25.09 Palmitate Regulates JMJD3 Epigenetic Alterations in Diabetic Macrophages

Posted on January 18, 2018

F. M. Davis1, A. Kimball1, A. Joshi1, A. Boniakowski1, S. Kunkel2, K. Gallagher1  2University Of Michigan,Pathology,Ann Arbor, MI, USA 1University Of Michigan,Surgery,Ann Arbor, MI, USA

Introduction: Macrophage (Mφ) plasticity allows for transition of Mφs from an inflammatory to a reparative phenotype and is critical for normal wound healing.  In pathologic conditions, such as type 2 diabetes (T2D), wounds fail to heal due to impaired resolution of inflammation. Although the mechanism(s) responsible for the persistent inflammatory Mφ phenotype in T2D wounds are unknown, growing evidence indicates that saturated fatty acids (SFAs) drive Mφ-mediated inflammation, possibly through an epigenetic mechanism. Given the excess SFAs in T2D, we hypothesized that SFAs induce epigenetic alterations in wound-Mφs and drive the inflammatory-Mφ phenotype in diabetic wound healing.

Methods:  Using our murine model of wound healing, bone marrow-derived macrophages (BMDMs) from control or diet-induced obese (DIO) mice (a mouse model of obesity and T2D) were cultured in standard fashion.  BMDMs were stimulated with either LPS (100 ng/mL), control or DIO serum, or palmitate (100 μM).  Expression of inflammatory genes (IL1β, IL12, TNFα) and JMJD3, an epigenetic enzyme known to control Mφ phenotype, were determined by qPCR. 

Results:  Control BMDMs incubated with DIO serum displayed a hyperinflammatory response to LPS suggesting that mediators in the diabetic serum may drive Mφs toward a proinflammatory phenotype.  Since SFAs are increased in T2Ds, with palmitate being the most abundant fatty acid, we stimulated BMDMs from DIO mice with palmitate and found significant increase in IL1β expression compared to controls (p<0.05).  Importantly, stimulation of BMDMs with palmitate was found to drive expression of the epigenetic enzyme, JMJD3, in the DIO setting (p<0.05).

Conclusion: In conclusion, these studies suggest that the diabetic milieu, specifically increased levels of the SFA palmitate, induces expression of the JMJD3 epigenetic enzyme in Mφs that controls inflammatory gene expression and cell function. 

25.07 Platelet Reactivity in Chronic Venous Insufficiency Patients Locally Enhanced in Incompetent Veins

Posted on January 18, 2018

Z. Z. Young1, D. Mix1, S. Toth1, R. Schmidt1, J. Ellis1, R. Glocker1, A. Doyle1, K. Raman1, M. Stoner1, S. Cameron1  1University Of Rochester Medical Center – Strong Memorial Hospital,Division Of Vascular Surgery,Rochester, NY, USA

Introduction:

Chronic venous insufficiency (CVI) is a major public health concern accounting for thousands of hospital and primary care encounters annually in the U.S. CVI causes leg pain, swelling, wounds, and has been linked to deep vein thrombosis (DVT). The etiology of CVI is incompletely understood but may involve alterations in endothelial cell and platelet function at regions of venous reflux where valves become incompetent. Our hypothesis is that platelet reactivity in CVI is altered locally in incompetent veins and globally in response to reflux which may be a risk factor for myocardial infarction (MI) and cerebrovascular accidents (CVA).

Methods:

Purified platelets (platelet rich plasma) were obtained from venous blood from control antecubital (AC) veins, CVI patient AC veins, CVI patient saphenous veins (SV) and platelet function assessed through the three platelet receptors for which prescription anti-platelet medications exist: Protease Activated Receptor (PAR1) (vorapaxar), Thromboxane (aspirin – indirectly), and P2Y12 (Plavix). The three receptor agonists used were: TRAP (PAR1), U46619 (Thromboxane), and ADP (P2Y12). Platelet activation was assessed by Fluorescence-activated cell sorting (FACS, P-selectin expression, Mean Fluorescence Intensity (MFI) SEM, performed in quadruplicate in each group).

Results:

Isolated, washed platelets from healthy volunteer subject antecubital veins (n=6), CVI patient antecubital veins (n=4) or incompetent saphenous veins (n=4) were included in analysis. Platelet reactivity in CVI patients is generally suppressed in the circulation, but enhanced locally in the incompetent vein (Figure 1). All three activation pathways were significantly different when comparing the CVI AC vein with CVI incompetent SV and Control AC veins (p<0.01). 

Conclusion:

Mechanical reflux of platelets locally may lead to enhanced activation and portend a risk for superficial vein thrombosis and further vein remodeling and potential progression to DVT. However, suppressed platelet function in the general circulation suggests a paradoxical and unrecognized compensatory mechanism, perhaps conferring a reduced risk of MI or stroke in select CVI patients.
 

25.08 Regulation of Neointimal Hyperplasia by the Short Chain Fatty Acid Butyrate

Posted on January 18, 2018

M. Nooromid1, L. Xiong1, K. Wun1, T. Jiang1, E. Chen1, O. Eskandari1, K. J. Ho1  1Northwestern University,Surgery,Chicago, IL, USA

Introduction:  The short chain fatty acids (SCFA) acetate, propionate and butyrate are produced primarily by the gut microbiome from metabolism of dietary fiber. SCFA serve as a source of energy but also act as signaling molecules. Our prior work in a rat model has demonstrated that butyrate potentially ameliorates neointimal hyperplasia development after arterial injury. To expand our understanding of how butyrate modulates neointimal hyperplasia, we utilized a mouse model of carotid artery ligation, dietary supplementation with butyrate, and knockout mice lacking free fatty acid 3 (FFAR3), a G protein-coupled receptor activated by butyrate. 

Methods:  21-week-old male C57BL6 mice were given drinking water supplemented with butyrate (.5 mg/mL) or control water for four weeks prior to undergoing left carotid artery ligation. Serum butyrate concentration was assessed by gas chromatography. Four weeks later, mice were sacrificed and bilateral carotid arteries were harvested for morphometric analysis. A separate cohort FFAR3 knockout mice also underwent left carotid artery ligation and similar morphometric vessel analysis. 

Results: Post-ligation common carotid arteries from butyrate-treated mice developed significantly less neointimal hyperplasia development than control-treated mice (neointima area .047 ± .008 mm2 control vs. .019 ± .005 mm2  butyrate; P=.03), which correlated inversely with changes in serum butyrate levels (2.3 ± .63 mg/mL control vs. 1.2 ± .12 mg/mL butyrate; P<.001). Interestingly, FFAR3 knockout mice demonstrated significantly decreased neointimal hyperplasia compared to wild-type mice (P=.03). 

