23.07 Improved Sepsis Survival in JAM-A-/- Mice is Associated with Enhanced Intestinal Mucosal Immunity

Posted on January 25, 2017

N. J. Klingensmith1, K. Fay1, C. Chen1, J. Lyons1, C. M. Coopersmith1  1Emory University School Of Medicine,Surgery,Atlanta, GA, USA

Background – Sepsis is associated with intestinal hyperpermeability which carries significant mortality. This permeability is augmented in mice lacking Junctional Adhesion Molecule A (JAM-A), an enterocyte tight junction protein. Our unpublished data reveal JAM-A-/- mice paradoxically have decreased systemic bacterial burden and improved sepsis survival following cecal ligation and puncture (CLP). At baseline, the intestinal epithelium, local immune system, and gut microbiome all interact to maintain homeostasis. How the immune system and microbiome respond to intestinal hyperpermeability in septic JAM-A-/- animals may provide a mechanism for their improved sepsis survival.

 

Methods – 6-12 week old male and female C57BL/6 (WT) and JAM-A-/- mice underwent CLP to induce sepsis and were sacrificed 24 hours later for functional studies. Mesenteric lymph nodes (MLN) and small bowel Peyer’s Patches (PP) were collected and B220+ cells were stained and counted by flow cytometry. Serum and small bowel homogenate IgA were measured by ELISA. Intestinal microbiome taxonomy were identified from fecal samples by 16S rRNA amplicon pyrosequencing followed by QIIME pipeline analysis. Statistical analysis was performed using a Student’s t-test or Mann-Whittney test depending on Gaussian distribution and a p-value of <0.05 was considered to be statistically significant.

 

Results-  Septic JAM-A-/- mice had a greater frequency of B cells compared to septic WT controls in their MLN (mean frequency, 25.2±2.3% vs 17.2±1.4%, p=0.009, n=10) and PP (mean frequency, 19.0±1.7% vs 12.8±2.2%, p=0.04, n=7). IgA within the intestine, a product of B cells, was elevated in septic JAM-A-/- animals compared to septic WT controls (mean, 241.7±37.9 vs 131.8±19.8 ng/mg of gut tissue, p=0.02, n=7-10) suggesting prior antigenic exposure. Furthermore, serum IgA concentration in septic JAM-A-/- mice was increased compared to septic WT mice (mean, 178.1±43.9 vs 39.5±6.9 ng/mL, p=0.005, n=11) providing a potential mechanism for decreased systemic bacterial burden in septic JAM-A-/- mice. Principle coordinate analysis of  weighted and unweighted UniFrac distances of 16S rRNA sequencing revealed separation between septic JAM-A-/- mice and septic WT animals (p<0.001, n=5-7). 

 

Conclusion – JAM-A-/- mice have greater populations of B cells in their MLN and small bowel PP that produce more IgA. The increased gut IgA may result in a shift toward less pathogenic microflora which are potentially cleared more easily in the event of systemic contamination, such as in the setting of sepsis. Greater antigen presentation, local immune priming, and alteration of the microflora may be the source of improved sepsis survival in JAM-A-/- mice.

23.06 Effects of Valproic Acid and Molecular Hydrogen on Phenotypes of Microglia Treated with Hypoxia

Posted on January 25, 2017

X. Wu1, P. Chang1, R. Zeng1, Y. Deng1, Y. Zhao1, W. Chong1  1The First Affiliated Hospital Of China Medical University,Emergency,Shenyang, LIAONING, China

Introduction: Secondary hit is regarded as a lethal complication of traumatic brain injury (TBI), due to severe inflammation response induced by ischemia and hypoxia. Recently, it has been demonstrated that macrophages are mainly involved in inflammation with two different functional phenotype (or polarization): ones that encourage inflammation are called M1 macrophages, whereas those that decrease inflammation are called M2 macrophages. In TBI, microglia have the similar phenotype conversion as macrophages. Researches show that valproic acid (VPA) and molecular hydrogen (H2) attenuate cerebral swelling and manifest nervous protection in animal model of TBI in uncertain ways. The hypothesis in this study was that hypoxia enhances microglia polarization toward the pro-inflammatory phenotype. Both VPA and H2 inhibit hypoxia-induced inflammatory effects on microglia.

Methods: Mouse BV2 microglia were divided into 4 groups: Sham (no hypoxia, no VPA), hypoxia group, VPA group and H2 group. Hypoxia and VPA groups were exposed to hypoxic conditions (0.5% O2, 10% CO2, and 89.5% N2) at 37°C in the absence or presence of VPA (3mM) for 2 hours. H2 group were treated with H2 (0.5%O2, 2%H2, 10%CO2, 87.5%N2) for 1 hour under same hypoxic condition. Enzyme-Linked Immunosorbent Assay (ELISA), flow cytometry and Real-Time PCR were performed to assess protein and mRNA levels of phenotypic markers in microglia respectively.

Results: Hypoxia increased mRNA levels of M1 markers and reduced mRNA levels of M2 markers in microglia (P<0.05). In addition, the ratio between the mRNA levels of M1 markers and M2 markers were also increased by hypoxia (P<0.05). VPA reduced protein and mRNA levels of M1 markers (P<0.05), decreased the ratio between the mRNA levels of M1 markers and M2 markers of microglia (P<0.05) compared with hypoxia group. H2 not only decreased protein and mRNA levels of M1 markers (P<0.05) but also increased cytokine secretion and mRNA levels of M2 markers in microglia (P<0.05), and lowered the ratio between the mRNA levels of M1 markers and M2 markers of microglia as well in comparison with hypoxia group (P<0.05).

Conclusion: Hypoxia enhances microglia polarization toward the pro-inflammatory phenotype. Both VPA and H2 attenuate hypoxia-induced pro-inflammatory effects on microglia.

23.05 IL-33 DRIVES TYPE 2 CYTOKINES THROUGH ILC2 IN THE LUNGS AFTER HEMORRHAGIC SHOCK/TRAUMA (HS/T)

Posted on January 25, 2017

J. Xu1, J. Guardado1, R. Hoffman1, H. R. Turnquist1, T. R. Billiar1  1UPMC,Surgery Department,Pittsburgh, PA, USA

Introduction: The immunosuppression/immune dysregulation that follows severe injury includes a strong bias towards type 2 immune responses. These responses, which are characterized by the production of the type 2 cytokines (IL-4, IL-5 and IL-13), are typically thought to be the result of polarization of CD4+ T cells towards a Th2 phenotype. However, polarization of naïve T cells can take several days while elevations of type 2 cytokine production is often detected early after injury. This lead us to hypothesize that IL-33, an alarmin released early after injury, and type II innate lymphoid cells (ILC2) , a recently described population of T cell receptor negative innate lymphocyte cells (most abundant in the lungs), contribute to the early type 2 immune responses after injury.

