancer stem cell marker, CD133 (18.5% ± 0.2 untreated cells vs. 11% ± 0.3 and 5.3% ± 0.1, PF or Y15, respectively, p< 0.02). FAK inhibition with PF or Y15 also significantly decreased sphere formation in both human NB PDXs in culture (Figure), indicating a decrease in cell “stemness”. 

Conclusion:
Inhibition of FAK with small molecule inhibitors decreased viability, proliferation, and cancer stem cell maintenance in human NB PDXs. Multiple clinical trials examining the safety and efficacy of FAK inhibitors are currently ongoing, although none have yet been undertaken in children or patients with NB. Our data indicate that FAK inhibitors warrant further exploration as a novel therapy for NB.
 

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