Conclusion: Butyrate has a protective effect on neointimal hyperplasia development after carotid ligation, but the role of FFAR3 on mediating this effect is unclear and likely complex. Future studies will focus on validating these findings in alternative murine models and exploring FFAR3 signaling in ex vivo studies. 

 

25.06 Systemic Delivery of Stem Cells to Atherosclerotic Lesions using Novel Nanocarrier in ApoE-/- Mice

Posted on January 18, 2018

Z. Liu1, M. M. Regueiro1, L. Zhang1, Y. Li1, S. Joel2, G. S. O’Connor2, S. Deo2, S. Daunert2, O. C. Velazquez1  1University Of Miami,Department Of Surgery, School Of Medicine,Miami, FL, USA 2University Of Miami,Department Of Biochemistry & Molecular Biology, School Of Medicine,Miami, FL, USA

Introduction:  Atherosclerosis is an inflammatory disease. Intercellular adhesion molecule-1 (ICAM-1) is induced on the surface of inflamed endothelial cells (iEC) in atherosclerosis. Integrin LFA-1 associates with ICAM-1, and the I-domain of LFA-1 (idLFA-1) mediates binding to ICAM-1. idLFA-1/ICAM-1 pair is thus an attractive target for mediating homing of therapeutic cells to atherosclerotic lesions where iEC are presented. In this study, we tested a systemic targeted cell delivery method by coating stem cell surface with novel nanocarrier composed of idLFA-1-dendrimer complex. The nanocarriers guide the coated stem cells homing to atherosclerotic lesion via molecular recognition and association with ICAM-1 highly expressed on iEC in aorta of ApoE-/- mice. The efficiency of novel nanocarriers in mediating interaction of nanocarrier-coated cells to iEC in vitro and home to inflamed aorta in vivo was investigated.

Methods:  Expression of ICAM-1 in iEC at aortic atherosclerotic lesion was examined by immunofluorescence (IF). Binding activity of recombinant human idLFA-1 to ICAM-1 was validated by testing association of Cy5-conjugated idLFA-1 with human aorta endothelial cells (HAEC) which pre-engineered to express high ICAM-1 (ICAM-1hi). Nanocarriers were created by conjugation of idLFA-1 or BSA (control) with acetylated generation 5 (Ac-G5)-dendrimers. DsRed+ human endothelial progenitor cells (EPC) were coated with idLFA-1-nanocarriers and BSA-nanocarriers, respectively. The efficiency of idLFA-1-nanocarriers in mediating interaction of DsRed+EPC to ICAM-1hi vs ICAM-1lo HAEC was tested using in vitro cell-cell binding assay. For in vivo testing, 1 x 106 luciferase (Luc)+ murine bone marrow-derived mesenchymal stem cells (MSC) coated with idLFA-1-nanocarriers or murine albumin-nanocarriers were infused twice via tail vein (i.v.) into ApoE-/- mice fed with high fat diet (HFD) (n=5/group). Aortas were harvested next day post the 2nd cell infusion. MSC homed to aorta were examined by IF using anti-Luc antibody and quantified based on fluorescence intensity in aorta sections.

Results: ICAM-1 is elevated in iEC at aortic atherosclerotic lesions of ApoE-/- mice. In vitro, Cy5-conjugated idLFA-1 preferentially binds to “inflamed” HAEC compared to control HAEC (p<0.01). idLFA-1-nanacarriers mediate increased interaction of DsRed+EPC to ICAM-1hi HAEC compared to ICAM-1lo HAEC  (>2 fold,  p<0.01). In vivo, idLFA-1-nanacarriers can successfully deliver more MSC to aorta in HFD fed ApoE-/- mice than control nanocarriers (>5 fold, p=0.03).

Conclusion: We demonstrated higher efficiency of idLFA-1-nanocarriers in delivering MSC to inflamed aorta in ApoE-/- mice. This work establishes a novel method for systemic cell delivery to inflamed aorta in animal model and paves way towards developing cell therapies for treatment of atherosclerosis and maybe other inflammatory diseases. 

 

25.05 TGFβ -1 is Differentially Expressed in Adult and Fetal Hearts After Myocardial Infarction

Posted on January 18, 2018

M. M. Hodges1, C. Zgheib1, J. Xu1, L. C. Dewberry1, S. A. Hilton1, J. Hu1, K. W. Liechty1  1University Of Colorado-Denver, Anschutz Medical Campus,Department Of General Surgery, Laboratory For Fetal And Regenerative Biology,Aurora, COLORADO, USA

Introduction: The fetal response to myocardial infarction (MI) is regenerative, whereas the adult response to MI is reparative and associated with increased fibrosis. Global gene expression analysis of ovine specific microarray demonstrated significant increase in extracellular matrix (ECM) related gene expression after MI in adult versus fetal ovine hearts. Transforming growth factor beta-1 (TGFB1) is a key regulator of ECM related gene expression in renal and pulmonary fibrosis; however, the expression of TGFB1 in the fetal response to MI has yet to be described. We hypothesize that expression of TGFB1 is downregulated in the regenerative, fetal model of myocardial regeneration following MI when compared to the reparative and fibrotic, adult response to MI.

Methods:  Myocardial infarction (MI) was induced in fetal (65-76 days gestation, n=19) and adult (7 months old, n=8) sheep via ligation of the left anterior descending coronary artery. Ovine specific microarray was used to evaluate expression of TGFB1 and related genes (collagen I, collagen III, elastin, and miR-21) in adult and fetal hearts, 3 and 30 days after MI. Gene expression in the infarct area (IA) was compared to gene expression in the remote zone. Student’s t-test was used for statistical comparisons, with p<0.05 considered statistically significant.

Results: Both 3 and 30 days after MI, TGFB1 gene expression was significantly upregulated in the IA of adult hearts (p=0.036 and p=0.003); however, there was no significant change in TGFB1 gene expression in the IA of fetal hearts. At 3 days, the increased TGFB1 gene expression in the IA of adult hearts was associated with a significant increase in collagen I (p=0.009), collagen III (p=0.0003), elastin (p=0.045), and miR-21 (p=0.009) gene expression.   While there was a significant increase in collagen III expression in the IA of fetal hearts 3 days after MI (p=0.008), there was no significant difference in the expression of collagen I, and there was a significant reduction in the expression of elastin (p=0.04). MiR-21 was undetectable in fetal hearts. The gene expression of collagen I (p=0.014), collagen III (p=0.047), elastin (p=0.027), and miR-21 (p=0.039) remained elevated in adult IAs 30 days after MI.  However, there was no significant difference in collagen I, collagen III, or elastin gene expression in the fetal IA 30 days after MI.