Methods:  IL-33, and the type 2 cytokines (IL-4, IL-5 and IL-13) levels were detected in the plasma of blunt trauma patients. C57BL/6, IL-33-/- and ILC2 deficient mice were subjected to resuscitated hemorrhagic shock + bilateral lower extremity injury (HS/T), ILC2 percentage and activation were assessed by flow cytometry of lung leukocytes using IL-5 as a representative type 2 cytokine. Mice deficient in ILC2 were generated by reconstituting irradiated wild type (WT) with bone marrow from staggerer (Rorasg/sg) mice.

Results: Severely injured human blunt trauma patients (n=493, average ISS=20.2) exhibited early elevations in plasma IL-33 which correlated positively with increases in IL-4, IL-5 and IL-13. In C57BL/6 mice, HS/T led to a significant increase in lung IL-5 (measured by flow), IL-33 (measured by ELISA) and ILC2 percentage (CD45+Lineage–CD25+ CD127+CD90.2+ ST2+Sca-1+ CD117int) in the lungs at 6 hrs (p<0.05, n=4 for each time point). IL-5+ ILC2 were easily identified in the lungs by 6hrs. However, the increases in both ILC2 percentage and ILC2 IL-5 expression were absent in IL-33-/- mice subjected to HS/T (n=4/group). IL-5 could also be detected in PMN (Ly6G+CD11b+) in the lungs of injured mice but this was suppressed by IL-33-/- or ILC2 deletion. Using cultured PMN, we confirmed that exogenous IL-5 could lead to the increase of PMN IL-5 expression, measured by both flow and PCR (n=4/group).

Conclusion: These data show that early IL-33 elevations correlate with type 2 cytokines levels in blunt trauma patients. Reverse translation experiments in mice establishes that IL-33 drives ILC2 activation and type 2 cytokine production in the lungs of injured mice within 6 hrs. Furthermore, ILC2 derived IL-5 appears to upregulate IL-5 in PMNs thus amplifying the local type 2 response.  Combined these data show that the shift towards type 2 responses occurs rapidly after injury involve innate lymphoid cells responding to IL-33.
 

23.04 Post-Shock Mesenteric Lymph Exosomes are Key Mediators of Acute Lung Injury

Posted on January 25, 2017

M. Kojima1, T. Chan1, J. Gimenes1, B. Eliceiri1, A. Baird1, T. Costantini1, R. Coimbra1  1University Of California – San Diego,Division Of Trauma, Surgical Critical Care, Burns And Acute Care Surgery/Department Of Surgery,San Diego, CA, USA

Introduction: Acute lung injury (ALI) is a common cause of morbidity in patients following severe injury. Studies have shown that mesenteric lymph (ML) carries gut-derived inflammatory mediators to the lung and serves as the inciting event in ALI. Alveolar macrophages (AM), a lung resident macrophage, play a critical role in the development of ALI. We have recently demonstrated that exosomes, nano-sized extracellular vesicles, are present in ML and have the ability to trigger NF-κB activation in human monocytic cells in vitro. We hypothesized that exosomes in post-injury ML induce pro-inflammatory cytokine production in AM and contributes to post-injury ALI. 

Methods: Male rats underwent cannulation of the femoral artery, jugular vein and ML duct prior to trauma/hemorrhagic shock (T/HS; mean arterial pressure 35 mmHg for 60 min), followed by resuscitation with shed blood and two times normal saline. The ML was collected before hemorrhagic shock (pre-shock) and after T/HS (post-T/HS) for isolation of exosomes by differential centrifugation. In vitro AM were stimulated with exosomes harvested from pre-shock or post-T/HS ML for 6 or 12 hours for measurement of cytokine production by quantitative reverse transcription PCR (qRT-PCR). ML Exosomes from each experimental group (2 x 109 exosomes/g) were also intravenously injected into male naïve C57BL/6 mice to assess in vivo biologic activity. Lung injury was evaluated by measuring histologic lung injury, vascular permeability and immune cell recruitment in bronchoalveolar lavage (BAL) and lung tissue. 

Results:  Exosomes released into post-T/HS ML increased the gene expression of pro-inflammatory cytokines (TNF-a and CINC-1; see figure), NF-kB (5-fold increase at 6h vs. pre-shock; p<0.001) and iNOS (4-fold increase at 12h vs. pre-shock; p<0.001) in AM in vitro. Compared to pre-shock ML exosomes, the in vivo injection of post-T/HS ML exosomes resulted in increased histological lung injury score, a 2-fold increase in Evan’s blue dye lung permeability (0.058 ± 0.003 to 0.117 ± 0.011 mg/g tissue; p<0.05) as well as an increase of wet-to-dry ratio (4.608 ± 0.071 to 5.202 ± 0.149; p<0.05). Exosomes in post-T/HS ML also induced increased recruitment of neutrophils (CD45+Ly-6G+) and macrophages (CD45+CD11c+) in BALF and lung parenchyma determined by flow cytometry (p<0.05 vs. pre-shock).   

Conclusion:  Exosomes released into post-T/HS ML caused an inflammatory response from AM in vitro and ALI in vivo. Our findings define the critical role of ML exosomes as a biologically active mediator of ALI after severe injury.

 

23.03 FER Gene Delivery Improves Survival by Enhancing Lung Immune Response Against Lethal Pneumonia

Posted on January 25, 2017

L. K. McCandless1, Y. S. Yin1, V. Dolgachev1, S. Panicker1, M. Suresh1, M. Hemmila1, K. Raghavendran1, D. Machado-Aranda1  1University Of Michigan,Division Of Acute Care Surgery/Department Of Surgery,Ann Arbor, MI, USA

Introduction: Alternative treatments against antibiotic-resistant bacteria are being strongly investigated. Potentially, transient manipulations of the genome could induce a more efficient immune response. A recent Genome-wide Association Study demonstrated correlation of FER gene expression with survival among pneumonia (PNA) patients. This was confirmed by electroporation-mediated delivery of FER gene in a lethal murine model of combined lung contusion (LC) and PNA. Neither study was conceived to determine the mechanisms for these favorable outcomes. We propose that FER improves survival by recruitment of activated immune cells primed to remove bacteria from the lung.

Methods: C57/BL6 mice were inoculated with 500 CFU of Klebsiella pneumoniae. At 1-hr they received plasmid DNA encoding human (pFER) gene via pharyngeal drop followed by 8 electroporation pulses (EP)  inducing its expression in the lung. We recorded survival data for pFER-EP and control groups (PNA-only; PNA/EP-empty vector and PNA/EP-Na+/K+-ATPase gene). In parallel experiments, animals were sacrificed at specific time points (24, 48, 72 hr), bronchial alveolar lavage (BAL) fluid and lung tissues were harvested; cellular subpopulations counted by flow cytometry; specific genes and signaling pathways were assessed by TaqMan/Western Blot and finally cytokines by ELISA.