Conclusion: TGFB1 has been described as a master regulator of the fibrotic response in adult hearts after MI. This is the first evaluation of TGFB1 expression in the regenerative, fetal response to MI. We have shown that TGFB1 expression is unchanged in the regenerative, fetal response to MI in contrast to the upregulation of TGFB1 seen in the adult response. While safe and effective therapies directed at reducing fibrosis after MI by decreasingTGFB1 are lacking, these results indicate that promising therapies may be developed by continuing to pursue TGFB1 as a therapeutic target.

 

25.03 Flomax® (Tamsulosin) Attenuates Abdominal Aortic Aneurysm Growth

Posted on January 18, 2018

W. Montgomery1, M. Spinosa1, J. Cullen1, M. Salmon1, G. Su1, T. Hassinger1, A. Sharma1, G. Lu1, A. Fashandi1, G. Ailawadi1, G. R. Upchurch1  1University Of Virginia,Surgery,Charlottesville, VA, USA

Introduction:
Flomax® (Tamsulosin) is traditionally prescribed to treat benign prostatic hyperplasia (BPH) in men over the age of 50 years old, the same demographic most susceptible to abdominal aortic aneurysm (AAA) formation. The goal of this study was to investigate the effect of Flomax® on AAA pathogenesis, as it was hypothesized that Flomax® would attenuate AAA growth.

Methods:
AAAs were induced in 8-12 week old C57BL/6 (Wild type: n=9-18/group) using a previously established topical elastase AAA model.  Osmotic pumps were implanted in each mouse 5 days prior to surgery to administer either a low dose (0.125 µg/day tamsulosin), high dose (0.250 µg/day tamsulosin), or vehicle treatments both with and without topical elastase application during AAA surgery.  Blood pressure in each group was measured preoperatively, and on postoperative days 7 and 14.  On postoperative day 14, aortic diameter was measured prior to harvest. Sample aorta were prepared for histology and cytokine analysis. Data was analyzed statistically using Fisher’s exact test or chi-squared test as appropriate with significance set to α<0.05.

Results:
Systolic blood pressure measurements were not significantly different preoperatively, on day 7 or 14 for all groups.  Mice treated with both low and high-dose of tamsulosin showed significantly decreased aortic diameter compared to the vehicle-treated control mice (p<0.001). Cytokine analysis demonstrated a significant down-regulation in pro-inflammatory cytokines (TNF-α, IL-1β, IL-7, IL-17, CXCL2, and IFN-γ) in both treatment groups compared with control (p<0.05). Histology exhibited a significant preservation of elastin integrity in both low and high-dose tamsulosin-treated groups (p=0.0041 and p=0.0018, respectively).

Conclusion:
Flomax®, an alpha 1 adrenergic receptor inhibitor, which is used commonly in BPH and affects smooth muscle cell vasorelaxation, attenuates AAA formation in association with increased elastin preservation and decreased pro-inflammatory cytokine production.  Further research is required to elucidate the mechanism by which tamsulosin inhibits AAA growth.  Flomax® (Tamsulosin) could represent a potential therapeutic medical treatment strategy for AAA disease.
 

25.04 NGF Upregulation by Diazoxide and Erythropoietin treatment attenuates Spinal Cord Ischemic Injury

Posted on January 18, 2018

K. Yamanaka1, M. Eldeiry1, M. Aftab1, J. Mares1, X. Meng1, M. J. Weyant1, J. C. Cleveland1, D. A. Fullerton1, T. B. Reece1  1University Of Colorado,The Department Of Surgery, Division Of Cardiothoracic Surgery,Aurora, COLORADO, USA

Introduction:
Paraplegia remains the most significant complication of thoracoabdominal aortic intervention. Despite the evolution of surgical adjuncts to protect the spinal cord, the optimal pharmacotherapy hasn’t been established yet. We previously reported that Diazoxide (DZ), potassium ATP-sensitive channels opener, upregulates the beta-common receptor subunit (βcR) of the erythropoietin (EPO) receptor, and enhances the neuroprotective efficacy of EPO through the upregulation of βcR. The aim of this study is to show the different mechanism of the combined treatment with DZ and EPO. We hypothesized that combined treatment with DZ and EPO attenuates spinal cord ischemia and reperfusion injury (SCIRI) though upregulation of Nerve Growth Factor (NGF).                    

Methods:
 DZ (pretreatment) was given to adult male C57/BL6 mice by oral gavage and EPO (before surgery) was intraperitoneally injected 32 hours after administration of DZ. Spinal cords were harvested after 0, 2, 4, 6 hours after injection of EPO. The expression of NGF was assessed by western blot analysis. After determining the optimal time, NGF expression was compared between DZ (pretreatment)+EPO (before surgery), DZ+PBS, PBS+EPO, PBS+PBS (ischemic control) at this optimal time. Five groups were studied: DZ+EPO (n=11), ischemic control (n=7), DZ+EPO+TrkA (NGF receptor) inhibitor (n=7), TrkA inhibitor (ischemic control + TrkA Inhibitor, n=7), and sham (without crossclamping, n=4). Spinal cord ischemia was induced by 4-minutes thoracic aortic cross-clamp. Functional scoring (Basso Mouse Score: BMS) was done at 12-hour intervals until 48 hours, and spinal cords were harvested for the evaluation of NGF expression and histological changes.

Results:
The expression of NGF was significantly upregulated 4 hours after administration of EPO. NGF expression in DZ+EPO 4 hours after administration of EPO was significantly higher than DZ+PBS, PBS+EPO and PBS+PBS. Combined treatment with DZ and EPO significantly preserved the motor function comparing all other groups. There was no significant difference between BMS of DZ+EPO+TrkA inhibitor and that of ischemic control. The level of NGF expression in mice with DZ and EPO, 48 hours after reperfusion, was significantly higher comparing with all other groups.

Conclusion:
Combined treatment with DZ and EPO attenuates SCIRI through upregulation of NGF. Better understanding of this mechanism may serve to further prevent ischemic complications for aortic intervention.
 