Results: After pFER-EP; 5-day survival was markedly improved compared to PNA-only control (80% vs 20%; p < 0.05). Early significant numbers of inflammatory monocytes were only detected in BAL fluid from pFER-EP animals exhibiting known antibacterial markers Toll Receptor- 2 and 4 (TLR2 and TLR4) respectively; being the later more predominant. Both BAL cells and lung tissues had higher protein expression of phosphorylated STAT3 (p-STAT3), a transcription factor critical in bacterial removal. The increased levels of p-STAT3 correlated with the decreased expression of its chaperone Heat-Shock Protein 90 (HSP90). However at 72 hr, HSP90 and other proteosome genes (NRLP2, NRF2) dramatically increased to counter-regulate p-STAT3, inducing monocyte apoptosis and avoiding damage to surrounding tissues.  BAL TLR4+ monocytes were found to highly express Nitric oxide synthase-2 (Nos-2), Resistin-like molecule α1 (Fizz1), Tumor Necrosis Factor-α (TNFα) and Interferon-γ  (IFNγ) all important against bacterial infection. Additionally, pFER-EP was able to rescue neutrophilic response in TLR4-/- mice, via increased production of strong chemoattractant KC and counterbalanced by decoy receptor for advance glycosylation (sRAGE), and independent from TNFα  and IFNγ levels.

Conclusion: Lung gene delivery of the FER improved pneumonia survival by early STAT3 phosphorylation and suppression of HSP90, in turn enhancing antibacterial TLR4+ monocytes. Additionally, FER expression can modulate neutrophilic response via KC and sRAGE cytokines independently from TLR4, TNFα and IFNγ, thus constituting a novel therapeutic strategy.  

23.02 Storage of Thawed Plasma Over 14-Days Does Not Affect Ability to Inhibit tPA-induced Fibrinolysis

Posted on January 25, 2017

B. R. Huebner1, C. C. Silliman1,3, R. Shepherd-Singh2, G. Stettler1, H. B. Moore1, G. Nunns1, A. Banerjee1, E. E. Moore1,2  1University Of Colorado Denver,Trauma Surgery,Aurora, CO, USA 2Denver Health Medical Center,Trauma Surgery,Aurora, CO, USA 3Bonfils Blood Center,Denver, CO, USA

Introduction:
Emerging evidence supports plasma-first resuscitation in the treatment of trauma-induced-coagulopathy (TIC). The logistics of plasma-first resuscitation require thawed plasma (TP) be readily available due to the thawing time of fresh frozen plasma (FFP) (30-minutes at 37°C). The current standard is storage of TP for up to 5 days at 4°C for use in major hemorrhage scenarios, but there remains a 2.2% outdated wastage rate accounting for 129,000 discarded units each year. This 5-day lifespan of TP prevents smaller hospital from having it readily available, and is based on factors V, VII, and VIII levels at outdate. However, the key plasma proteins in attenuating fibrinolysis and correction of TIC remain unknown as does their change in activity level over the thawed storage time. We hypothesize that TP retains the ability to inhibit tPA-induced fibrinolysis at 14 days storage at 1-4°C.
 

Methods:
Pooled FFP was thawed and stored at 1-4°C at the following intervals: 14, 10, 7, 5, 3, and 1-day prior to the experiment. Healthy volunteers participated: 80% men, not pregnant or taking any medications at the time of blood draw (n=5). Blood was drawn into citrated tubes followed by a 50% dilution of whole blood (WB) with the above TP intervals as well as FFP, normal saline (NS), albumin, and WB control. Samples underwent citrated tPA-modified (75ng/ml) thromboelastography (TEG). TEG parameters including R-time, angle, maximum amplitude (MA), and LY30 were examined.
 

Results:

14-day TP retained the ability to inhibit tPA-induced hyperfibrinolysis (median LY30% 8.4) similar to FFP (6.4%), WB (14.6%) and superior to albumin (59.3%, p<0.00001) and NS (58.1%, p<0.00001) (figure 1). All TP intervals demonstrated similar clot characteristics to FFP including rate of clot initiation (R-time), rate of clot formation (angle), maximum clot strength (MA), and resistance to clot breakdown (LY30, figure 1) on tPA-challenge TEG. 14-day TP retained superior clot strength (median MA, 61.5mm) to albumin (21.6, p=0.001) and normal saline (32.2, p=0.0003). 14-day TP also had a faster rate of clot formation (median anlge, 66.2°) compared to albumin (34.8°, p=0.002) and normal saline (41.6°, p=0.006).

 

Conclusion:
TP plasma retained the ability to inhibit tPA-induced fibrinolysis as well as speed of clot formation (angle) and strength of clot formation (MA) over 14-day storage at 1-4°C. A clinical trial is needed to validate these in vitro results but consideration should be made to increasing the storage life of TP.

 

23.01 Microparticles from Aged pRBC Units Stimulate Thrombin Generation and Cause Pulmonary Vascular Congestion

Posted on January 25, 2017

Y. Kim1, B. T. Xia1, A. D. Jung1, A. L. Chang1, R. M. Schuster1, R. A. Veile1, M. D. Goodman1, T. A. Pritts1  1University Of Cincinnati,Surgery,Cincinnati, OH, USA

Introduction:  During storage, packed red blood cells undergo a series of physical, metabolic, and chemical changes collectively known as the red blood cell storage lesion. One key component of the red blood cell storage lesion is the accumulation of microparticles, which are submicron vesicles shed from erythrocytes as part of the aging process. Previous studies from our laboratory indicate that transfusion of these microparticles leads to lung injury, but the mechanism underlying this process is unknown. In the present study, we hypothesized that microparticles from aged packed red blood cell units induce procoagulant activity via accelerated thrombin generation. 

Methods:  Microparticles were isolated from leukoreduced, platelet-depleted, stored murine erythrocytes via ultracentrifugation. These microparticles were added to microplate wells supplemented with Factor Xa-Va mixture, calcium, thrombin inhibitors, and purified prothrombin and the activity of the generated thrombin substrate was measured. We also investigated the effects of microparticle transfusion on the murine pulmonary vasculature.  In separate experiments, mice were transfused with erythrocyte-derived microparticles or an equivalent volume of vehicle. After 1, 4, 8, or 24 hours, lungs were harvested and analyzed under light microscopy or Martius Scarlet Blue (MSB) staining. Blood was harvested from a separate set of mice following injection with either microparticles or saline vehicle and tested for fibrinogen levels.

Results: Erythrocyte-derived microparticles demonstrated a nearly hundredfold greater conversion of prothrombin to thrombin than controls (66.60±0.03 vs 0.70±0.01 peak OD; p<0.0001). Fibrinogen levels were significantly lower in mice injected with microparticles compared with saline vehicle (14.0 vs 16.5 μg/mL, p<0.05), suggesting conversion of fibrinogen to fibrin. Mice injected with erythrocyte-derived microparticles exhibited a significantly greater number of congested pulmonary vessels at 1 hour (10.0 vs 6.5 vessels/HPF) and 4 hours (10.3 vs 6.4 vessels/HPF) as compared to control mice (p<0.05 each) and elevated pulmonary fibrin deposition at 24 hours as determined by MSB staining (3.5±1.5 vs zero deposits, p<0.0001).