25.01 Human Venous Valve Disease Caused By Mutations In FOXC2 And GJC2

Posted on January 18, 2018

O. Lyons1, P. Saha1, C. Seet1, A. Kuchta2, A. Arnold2, S. Grover4, V. Rashbrook1, A. Sabine5, G. Vizcay-Barrena3, A. Patel1, F. Ludwinski1, S. Padayachee2, T. Kume6, B. Kwak7, G. Brice8, S. Mansour8, P. Ostergaard9, P. Mortimer9, S. Jeffery9, N. Brown10, T. Makinen11, T. Petrova5, B. Modarai1, A. Smith1  1King’s College London,Academic Department Of Surgery, Cardiovascular Division, BHF Centre Of Research Excellence,London, LONDON, United Kingdom 2Guy’s & St Thomas’ NHS Foundation Trust,Ultrasonic Angiology,London, LONDON, United Kingdom 3King’s College London,Centre For Ultrastructural Imaging,London, LONDON, United Kingdom 4Division Of Hemostasis And Thrombosis,Beth Israel Deaconess Medical Centre,Boston, MA, USA 5Ludwig Institute For Cancer Research And Division Of Experimental Pathology,Department Of Fundamental Oncology, Centre Hospitalier Universitaire Vaudois And University Of Lausanne, Epalinges, Switzerland,Lausanne, LAUSANNE, Switzerland 6Feinberg Cardiovascular Research Institute,Northwestern University School Of Medicine,Evanston, IL, USA 7Department Of Pathology And Immunology,University Of Geneva,Geneva, GENEVA, Switzerland 8South West Thames Regional Genetics Service,St George’s Hospital,London, LONDON, United Kingdom 9Cardiovascular And Cell Sciences Institute,St George’s Hospital,London, LONDON, United Kingdom 10Institute Of Medical And Biomedical Education,St George’s Hospital,London, LONDON, United Kingdom 11Rudbeck Laboratory,Department Of Immunology, Genetics And Pathology, Uppsala University,Uppsala, UPPSALA, Sweden

Introduction:

Venous valves (VVs) prevent blood reflux that can give rise to chronic venous hypertension and ulceration. Patients with mutations in the genes encoding the transcription factor Foxc2 and gap junction protein, connexin47 (Cx47), have venous reflux. We sought to examine VV phenotypes in these patients and to use a murine model to elucidate the function of these proteins in VV development.

Methods:

Human VV number and length were quantified by ultrasound. Murine VV phenotype (marked by expression of Prox1) was examined using confocal microscopy, in wild-type and mice with complete or conditional deletion of genes expressing transcription factors and connexins.

Results:

Patients with Foxc2/Cx47 mutations had reduced valve number (P<0.0005) and shorter valves (P<0.0005). VV initiation in mice was marked by elongation/reorientation of Prox1hi endothelia by postnatal day 0. Expression of Foxc2 and Nfatc1, and the gap junction proteins, Cx47, Cx43 and Cx37, were temporo-spatially regulated during this process. Combined Foxc2 deletion with calcineurin-NFAT inhibition disrupted endothelial organisation, suggesting co-operative Foxc2-NFATc1 patterning. Deletion/knockout of each of the connexins also disrupted endothelial organisation. Specific deletion of endothelial Foxc2 had no effect on VV maintenance.

Conclusion:

Patients with mutations in Foxc2 and Cx47 have globally reduced VV numbers and shorter VV leaflets. Foxc2 and Nfatc1 likely cooperate to organise the initial ring of VV-forming cells. Connexins are critical for early organisation of valve-forming cells at P0 and failure of this process may underlie abnormal VVs identified in patients with mutated Cx47. Foxc2, in endothelia, is not required for valve maintenance.  

25.02 Pre-Operative Protein/Methionine Restriction Attenuates the Surgical Response to Injury

Posted on January 18, 2018

K. Trocha1,2,3, P. Kip1,3, M. MacArthur2, M. Tao1, J. R. Mitchell2, K. Ozaki1  1Brigham And Women’s Hospital,Vascular Surgery,Boston, MA, USA 2Harvard School Of Public Health,Genetics & Complex Diseases,Boston, MA, USA 3Leiden University Medical Center,Surgery,Leiden, , Netherlands

Introduction: Short-term pre-operative dietary restriction holds promise as a translatable approach to mitigate surgical complications. One of the regulators responsible for these benefits is hydrogen sulfide (H2S), an anti-inflammatory gaseous signaling molecule produced endogenously by the enzyme cystathionine γ-lyase (CGL). Recently we have shown that short-term methionine (MR) and protein restriction (PR) upregulates H2S production reducing ischemia-reperfusion damage and attenuating the surgical stress response, showing great potential for clinical translation. However, short-term restriction of protein or amino acids could potentially impair wound healing in surgical patients, we therefore hypothesized that short-term pre-operative protein restriction (PR) or methionine restriction (MR) would not impair wound healing and by genetically upregulating H2S, mimicking PR and MR that this gaseous transmitter would protect against ischemia reperfusion injury.

Methods:  Surgical outcomes were tested using a bilateral 35-minute renal ischemia-reperfusion model and a Mcfarlane wound healing model, where a pedicle flap measuring 2.5 x 1.25cm with a silicone flap placed inferior to flap to prevent angiogenesis from bellow was performed on the dorsum of the mouse and wound healing was observed for 7 days post operatively. Dietary interventions included 7 days of either an isocaloric protein restricted (0%) diet or 3 weeks of an isocaloric methionine restricted diet. A CGL transgenic overexpressing mouse model (10-12 weeks, C57BL/6 background) of increased endogenous H2S production was used to test the potential of increased endogenous H2S to protect from a severe 35-minute renal ischemia. 

Results: PR mice revealed a trend towards accelerated wound healing, with no difference in percentage of viable skin 7 days after surgery (Figure A). MR mice displayed accelerated wound healing 1-4 days after surgery compared to controls while after 7 days, as with PR mice, no difference in percentage viable skin was observed (Figure B). Also, 75% of CGLtg mice survived at post operative day 3 following bilateral renal ischemia, compared to a 100% mortality of littermate controls (p<0.05,Figure C), thereby elucidating the benefits of H2S upregulation on ischemia-reperfusion damage and survival.

Conclusion: Reducing protein intake or specific amino acids prior to surgery without reducing calories in pre-clinical models protects against ischemia-reperfusion injury and may protect against surgical stress. Improving mortality via the reduction of ischemia reperfusion damage using a CGLtg mouse we elaborate on our previous work that these dietary benefits are largely secondary to upregulation of endogenous H2S. Here we show that such brief dietary-manipulations stand as simple strategies toward improving the host response to surgical injury and they do not inhibit or impair wound healing in mice, thereby further enhancing clinical applicability. 
 

24.08 Mesenchymal Stem Cell Secretome Improves Donor Heart Function Following Ex-vivo Cold Storage

Posted on January 18, 2018

M. Wang1, Q. Li1, Y. Yang1, L. Yan1, M. Turrentine1, I. Wang1  1Indiana University School Of Medicine,Cardiothoracic/Surgery,Indianapolis, IN, USA

Introduction:  Heart transplantation is the only gold standard of treatments for end-stage heart failure, but its use is limited by extreme shortage of donor organs. The current cold organ preservation limits the maximal storage to 4-6 hours, beyond which the ischemia/reperfusion (I/R) injury deteriorates graft and patient outcomes. Such time constraint lowers utilization of donor organs. Amelioration of I/R injury, therefore, will prolong preservation time and potentially increase donor heart utilization. Although mesenchymal stem cell (MSC)-derived paracrine actions are mainly responsible for MSC-mediated cardiac protection, currently, no study has reported using stem cell-derived secretome to mitigate ischemic injury in donor hearts during preservation. We aim to evaluate potential amelioration of I/R-damaged myocardial function by MSC conditioned medium (CM) using in vivo murine heterotopic heart transplantation model.

Methods:  The CM were obtained from cultivation of human bone marrow-MSCs (3X104/cm2) in serum-free media for 72 hours. Donor hearts from C57BL/6 male mice were stored in University of Wisconsin solution at 0-4°C and randomly divided into three groups: 1) <1hr-cold storage/ischemia (control, n=3); 2) 6hr-cold storage/ischemia (6hr-I+vehicle, n=6); and 3) 6hr-cold storage/ischemia + MSC CM (6hr-I+CM, n=5). These preserved mouse hearts were then implanted into C57BL/6 male recipient mice using cervical heterotopic heart transplantation. At 24-hour post implantation, myocardial function (LVDP, heart rate, and +/-dP/dT) was detected in transplanted hearts by Millar pressure catheter. Native heart rate was recorded by ECG and pulse pressure difference of abdominal artery was measured in recipient mice. p<0.05=statistically significant.

Results: Six-hour cold ischemia significantly impaired myocardial function in heterotopically implanted hearts stored with vehicle vs. control: Fig A. prolonged delay in re-beating time (time of the implanted heart resumed heart beat after unclamping the vessel; B. decreased RPP; C. decreased heart rate; D. reduced dP/dt; and E. impaired –dP/dt. The addition of CM as an adjunct to preservation solution reversed detrimental effects of cold ischemia on myocardial function, as shown by restored parameters in Fig. A-E. There were no pulse pressure differences seen in the native hearts among the 3 groups, providing additional experimental control to exclude potential procedural variability. 

Conclusion: Our results represent the first evidence that using MSC CM during ischemic cold storage confers improved myocardial preservation, suggesting protective role of the MSC secretome that may allow optimization of current storage methods to improve organ function and patient recovery.
 

24.09 Pioglitazone Reduces Hepatocarcinogenesis in a Rodent Model of Cirrhosis

Posted on January 18, 2018

S. Li1, S. Ghosal1, G. Arora1, D. J. Erstad1, M. Lanuti2, K. K. Tanabe1, B. Fuchs1  1Massachusetts General Hospital,Surgical Oncology,Boston, MA, USA 2Massachusetts General Hospital,Thoracic Surgery,Boston, MA, USA

Introduction:  Advanced hepatocellular carcinoma (HCC) is a leading cause of mortality worldwide with limited treatment options. There is a readily identifiable cohort of cirrhosis patients at risk and they are ideal candidates for chemoprevention. Anti-hyperglycemic agents have garnered interest for their chemo-preventive effects. Pioglitazone, a selective PPAR-y agonist, has been shown to reduce non-alcoholic steatohepatitis (NASH), but its role as an anti-fibrotic and chemo-preventive agent has yet to be elucidated. The hypothesis of this study is that Pioglitazone reduces cirrhosis and subsequent HCC development in rats with diethylnitrosamine (DEN)-induced cirrhosis.

Methods:  Male Wistar received DEN 50mg/kg by intraperitoneal injection. DEN injury reliably recapitulates human HCC development with induction of hepatic fibrosis at 8 weeks, cirrhosis at 12 weeks, and HCC by 18 weeks. DEN-injured rats were randomized to receive oral gavage of pioglitazone at 3mg/kg/day (n=9) or vehicle control (n=9). Initiation of pioglitazone coincided with the development of liver fibrosis at 8 weeks. All animals were sacrificed at 18 weeks.

Results: As expected, repeated injections of DEN in rats resulted in progressive fibrosis, cirrhosis, followed by HCC formation. Treatment with pioglitazone resulted in a 56% reduction of surface nodules relative to treatment with vehicle (7.4±4.9 vs. 17±7; p<0.005). Pioglitazone treatment resulted primarily in a reduction of nodules<8mm compared to vehicle (6.3±1.5 vs. 15.14±2.5; p<0.001). Liver sections were stained by picrosirius red to assess fibrosis. Pioglitazone significantly reduced collagen deposition in DEN-injured rats (collagen proportional area = 3.2±1.8% vs. 9.2±2%; p<0.035). This histology was confirmed by gene expression analysis with reductions in COL1A1, α-SMA, TGF-β, and TIMP1. Pioglitazone treatment resulted in an upregulation of Adiponectin, which has been shown to antagonize carcinogenesis. Pioglitazone treatment also increased AMPK signaling, a well-recognized target for anti-tumor drug discovery as well as a down regulation of the mitogenic MAPK pathway. Daily pioglitazone dosing signifcantly reduced the gene expression of progenitor cell activation including CD44, RAGE, and DLK1. 

Conclusion: Overall our data supports the hypothesis that the anti-diabetic agent pioglitazone may be repurposed as a drug to reduce fibrosis and prevent HCC. This could be beneficiary in patient management given the low cost as well as minimal side effects.

24.10 Histone Deacetylase Inhibition Mitigates Ischemia/Reperfusion & Acetaminophen-Induced Liver Injury

Posted on January 18, 2018

S. J. Concors1,3, D. R. Murken1, D. D. Aufhauser1, Z. Wang1, G. Ge1, W. W. Hancock2,4, M. H. Levine1,3  1University Of Pennsylvania,Surgery,Philadelphia, PA, USA 2University Of Pennsylvania,Pathology And Laboratory Medicine,Philadelphia, PA, USA 3Children’s Hospital Of Philadelphia,Surgery,Philadelphia, PA, USA 4Children’s Hospital Of Philadelphia,Pathology And Laboratory Medicine,Philadelphia, PA, USA

Introduction: Ischemia/reperfusion injury (IRI) leads to significant morbidity and mortality in liver transplantation. Understanding the physiology of IRI is necessary to develop methods of prevention and treatment. Histone deacetylases (HDACs) regulate diverse cellular processes. We have previously shown protection from renal IRI with HDAC inhibition. We wished to investigate the effects of HDAC inhibition in liver IRI, and if this protective effect is translatable to other mechanisms of liver injury.