Conclusion: Erythrocyte-derived microparticles directly propagate the conversion of prothrombin to thrombin in our murine model. Microparticle treatment in vivo led to decreased fibrinogen and increased pulmonary vessel congestion and fibrin deposition. Microparticles from aged packed red blood cell units may damage injure recipient lungs through accelerated thrombin conversion. 

 

22.10 Fluorescence Guided Surgery May Enhance Localization of Residual Disease During NSCLC Surgery

Posted on January 25, 2017

J. D. Predina1, A. Newton1, J. Keating1, O. Venegas1, S. Singhal1  1University Of Pennsylvania,Philadelphia, PA, USA

Introduction:  Postoperative cancer recurrences occur in 40% of patients undergoing resection for NSCLC.  Such recurrence rates are partially attributable to limitations in intraoperative tools that assist the surgeon in disease identification.  We hypothesized that intraoperative imaging using a fluorescent imaging agent, 5-aminolevulinic acid (5-ALA), could enhance intraoperative identification of NSCLC cancer deposits that would otherwise be missed.

Methods:  The murine NSCLC line, TC1, was cultured in vitro and exposed 1 mM concentrations of 5-ALA for 0 to 24 hours.  Fluorescence was assessed with flow cytometry.  Next, in vivo feasibility of systemic 5-ALA was tested using a small animal model of NSCLC cancer surgery (n=25).   

Results: In vitro, TC1 exhibited high levels of fluorescence after 2 hours of exposure to 5-ALA.  Additional exposure did not augment signal.  Involving in vivo studies, systemic administration of 5-ALA helped identify the presence of residual tumor cells after surgery in 17/25 resections.  The mean residual tumor size was 1.8mm, with a mean fluorescence signal-to-background ratio (SBR) of 4.1.  Of note, minimal fluorescence was noted within thoracic structures including lung parenchyma, pericardium and pleura.  

Conclusion: Systemic 5-ALA reliably accumulates in murine models of NSCLC and helps identify residual disease deposits.  This data supports additional pre-clinical studies which will set the basis for a Human Trial utilizing 5-ALA to enhance NSCLC resection.  

 

22.09 Effect of collagen substrates on oxidative stress-induced changes in SMC phenotype in BAV aortopathy.

Posted on January 25, 2017

P. G. Chan1, M. Billaud1, J. Phillippi1, T. Gleason1  1University Of Pittsburgh,Cardiothoracic Surgery,Pittsburgh, PA, USA

Introduction: Bicuspid aortic valve (BAV) is the most common congenital cardiac malformation and is associated with ascending aortopathy in form of dissection or aneurysmal disease which involve extensive collagen remodeling.  Our prior studies have uncovered that the smooth muscle cells (SMC) from the ascending aorta of BAV specimens have decreased oxidative stress defense compared to SMCs from patients with tricuspid aortic valve (TAV).  We hypothesize that oxidative stress-induced alterations in SMC phenotype can be alleviated by collagen substrates in TAV SMCs but not in BAV SMCs.

Methods: Aortic tissue was obtained from patients undergoing aortic surgery or heart transplantation with IRB approval and informed patient consent.  SMCs from TAV (n=2) and BAV (n=3) patients were harvested using previously established protocols.  SMCs were seeded at a density of 2.8×103/cm2 on tissue culture polystyrene (TCP) in the presence or absence of type 1 collagen substrate.  SMCs were cultured in the presence or absence of 20µM tert-butyl hydroperoxide (tBHP) and monitored in three random fields per well using phase contrast time-lapse microscopy in a stage-top incubation chamber for up to 12 hours.  Post-imaging, the aspect ratios (long axis/short axis) of cells (>17 cells/well) were measured at period time points as a means to assess SMC phenotype.

Results: Aspect ratio for SMCs isolated decreased in the presence of tBHP from 4.4±0.81 to 2.4±0.17 (p<0.001) for TAV SMCs and from 5.0±0.46 to 2.3±0.52 (p<0.001) for BAV SMCs.  When cultured on Type 1 collagen coated wells, TAV SMCs maintained a high aspect ratio despite exposure to oxidative stress.  TAV SMCs increased in aspect ratio on collagen to 6.2±1.55 which was relatively maintained at 4.8±0.83 (p=0.557) when exposed to tBHP.  The aspect ratio of BAV SMCs cultured on collagen was found to be decreased under tBHP treatment (3.88±0.38 vs. 2.1±0.32, p<.001) (Figure 1).

Conclusion: Oxidative stress was found to alter SMC phenotype by decreasing aspect ratio. Culture on collagen prevented oxidative stress induced alteration for TAV SMCs, but not for BAV SMCs.  These results provide further support that BAV SMCs have impaired basal oxidative stress response compared to TAV SMCs despite modulating the extracellular milieu.  Ongoing work is focused on how the extracellular matrix interplays with oxidative stress mediated effects on SMC function in BAV aortopathy.  Ultimately, with this knowledge, we hope to develop therapeutics for patients who are at risk for aneurysmal progression, delineate the molecular mechanisms associated with BAV aortopathy, and improve rupture risk predictions and recommendations for surgical or medical intervention.

22.08 Role of Cardiac Progenitor Cells in Macrophage Polarization During Fetal Cardiac Regeneration post-MI

Posted on January 25, 2017

C. Zgheib1, M. H. Hodges1, M. Allukian2, J. Xu1, K. L. Spiller3, J. H. Gorman2, R. C. Gorman2, K. W. Liechty1  1Laboratory For Fetal And Regenerative Biology, Department Of Surgery, School Of Medicine, University Of Colorado Denver – Anschutz Medical Campus And Colorado Children’s Hospital,Aurora, COLORADO, USA 2The University Of Pennsylvania School Of Medicine,Surgery,Philadelphia, PA, USA 3Drexel University, School Of Biomedical Engineering,Philadelphia, PA, USA

Introduction: Myocardial infarction (MI) remains the leading cause of death in the US. In contrast to the fibrotic response observed in adults, we have previously shown that the fetal response to MI is regenerative and is associated with increased angiogenesis and decreased inflammation. This cardiac regeneration requires both recruitment of cardiac progenitor cells (CPC) to replace lost myocardium as well as restoration of blood supply to support these recruited cells. Macrophages are also known to contribute to this repair process; however, the contribution of macrophages to tissue repair depends on their phenotype: M1 macrophages are pro-inflammatory and initiate angiogenesis; M2a macrophages are pro-fibrotic and contribute to blood vessel maturation; and M2c macrophages are pro-angiogenic, anti-inflammatory, and contribute to tissue remodeling. However, the relationship between CPCs and macrophages during the regenerative process has yet to be defined. Thus, we propose that CPCs promote fetal cardiac regeneration after MI by regulating the polarization of macrophages.