Methods: Male wild type C57BL/6 (WT) mice were treated with pan-HDAC inhibitor trichostain A (TSA), class I HDAC inhibitor MS-275, HDAC6 inhibitor Tubastatin-A (TubA), Hsp90 inhibitor 17AAG, or DMSO at 16 and 1 h pre-IRI. Whole-body HDAC-1, -2, -6, and -8 deficient and tamoxifen-treated WT male mice were also used. Mice were subjected to 70% liver ischemia for 60 min under strict temperature control.  For the acetaminophen (APAP) toxicity experiment, WT mice were treated with DMSO or TubA at 16 h and again just prior to administration of a sublethal dose of APAP (500mg/kg). In both experiments, AST and ALT levels were assessed 24 hours after injury, as well as histopathologic examination and Suzuki liver injury scale scoring at 48 hours post injury. 

Results: TSA- and TubA-treated mice developed significantly less hepatocellular injury after liver IRI than controls with significantly lower AST/ALT levels(Fig 1A); Tub-A developed significantly less liver injury on histopathology.  H1-/- mice developed significantly less injury after IRI compared to controls, but HDAC6 gene deletion did not mitigate IRI (Fig 1B).  TubA also mitigated APAP-induced liver injury (Fig 1C).

Conclusion:Pan-HDAC inhibition mitigates liver injury after IRI in mice. Selective inhibition of HDAC1 via inducible gene deletion and pharmacologic inhibition of HDAC6 both replicate this protection. These findings are distinct from the pattern of HDAC involvement in renal IRI and suggest tissue-specific roles for HDACs in IRI response. Pharmacologic inhibition of HDAC6 also mitigated APAP-induced hepatocellular injury suggesting that the protective effects of TubA are not injury-type specific. 

 

24.07 Supplemental Estrogen Mitigates Liver Ischemia/Reperfusion Injury in Male and Female Mice

Posted on January 18, 2018

S. J. Concors1,3, D. R. Murken1, D. D. Aufhauser1, Z. Wang1, G. Ge1, W. W. Hancock2,4, M. H. Levine1,3  1Hospital Of The University Of Pennsylvania,Surgery,Philadelphia, PA, USA 2Hospital Of The University Of Pennsylvania,Pathology And Laboratory Medicine,Philadelphia, PA, USA 3Children’s Hospital Of Philadelphia,Surgery,Philadelphia, PA, USA 4Children’s Hospital Of Philadelphia,Pathology And Laboratory Medicine,Philadelphia, PA, USA

Introduction: Hepatic ischemia/reperfusion injury (IRI) complicates liver transplantation and other surgical scenarios and can lead to early allograft dysfunction and liver failure. A better understanding of the molecular mechanisms of IRI may facilitate development of strategies to prevent and treat IRI. We have previously reported female mice tolerate renal IRI with profound reduction in injury compared to male mice and have demonstrated that supplemental estrogen mitigates renal IRI in female mice. We wished to investigate whether estrogen may provide similar protection in liver IRI.

Methods: 8-12 week old C57BL/6 iM (intact male) and iF (intact female) mice were subjected to 70% liver ischemia for 60 minutes under strict temperature control with assessment of plasma AST/ALT levels at 24 h post-IRI. Male (nM) and female (nF) mice were neutered at 3 wks of age and then subjected to liver IRI at age 8-12 wks. iM, iF, nM, and nF mice were treated with supplemental estradiol (+E2) 16 hours and 1 hour before IRI.  Estrogen receptor alpha knock out (ESRaKO) female mice were also subjected to this liver IRI model.

Results: iF mice displayed a trend towards reduced liver injury after liver IRI compared to iM mice (Fig 1A). nM and nF mice displayed increased liver injury compared to iM and iF mice, respectively (Fig 1B). Supplemental estradiol administration mitigated liver IRI in both genders (Fig 1C).  ESRaKO mice exhibited significantly worse transaminase levels and decreased survival after IRI (Fig 1D).

Conclusion: Neutering mice of either sex leads to impaired tolerance of liver IRI.  Supplemental estradiol has a protective effect on liver IRI in both hormonally intact and neutered M, as well as females.  These findings are distinct from patterns observed in setting of renal IRI, suggesting tissue-specific injury pathways. This has implications for therapeutic intervention as a wide range of hormonal therapies with either estrogen or testosterone, or both, may be useful in the treatment of liver IRI.  

 

24.06 Human Hepatocyte Transplantation Corrects The Metabolic Liver Disorder Arginase Deficiency

Posted on January 18, 2018

S. Angarita1, B. Truong1, M. Lin1, S. Khoja1, A. Lam1, S. Duarte1, G. S. Lipshutz1  1David Geffen School Of Medicine,Surgery,Los Angeles, CALIFORNIA, USA

Introduction: Hepatocytes perform a variety of functions including protein synthesis, detoxification, and biliary excretion. Inherited abnormalities of any one of the multitude of enzymes in the liver can lead to a metabolic disorder. Liver transplantation (LT) can cure patients with liver-based metabolic disorders. However, LT is dependent on the availability of donor livers which are particularly limited for infants and neonates due to organ scarcity and size limitations. We are presently exploring the transplantation of isolated primary hepatocytes as a minimally invasive alternative to LT. In these studies, we demonstrate that human hepatocytes can be used to treat arginase deficiency, an inherited disorder of nitrogen metabolism typically presenting in the first two years of life, by hepatocyte repopulation of the liver in a murine model of the disorder.

Methods: Conditional arginase deficient mice were mated with a mouse model deficient in fumarylacetoacetate hydrolase (FAH) and with Rag2 and IL2-Rγ mutations. The FAH knockout results in a defect in the tyrosine catabolic pathway, resulting in highly reactive toxic metabolites that cause hepatocellular injury. Toxicity can be prevented by oral administration of 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione (NTBC), which blocks the tyrosine pathway upstream. This enzymatic defect provides a selection advantage for transplanted cells. On day -1, a uroplasminogen-expressing adenoviral vector was administered IV. On day 0, human hepatocytes were isolated and 1×106 were administered by intrasplenic injection (n=12). As the initial number of administered hepatocytes was too low to prevent hepatotoxicity-induced mortality, NTBC was added to the drinking water of the mice at regular intervals with eventual withdrawal for 3 weeks at a time. Ninety days after hepatocyte transplantation, 2×1011 genome copies of AAV-TBG-Cre recombinase was administered IV to knock out endogenous arginase expression in hepatocyte-transplanted mice and controls. Survival was followed; amino acids and ammonia were studied 30 days later.

Results: All control mice (n=16) died by day 34 (26.2 ± 3.4) while all except one human hepatocyte transplanted mice survived. Ammonia and amino acids were analyzed in both groups comparing day 60 (30 days before arginase knockout) and day 150 (30 days after AAV-TBG-Cre administration). Ammonia, arginine and glutamine (elevated in urea cycle dysfunction) were well-controlled in the transplanted group, while in the controls they were markedly abnormal.

Conclusion: These studies demonstrate that human hepatocyte repopulation in the murine liver can result in cure of arginase deficiency, and likely is also applicable to other metabolic disorders that are currently treated by LT. With success of these proof of principal studies, we are presently exploring methods that may allow for selective advantage of transplanted hepatocytes in humans with such disorders.

 

24.03 IL-1 Signaling Induces Stemness To Mediate Chemoresistance in Pancreatic Ductal Adenocarcinoma

Posted on January 18, 2018

A. R. Dosch1, C. Roberts1, M. VanSaun1, S. Banerjee1, P. Lamichhane1, A. Gaidarski1, N. Nagathihalli1, D. Dai1, F. Messaggio1, N. B. Merchant1  1University Of Miami,Department Of Surgical Oncology,Miami, FL, USA

Introduction:
Pancreatic cancer (PDAC) remains a major therapeutic challenge due to its innate and acquired chemoresistance. PDAC tumors are heterogeneous entities which containing tumor cells, immune cells, cancer associated fibroblasts (CAF) and cancer stem cells (CSC). Cytokines produced within the tumor microenvironment (TME) are a prominent mechanism for the activation and maintenance of the CSC phenotype. IL-1α and IL-1β are produced by a variety of cells in the TME, including CAFs and inflammatory cells. These molecules are potent upstream mediators of the transcription factor NF-κB which has been shown to increase PDAC CD133+ overexpression, a marker of CSC differentiation. CD133+ cells confer chemoresistance and promote tumor progression through multiple downstream targets implicated in self-renewal, pluripotency, and epithelial-to-mesenchymal transition (EMT). The purpose of this study is to elucidate the molecular regulation of IL-1 signaling that contributes to the CSC phenotype and mediates therapeutic resistance in PDAC.

Methods:
The role of IL-1α and IL-1β signaling was determined in human PDAC cells lines Capan1, MiaPaCa-2, and Panc-1. Total level of IL-1α/β and expression of IL-1 receptor was determined in vivo in Ptf1acre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice and compared with control tissue using cytokine array kit and immunohistochemistry, respectively. ELISA was used to quantify IL-1α/β expression in PDAC cells, inflammatory cells, and CAFs. Expression of target genes related to pluripotency and EMT were quantified using qPCR. Flow cytometry and immunofluorescence were used to delineate changes in CD133 expression in response to IL-1 treatment.

Results:
Our results demonstrate that CAFs secrete greater amounts of IL-1β compared with tumor cells or tumor associated macrophages. Expression of IL-1R was significantly increased in PDAC tumor cells when compared with normal ductal cells in PKT mice. Exogenous IL-1α and IL-1β stimulation activated the ERK1/2 and NF-κB pathways, which correlated with upregulation of pluripotent gene expression including Sox2, Nanog, Oct4, as well as enhanced transcription of the metastatic gene Snai1. Additionally, IL-1 significantly increased percentage of CD133+ cells in PDAC cell lines. Inhibition of the NF-κB or MEK pathway decreased expression of EMT-related genes, CD133+ cells, and decreased cell invasion.

Conclusion:
The inflammatory cytokines IL-1α and IL-1β regulate pluripotent genes essential for EMT and CSC differentiation in the TME. These data show a novel therapeutic potential of targeting IL-1 signaling through MEK inhibition and/or NF-κB in effectively reversing EMT and reducing the CSC population to enhance therapeutic response in PDAC. These results may broadly applicable to many types of cancers due to the commonality of CSCs and the mechanisms regulating pluripotent gene expression.
 

24.04 Adenosquamous Carcinoma of the Pancreas: A Translational Approach

Posted on January 18, 2018

M. C. Hernandez1, J. Leiting1, L. Yang2, J. R. Bergquist1, M. J. Truty1  1Mayo Clinic,Department Of Surgery,Rochester, MN, USA 2Center For Individualized Medicine,Biomarker Discovery Program,Rochester, MN, USA

Introduction:

Adenosquamous carcinoma of the pancreas (ASCP) is a rare and lethal histologic subtype of pancreatic cancer. ASCPs are defined by a mix of at least 30% malignant squamous cell carcinoma and coexisting ductal adenocarcinoma. Normal pancreas tissue has no benign squamous epithelial components. Thus the origin of this tumor is uncertain. Postoperative recurrence rates are high and ASCP demonstrates significantly worse overall survival, even compared to ductal adenocarcinoma. The low prevalence of ASCPs makes research studies and clinical trials exploiting unique features of this tumor difficult. We aimed to generate and amplify patient-derived ASCP malignant tissue in order to (1) genomically characterize  (2) functionally assess for sensitivity to MTH1 inhibition and (3) correlate functional assay sensitivity with therapeutic response using tumor bearing ASCP mice.

Methods:

Patient derived xenografts (PDX) were generated from surgical resection of patient tumor tissue in NOD SCID mice. All patient and derived PDX tumors were histologically (H&E and IHC) confirmed. We performed whole genome mate pair sequencing (MPseq) on PDX tissues. Western blot and immunohistochemistry for the presence of MTH1 enzyme was performed to identify possible sensitivity to MTH1 inhibition. Cells were cultured using a hanging drop technique and treated with cytotoxic and targeted therapies. Cell viability was assessed using daily cell counts and Prestoblue dye.

Results:

Five ASCP PDX models were created with 100% initial engraftment rate and >90% engraftment ratio. Immunohistochemistry for p63 (squamous) and mucin components demonstrated the ASCP phenotype. MPseq revealed distinct patterns of aneuploidy and all losses in 17p, 18q and 21q. Each predicted homozygous loss of p16 (CDKN2A) (9p21.3) and heterozygous losses of both TP53 and SMAD4. Two models also predicted double and single losses of PTEN.

Western blot and immunohistochemistry revealed variable MTH1 expression. Cellular spheroids and 2D cultures demonstrated cytostatic sensitivity to the combination of gemcitabine and oxaliplatin as well as the sensitivity to high MTH1 expression tumors and insensitivity to low MTH1 expression tumors. These findings were confirmed with in-vivo treatment studies in tumor bearing PDX models of ASCP.

Conclusion:

ASCP is a rare but more malignant phenotype compared to pancreatic adenocarcinoma. We have generated the world’s first successful models of ASCP and demonstrate variable expression of MTH1. Whole genome sequencing reveals common genomic aberrations. Functional assays using three dimensional organoids demonstrate cytotoxic as well as targeted monotherapy responses. This correlated with therapeutic response in tumor bearing PDX models.
 