Methods: To test this hypothesis, 20% apical infarcts were created in fetal sheep by LAD ligation. Subsets of these infarcts were injected with a lentivirus containing the SDF-1α inhibitor (SDFi) transgene or an empty vector control. Hearts were harvested 3 or 30 days following infarction. Real time PCR was used to analyze the expression of macrophage phenotypes markers: IL-1β (M10, CD206 (M2a), and CD163 (M2c). 

Results: Our results revealed a significant upregulation of IL-1β, CD206, and CD163 gene expression in the fetal hearts 3 days post-MI; this upregulation returned to baseline at 30 days. In contrast, fetal hearts treated with SDFi demonstrated a significant increase in the expression of all macrophage markers 30 days post-MI, when compared to both untreated fetal hearts and treated hearts 3 days post-MI. These results suggest an early increase in the expression of M1, M2a, and M2c macrophage markers in the fetal hearts 3 days post-MI, with reversal of this upregulation following myocardial regeneration 30 days post-MI. Blocking CPC recruitment by inhibition of SDF-1α was associated with a persistent elevation in M1, M2a, and M2c gene expression, consistent with prolonged inflammation, fibrosis, and remodeling.

Conclusion: Our results suggest that following MI, CPCs promote fetal cardiac regeneration via the modulation of macrophage phenotype. Strategies to manipulate macrophage phenotype may represent a therapeutic target to promote regeneration in adult hearts after MI.

22.07 Reduced Immunogenicity Observed in Multi-Transgene Heart Xenotransplant Model with Suppressed MCP-1

Posted on January 25, 2017

J. L. Chan1, A. K. Singh1, P. C. Corcoran1, M. L. Thomas2, B. G. Lewis2, K. A. Horvath1, M. M. Mohiuddin1  1National Institutes Of Health,Cardiothoracic Surgery Research Program/National Heart, Lung And Blood Institute,Bethesda, MD, USA 2National Institutes Of Health,Division Of Veterinary Resources/Office Of Research Services,Bethesda, MD, USA

Introduction:
Considerable progress has been achieved in xenotransplantation with genetic engineering and the inclusion of additional humanized transgenes appears to be associated with improved outcomes. Monocyte chemoattractant protein-1 (MCP-1), a potent chemotactic factor, has been implicated in transplant immune interaction and rejection. We report the novel use of six-transgene cardiac xenografts and characterize their impact on MCP-1.

Methods:
Heterotopic cardiac xenotransplantations were performed on baboons with swine donor hearts expressing three-gene modifications (GTKO.CD46.TBM) or six-gene modifications (GTKO.CD46.EPCR.DAF with TFPI.CD47 or CD39.TBM). Standard immunosuppressive therapy was administered. MCP-1 levels were measured by fluorescence intensity (FI) using Luminex assay.

Results:
No significant difference in MCP-1 levels were identified preoperatively. Within 48 hours of transplantation, recipients with six-gene modifications (n=3) had a significant reduction in MCP-1 compared to the three-gene cohort (n=3) (1376FI vs. 1928FI, p=0.03). The six-gene group continued to demonstrate decreased MCP-1 in the immediate 30-day postoperative period (1403FI vs. 1916FI, p=0.02) and remained sustained after three months (1159FI vs. 1761FI, p=0.004). Analysis of additional cytokines (IFNγ, TNFα, IL-6) and transmyocardial biopsy histology paralleled findings of a reduced inflammatory response in six-gene recipients. Elevations of MCP-1 to >10% above baseline were associated with transplant rejection within 30 days (odds ratio: 7.40, p=0.01).

Conclusion:
Expression of additional humanized factors in the six-gene cardiac xenografts, including DAF (complement inhibition), CD47 (cellular immune suppression), and CD39 (effector immune modulation), is associated with suppression of MCP-1 production. Decreased MCP-1 levels observed with use of these expanded multi-transgenic donors may reflect superior inflammatory regulation, reduced immunogenicity, and improved xenotransplantation outcomes.

22.05 Calpain Inhibition Decreases Oxidative Stress Via Mitochondrial Pathways in Ischemic Myocardium

Posted on January 25, 2017

B. A. Potz1, L. A. Scrimgeour1, R. T. Clements1, F. W. Sellke1  1Brown University School Of Medicine,Cardiothoracic Surgery Research,Providence, RI, USA

Introduction: Calpain is an intracellular calcium depedent protease whose activity gets overexpressed in cells during times of stress.  Calpain overexpression has been linked to myocardial ischemic disease and organ dysfunction in patients with metabolic syndrome.  Calpain overexpression is implicated in mitochondrial damage leading to tissue oxidative stress.  The purpose of this study was to investigate the potential ability of calpain inhibition on mitochondrial impairment and oxidative stress in a swine model of chronic coronary ischemia in the setting of metabolic syndrome. 

Methods:   Yorkshire swine were divided into 3 groups, fed a high cholesterol diet for 4 weeks, then underwent surgical placement of an ameroid constrictor to their left circumflex artery. Three weeks later animals received either: no drug, high cholesterol control group (CON; n= 8); a low dose calpain inhibitor (0.12 mg/kg; LCI, n= 9); or high dose calpain inhibitor (0.25 mg/kg; HCI, n= 8).  The diets and CI was continued for 5 weeks then the heart was harvested for analysis.  OxyBlot which measures protein carbonyl content (Billerica, MA) was used to determine oxidative stress.  Western Blot was used to identify protein expression (all data was normalized to GAPDH for loading control).

Results:  Calpain inhibition was associated with decreased oxidative stress compared to the control group in the ischemic tissue. There was no change in oxidative stress between groups in the nonischemic tissue. [Figure 1A] In the ischemic myocardial tissue, calpain inhibition was associated with increased expression of the following mitochondrial proteins compared to the control group: the mitochondrial antioxidant protein superoxide dismutase 1; the electron transport chain protein succinate:quinone oxidoreductase; and  the citric acid cycle protein pyruvate dehydrogenase. There was no change in expression between groups prohibitin 1 (p=0.13) and cytochrome c (p=0.15).  [Figure 1B]

Conclusions:  In the setting of metabolic syndrome, CI improved oxidative stress in the ischemic myocardial tissue and was associated with increased expression of mitochondrial proteins involved in 1) reducing superoxide radicals and 2) promoting the citric acid cycle and the electron transport chain.  Calpain inhibition had no effect on oxidative stress in the non-ischemic myocardium. The results of the present study demonstrate the potential ability of calpain inhibition to improve myocardial oxidative stress and suggests that the mechanism through which this beneficial effect is taking place is through the mitochondria.  

22.04 Macrophages Differentially Regulate Collagen Expression in Fetal versus Adult Cardiac Fibroblasts

Posted on January 25, 2017

M. M. Hodges1, C. Zgheib1, J. Xu1, J. Hu1, K. W. Liechty1  1University Of Colorado Denver,Department Of General Surgery, Laboratory For Fetal And Regenerative Biology,Aurora, CO, USA

Introduction:  Ischemic heart disease remains the leading cause of death worldwide.  Despite tremendous advances in the medical management of ischemic cardiomyopathy, the 5-year mortality rate following a diagnosis of heart failure exceeds 50%. In our novel, ovine model of myocardial regeneration after myocardial infarction (MI), we have previously shown that the fetal response to MI is regenerative, whereas the adult response to MI is reparative and is associated with decreased angiogenesis, increased inflammation, and increased fibrosis. Macrophages have been shown to play key roles in regulation of angiogenesis and inflammation; however, the impact that macrophages have on the development of fibrosis and scar formation after MI has yet to be described. We hypothesize that macrophages differentially regulate gene expression of collagen, matrix metalloproteinases (MMP-2 and MMP-9), and transforming growth factors (TGFβ-1 and TGFβ-3) in fetal and adult cardiac fibroblasts.

Methods: To test this hypothesis, cardiac fibroblasts were isolated from the left ventricle of adult and fetal sheep. After isolation, these cardiac fibroblasts were co-cultured for 24 hours with macrophages from the RAW 264.7 cell line.  Quantitative polymerase chain reaction was used to quantify the gene expression of collagen 1α2, collagen 3α1, MMP-2, MMP-9, TGFβ-1, and TGFβ-3.

Results: When compared to adult cardiac fibroblasts, fetal cardiac fibroblasts have significantly upregulated expression of both col1α2 and col3α1, as well as significantly upregulated expression of MMP-2, MMP-9, TGFβ-1, and TGFβ-3.  After co-culture with macrophages, adult cardiac fibroblasts demonstrated significant upregulation of collagen 1α2, which was associated with a significant upregulation in TGFβ-3 and downregulation in MMP-9 gene expression. Conversely, after co-culture with macrophages, fetal cardiac fibroblasts demonstrate a significant downregulation in both col1α2 and col3α1, as well as a significant downregulation in MMP-2, MMP-9, TGFβ-1, and TGFβ-3. These results suggest a differential response of fetal and adult cardiac fibroblasts to co-culture with macrophages.  

Conclusion: Our results suggest that macrophages may play an integral role in regulating the differential responses of the fetus and adult following MI. Additionally, our results suggest that fetal cardiac fibroblasts and adult cardiac fibroblasts are characterized by baseline phenotypic differences that may further contribute to the regenerative response observed in the fetus after MI, compared to the reparative response observed in the adult after MI. A more in-depth understanding of both the phenotypic differences between adult and fetal cardiac fibroblasts, as well as the varying responses following co-culture with macrophages will enable a more complete understanding of the role macrophages play in regulating fibrosis after MI.

22.03 Role of COX-2 in Microvascular Reactivity of Peripheral Arterioles in Diabetic Patients after CPB

Posted on January 25, 2017

K. Anderson1, J. Feng1, Y. Liu1, A. K. Singh1, A. Ehsan1, F. W. Sellke1  1Rhode Island Hospital,Cardiothoracic Surgery/Surgery/Brown Medical Scholl,Providence, RI, USA

Introduction: Diabetic patients are associated with impaired peripheral microvascular function after cardiopulmonary bypass (CPB) and cardiac surgery.  We hypothesized that upregulation inducible cyclooxygenase 2 (COX-2) contributes to altered microvascular reactivity of peripheral arterioles in diabetic patients undergoing CPB and cardiac surgery.

Methods: Skeletal muscle tissue samples of diabetic (DM) and non-diabetic (ND) patients (n = 5-6/group) undergoing cardiac surgery were harvested before and after CPB.  Peripheral arterioles were dissected from the harvested skeletal muscle tissue samples. The isolated arterioles (80-180µm) were cannulated and pressurized and changes in diameter were measured with video microscopy.  In-vitro relaxation responses of pre-contracted arterioles were examined in the presence of the endothelium-dependent vasodilator bradykinin (10-10 to 10-6M) and in the presence or absence of the selective COX-2 inhibitor NS398 (10-6M). 

Results:The post-CPB protein levels of the inducible COX-2 were increased significantly compared with pre-CPB values in both DM and ND groups (P<0.05), whereas, this increase was higher in DM than that of non-diabetics (P<0.05). In the DM arterioles, not the ND vessels, bradykinin-induced relaxation response was inhibited in the presence of the specific COX-2 inhibitor NS398 at baseline (P<0.05). After CPB, bradykinin-induced relaxation response of the DM and ND arterioles was inhibited in the presence of the specific COX-2 inhibitor NS398, but this effect was more pronounced in the diabetic patients (P<0.05).

Conclusion: Diabetes and CPB are associated with upregulation in COX-2 expression/activation in human peripheral microvasculature. This alteration may lead to altered peripheral microvascular reactivity in diabetic patients undergoing cardiac surgery.

 

22.02 Both HMGB1 and DNA Released from Ischemic Myocardium Are Required to Cause Reperfusion Injury

Posted on January 25, 2017

E. J. Charles1, Y. Tian2, J. H. Mehaffey1, D. Wu1, I. L. Kron1, Z. Yang1  1University Of Virginia,Surgery,Charlottesville, VA, Virgin Islands, U.S. 2Tianjin Medical University General Hospital,Cardiovascular Surgery,Tianjin, , China

Introduction: Damage-associated molecular patterns such as high mobility group box 1 (HMGB1) and mitochondrial DNA (mtDNA) may play critical roles in mediating myocardial ischemia-reperfusion (IR) injury.  We hypothesized that HMGB1 and cell-free mtDNA collectively released from ischemia myocardium would lead to activation of splenic leukocytes and cause reperfusion injury.

Methods: Levels of HMGB1 and cell-free mtDNA were measured in plasma and cardiac perfusate obtained from C57BL6 mice (n=4-8/group) that underwent sham surgery or 10, 20, or 40-minute occlusions of the left coronary artery (LCA) without reperfusion.  Perfusate was obtained via antegrade coronary perfusion with phosphate-buffered saline. Levels were measured using SYTOX® Green florescence and Western blot. Separate C57BL6 mice (n=4-8/group) underwent 20 minutes of LCA occlusion followed by 60 minutes of reperfusion.  Resultant myocardial infarct size was measured with TTC and Phthalo blue staining.  Treated groups received recombinant HMGB1 (0.1μg/g/2μl; rtHMGB1), mtDNA obtained from naive C57BL6 mouse livers (0.5ug/g/2μl; mtDNA), or both rtHMGB1 and mtDNA (H+D) 5 minutes prior to reperfusion via external jugular vein injection.  An additional group of mice underwent splenectomy prior to LCA occlusion and received both rtHMGB1 and mtDNA (H+D+Splx).

Results: Plasma levels of HMGB1 and mtDNA were low and not significantly different between mice undergoing sham or LCA occlusion without reperfusion.  However, cardiac perfusate levels were significantly increased in ischemic hearts after 40 minutes of LCA occlusion (p<0.05 vs. other groups).  Infarct size as a percentage of left ventricular risk region after 40 minutes of LCA occlusion without reperfusion was 23.9±5.4%, compared to 0.0% after 20-minute occlusion (p<0.05). In mice undergoing 20 minutes of LCA occlusion followed by 60 minutes of reperfusion, injection of rtHMGB1 or mtDNA did not exacerbate infarct size compared to control mice (p>0.99).  However, H+D mice had significantly larger infarct size (21.2±4.9%), compared with control, rtHMGB1, and mtDNA (all p<0.01, Figure 1). This increased infarct size was attenuated by splenectomy (H+D+Splx: 5.3±2.1%, p=0.02 vs H+D).  There were no significant differences between groups in size of risk region as a percentage of left ventricular mass.

Conclusion: The release of both HMGB1 and mtDNA together from ischemic myocardium is critical to cause reperfusion injury and leads to increased infarct size.  Blocking the effects HMGB1 and/or mtDNA on splenic leukocyte activation may provide a therapeutic option for attenuating myocardial IR injury.

 

22.01 Divergent Roles of Alveolar Macrophages in Neutrophil-Driven Lung Injury

Posted on January 25, 2017

S. Chiu1, M. Akbarpour1, A. McQuattie-Pimentel2, K. Anekalla2, P. Reyfman2, A. Misharin2, H. Perlman2, G. S. Budinger2, A. Bharat1  1Northwestern University,Surgery,Chicago, IL, USA 2Northwestern University,Medicine,Chicago, IL, USA

Introduction:  Pathogen- and damage- associated molecular patterns (PAMPs and DAMPs) stimulate neutrophil recruitment and initiate injury in the lung. It has been suggested that monocytes recruit neutrophils in response to PAMPs while alveolar macrophages (AM) mediate DAMP-driven lung injury, such as ischemia-reperfusion (IR) following pulmonary transplant. Here, we utilize a novel multi-color flow panel to demonstrate that, contrary to the contemporary paradigm, AM ameliorate neutrophil infiltration following lung transplant, but are responsible for neutrophil recruitment in response to PAMPs. 

Methods:  Intratracheal instillation of lipopolysaccharide (LPS) was used to induce PAMP-driven lung injury. Murine single lung allogeneic transplant utilizing wild-type C57BL/6J and Balb/C was performed to induce DAMP-driven lung injury. Clodronate liposomes were used to deplete alveolar macrophages. EdU was injected at the time of transplant to quantify the number of cells entering S phase and undergoing cell division. Flow cytometry was used to measure cell populations and fluorescence-activated cell sorting to isolate alveolar macrophages. Next Generation RNA Sequencing was utilized to compare the transcriptomes of pre- and post-transplant AM. 

Results: After lung transplant or intratracheal LPS instillation, there was significant neutrophil infiltration at 24 hours (>3 fold increase, *p<0.001, Figure A&B). Following transplantation, donor AM proliferated in vivo, resulting in serial increase in AM cell counts (1.7 fold increase) and proportion of cells that had undergone cell division (1.6-fold increase). The increase in cell count was not due to infiltration by recipient AM, since all AM in the allograft remained of donor origin. Transcriptional profiling revealed that post-transplant AM upregulated genes involved in cell division and proliferation. However, genes responsible for activation of innate immunity, neutrophil recruitment, and pattern recognition receptor response were downregulated (Figure A). Depletion of AM in donor lungs prior to transplant lead to increased neutrophil infiltration following IR (Intact AM: 1.1 x 10^6 vs. Depleted AM: 1.5 x10^6, Figure A). In contrast, the robust neutrophil recruitment after intratracheal LPS was abrogated by depletion of AM (*p<0.001, Figure B).

Conclusion: Alveolar macrophages proliferate following lung transplant and downregulate genes involved in immune activation. Hence, they may play a role in ameliorating IR injury. Contrastingly, they augment the immune response and recruit neutrophils in response to PAMPs, demonstrating their role in pulmonary mucosal immunity.

 

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

Posted on January 25, 2017

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

Introduction:

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

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

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

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

21.07 Evaluating the Efficacy of Different Types of Stem Cells in Preserving Gut Barrier Function in NEC

Posted on January 25, 2017

C. McCulloh1, J. Olson1, Y. Wang1, J. Vu1, S. Gartner1, G. E. Besner1  1Nationwide Children’s Hospital,Columbus, OH, USA

Introduction:
Necrotizing enterocolitis (NEC) is a leading cause of gastrointestinal morbidity and mortality in premature infants.  Central to NEC is the increased permeability of intestinal mucosa, leading to bacterial translocation and fulminant infection.  We have previously shown that administration of stem cells (SC) can reduce the incidence and severity of NEC, however no study has compared the ability of different stem cells to restore mucosal integrity.   Our goal was to investigate the efficacy of four different types of stem cells in preservation of gut barrier function during NEC.

Methods:
The following types of stem cells were compared: (1) amniotic fluid-derived mesenchymal SC (AF-MSC); (2) bone marrow-derived mesenchymal SC (BM-MSC); (3) amniotic fluid-derived neural SC (AF-NSC); and (4) enteric neural SC (E-NSC).  SC lines were derived from Lewis rats, cultured, and verified via flow cytometry.  MSC multipotency was confirmed via adipogenic and osteogenic differentiation.  Sprague Dawley rat pups were delivered prematurely via C-section.  Each pup received an intraperitoneal (IP) injection of 2×106 SC in PBS.  Control pups received the same volume of PBS IP.  Pups were subjected to repeated stress (hypoxia and hypothermia three times daily, hypercaloric gavage feeds every 4h, and a single enteral dose of LPS on day one) to induce experimental NEC.  Control pups were breastfed and not subjected to experimental NEC. After 48h all animals received a single enteral dose of fluorescein isothiocyanante-labeled dextran (FD70, molecular weight 70,000, 750mg/ml) and sacrificed 4h later.  Serum was collected and the concentration of FD70 measured in a fluorescent plate reader with filter cutoffs 492/518nm.

Results:
Compared to breastfed, unstressed pups which had intact gut barrier function and normal intestinal permeability (serum FD70 concentration 2.22 ± 0.271 µg/mL), pups exposed to experimental NEC that received PBS had impaired gut barrier function with significantly increased intestinal permeability (18.6±4.25 µg/mL, p = 0.047) (Figure 1).  Compared to treatment with PBS, pups treated with SC had significantly reduced intestinal permeability: AF-MSC (9.45 ± 1.36 µg/mL, p = 0.017); BM-MSC (6.73 ± 2.74 µg/mL, p = 0.049); AF-NSC (8.052 ± 1.31 µg/mL, p = 0.0496); and E-NSC (6.60 ± 1.46 µg/mL, p = 0.033).  Values are shown as mean ± SEM.

Conclusion:
Stem cells improve gut barrier function in experimental NEC. Although all four types of stem cells reduce permeability equivalently, stem cells derived from amniotic fluid (AF) may be preferable due to the availability of AF at delivery and the ease of culture expansion, enhancing the potential for clinical translation.

21.05 H2S Improves Intestinal Perfusion and Integrity After Ischemia by Nitric Oxide Dependent Pathways

Posted on January 25, 2017

A. Jensen1, N. Drucker1, S. Khaneki1, T. Markel1  1Indiana University School Of Medicine,Surgery,Indianapolis, IN, USA

Introduction: Hydrogen Sulfide (H2S) is an endogenous gasotransmitter that has recently been found to exert beneficial vasodilatory properties. Therefore, hydrogen sulfide may be a novel therapeutic option in the setting of intestinal ischemia and reperfusion injury (I/R). We hypothesized that: 1) H2S would improve post-ischemic mesenteric perfusion and preserve intestinal histological architecture compared to vehicle following intestinal I/R, and 2) the benefits of H2S therapy would be mediated through nitric oxide dependent pathways.

Methods: Adult male C57Bl6J wild type (WT) and eNOS KO (eNOS KO) mice were anesthetized with isoflurane and a midline laparotomy was performed. The intestines were eviscerated, the small bowel mesenteric root identified, and baseline intestinal perfusion was determined using Laser Doppler Imaging. Intestinal ischemia was established by temporarily occluding the superior mesenteric artery for 60 minutes with a non-crushing vascular clamp. Following ischemia, the clamp was removed and the intestines were allowed to recover. Prior to abdominal closure, 250ul of 200nM sodium hydrosulfide (NaHS; an H2S donor) or 250ul of PBS vehicle was injected into the peritoneum.  Animals were allowed to recover for 24 hours after which time they were reanesthetized and their mesenteric perfusion reassessed. Perfusion was expressed as percentage of baseline. Following perfusion analysis, animals were sacrificed and intestines explanted and preserved in 4% paraformaldehyde. Intestinal segments were paraffin embedded, sectioned, and stained with H&E.  Sections were then graded based on a previously reported histologic injury scale.  Perfusion and histology scores were compared using Mann-Whitney test. P-values less than 0.05 were significant. 

Results:Hydrogen sulfide improved mesenteric perfusion (WT I/R + NaHS 71.0±6.6%, WT I/R + Vehicle 25.6±6.0%, p<0.01 (A)) following intestinal ischemia and reperfusion injury. In the setting of eNOS ablation, there was no improvement in mesenteric perfusion (eNOS KO I/R + 200nM NaHS 24.7±5.4%, eNOS KO I/R + Vehicle 32.5±8.1%, p=0.59, (A)). Histology injury scores were markedly improved in hydrogen sulfide treated WT animals (WT I/R + 200nM NaHS  1.1±0.3 vs. WT I/R + Vehicle 3.5±0.4, p<0.01(B)). Additionally there was no improvement in histology injury scores in the setting of eNOS ablation (eNOS I/R + 200nM NaHS 2.7±0.2 vs. eNOS KO I/R + Vehicle 2.7±0.3, p=0.84(B)). 

Conclusion:The use of hydrogen sulfide following intestinal I/R improves mesenteric perfusion and intestinal histology.  The benefits of H2S therapy appear to be mediated through nitric oxide dependent pathways.  Further studies are needed to define what other downstream mediators may be involved with hydrogen sulfide therapy prior to widespread clinical implementation.   

 

21.04 Protease Tailored Flourogenic Substrates Outperform CEA in Identifying Mucinous Pancreatic Cysts

Posted on January 25, 2017

D. A. Dominguez1, S. Ivry3, S. Hatcher3, E. Gilbert2, S. Kumar2, W. Park6, M. Schmidt7, R. Brand4, A. O’Donoghue5, K. Kirkwood2, C. Craik3  1UCSF East Bay,Department Of General Surgery,Oakland, CA, USA 2UCSF,Department Of General Surgery,San Francisco, CA, USA 3UCSF,Department Of Pharmaceutical Chemistry,San Francisco, CA, USA 4University Of Pittsburgh,Department Of Gastroenterology,Pittsburgh, PA, USA 5UCSD,Department Of Pharmacy And Pharmaceutical Sciences,San Diego, CA, USA 6Stanford University,Department Of Gastroenterology,Palo Alto, CA, USA 7Indiana University,Department Of Surgery,Indianapolis, IN, USA

Introduction:  Risk stratification of pancreatic cystic lesions remains an area of great clinical uncertainty.  Measurement of cyst fluid CEA is used to predict which cysts are mucinous, and therefore may have malignant potential, but its accuracy limits its usefulness.  Due to this lack of definitive pre-operative characterization, some patients will die from undiagnosed cancers, while others undergo unnecessary pancreatic resections with significant morbidity.  Dysregulation of protease expression and activity has been previously reported in mucinous pancreatic cyst fluid.  We tested the hypothesis that differences in proteolytic activity between mucinous (MUC) and non-mucinous (NON-MUC) cysts could be used to improve our ability to identify pre-malignant pancreatic tumors.

Methods:  We first analyzed cyst fluid from a cohort of human pancreatic neoplasms (MUC, n=16; NON-MUC, n=7) using multiplex substrate-profiling by mass spectrometry (MSP-MS), which is an unbiased, comprehensive technology for analyzing patterns of proteolytic activity. We found that aspartyl protease activity was unique to MUC pancreatic cysts. Next, using shotgun proteomic analysis, we identified 2 proteases (Prot1, Prot2), that were selectively expressed in MUC cyst fluid.  Specific cleavage profiles were used to design a selective fluorescent substrate for each protease. Substrates were then used to assess the activity of each protease in a larger patient cohort (MUC, n=71; NON-MUC, n=39); ROC curves were generated for each substrate.  Performance was compared with CEA values (CLIA lab) using >= 192 ng/mL as a cut off for MUC cysts. 

Results:  Receiver operator characteristic (ROC) curves for Prot1 and Prot2 exhibited an area under the curve (AUC) of 0.98 and 0.82, respectively. Prot1 demonstrated a sensitivity of 93% and specificity of 100%.  Prot2 had a sensitivity of 70% and a specificity of 92%.  Testing required < 0.2 mL cyst fluid.  CEA had an AUC of 0.86, and a sensitivity and specificity of 65% and 94%, respectively.

Conclusion:  MSP-MS is a powerful technology to examine dysregulated proteolysis in complex biological fluids.  The resultant fluorogenic substrates outperformed CEA, the highest of which had a sensitivity of 93% and a specificity of 100%, for the detection of MUC pancreatic cysts.  Our fluorescent assay has the potential to be a rapid, inexpensive, and highly accurate predictor of MUC pancreatic cysts. 

 

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