24.05 Pancreas-Specific Arid1a Deletion Induces Intraductal Papillary Mucinous Neoplasms in Mice

Posted on January 18, 2018

I. Nassour1,2, X. Sun1, S. Zhang1, X. Luo1, L. H. Nguyen1, L. Li1, L. Peng3, J. Shen4, H. Zhu1, S. Wang1,2  1University Of Texas Southwestern Medical Center,Children’s Research Institute,Dallas, TX, USA 2University Of Texas Southwestern Medical Center,Division Of Surgical Oncology,Dallas, TX, USA 3University Of Texas Southwestern Medical Center,Department Of Pathology,Dallas, TX, USA 4Stanford University,Department Of Pathology,Palo Alto, CA, USA

Introduction: Intraductal papillary mucinous neoplasms (IPMN) are precursors to pancreatic ductal adenocarcinoma (PDAC). While activating KRAS mutations are the most common alterations found in PDAC and IPMN, ARID1A, which is a component of the SWI/SNF chromatin remodeling complex, is also commonly mutated. However, the functional effects of ARID1A mutations in pancreas tumorigenesis are not known. Understanding molecular mechanisms that drive IPMN progression may lead to the development of novel therapies that prevent IPMN transformation into PDAC.

Methods: We generated transgenic mice that had pancreas-specific activating Kras mutations and Arid1a deletion (KrasG12D; Ptf1a-Cre; Arid1af/f, or “KCA” mice). We identified 35 human IPMN resection samples, performed immunohistochemistry staining for ARID1A, and graded the expression intensity. We knocked down ARID1A and MYC in human pancreatic ductal epithelial (HPDE) cells with siRNA. siScramble was used as the negative control. To measure protein synthesis, we used an assay based on the incorporation of modified puromycin molecules (O-propargyl-puromycin (OPP)) into nascent peptides. OPP was then fluorescently labelled and quantified by fluorescence-activated cell sorting. Fluorescence level was proportional to the extent of cellular protein synthesis.

Results: Mice with only pancreas-specific activating Kras mutations (KrasG12D; Ptf1a-Cre) had significant pancreatitis and pancreatic intraepithelial neoplasia, which is another type of PDAC precursor, consistent with previous reports. In contrast, KCA mice developed macroscopic mucinous pancreatic cysts at 100% penetrance. Histologic and biochemical assessment showed that these cysts most resembled gastric subtype IPMN. Evaluation of human IPMN samples revealed that gastric subtype IPMN frequently had little to no ARID1A expression while other subtypes had high expression, confirming the fidelity of our model (Fig. A). RNA-seq from Arid1a null pancreas showed significant upregulation of gene networks involved with MYC activity and protein translation. Knocking down ARID1A (siARID1A) in HPDE cells induced increased MYC expression (Fig. B) and protein synthesis (Fig. C). Concurrent knockdown of MYC (siMYC) and ARID1A abrogated the increase in protein synthesis (Fig. D).

Conclusions: Pancreas-specific ARID1A deletion resulted in the formation of gastric subtype IPMN and increased protein synthesis that was mediated through elevated MYC activity (Fig. E). These data suggest that translation is a potential therapeutic target to block the formation and progression of gastric subtype IPMN.

 

24.01 Induction of Persistent Tolerance of Lung Transplants by IL-2 complex-stimulated Regulatory T Cells

Posted on January 18, 2018

W. Jungraithmayr1,2, Y. Yamada3, D. Impellizzieri2, J. Jang3, I. Inci3, O. Boyman2, W. Weder3  1Brandenburg Medical School,Department Of Thoracic Surgery,Neuruppin, BRANDENBURG, Germany 2University Hospital Zurich,Department Of Immunology,Zurich, ZURICH, Switzerland 3University Hospital Zurich,Department Of Thoracic Surgery,Zurich, ZURICH, Switzerland

Introduction
Transplant (Tx) tolerance is a state of anergy by the recipient to Tx-related alloantigen. Lung allo-Tx has the worst allograft survival outcome when compared to all other transplantable solid organs. Interleukin (IL-) 2 promotes rejection through enhanced T cell-cytotoxicity. However, when binding IL-2 in complex (cplx) with a neutralizing anti-IL-2 antibody, it can induce expansion of regulatory T (Treg) cells. We therefore evaluated here the impact on allograft outcome of IL-2 cplx on experimental mouse lung Tx outcome.

Methods
Single lung Tx was performed from BALB/c to C57BL/6 mice to induce full allo-rejection. Recipients received intraperitoneal injections of either PBS (control) or IL-2cx with anti-IL-2 antibody clone JES6-1 on three consecutive days before Tx. The outcomes of transplants were analyzed on day 5, 15, 28 and 56 post-transplant by flow cytometry, lung functionality (oxygenation and compliance) and histology.

Results
Acute allo-Tx rejection (AR) was virtually absent macroscopically and histologically in IL-2cx-treated animals on day 5, 15, 28 and 56 compared to controls. AR scores of allo-transplants of IL-2cx-treated mice were significantly lower vs. control (day 5, p=0.02; day 14, p<0.001; day 28, p=0.007; day 56, p=0.04). Foxp3+CD4+CD25+ Treg cells in IL-2 cx-treated mice were significantly higher within allotransplants on all time point, as it was in contralateral naïve lungs and spleens on day 5 or 15 (p<0.05). IL-2cx-treated allograft on day 5 revealed better compliance (5.2±2.2 vs 1.6±0.8 µl/cmH2O, p=0.04) and a better oxygenation (253±136 vs 24±4 mmHg, p=0.04) compared to the control.    

Conclusion
Treatment of recipients using IL-2cx before lung Tx leads to Treg expansion, prevents from AR and maintains a viable engrafted lung up to 56 days post lung Tx. These data provide insight into the mechanism of adaptive tolerance induction in Tx and may have implications for immune protocols for Treg modulation in human lung transplantation.

24.02 Active K-RAS Mutant Gene detected in Pancreatic Juice from Patients with Peri-Ampullary Neoplasms

Posted on January 18, 2018

J. A. Reza1, S. Litherland2, X. Zhu5, P. Veldhuis4, A. J. Almodovar6, N. Fanaian3, J. P. Arnoletti1,2,4  1Florida Hospital,Center For Specialized Surgery,Orlando, FLORIDA, USA 2Florida Hospital Cancer Institute,Translational Research,Orlando, FLORIDA, USA 3Florida Hospital,Center For Diagnostic Pathology,Orlando, FLORIDA, USA 4Florida Hospital,Institute For Surgical Advancement,Orlando, FLORIDA, USA 5Florida Hospital,Center For Interventional Endoscopy,Orlando, FLORIDA, USA 6Florida Hospital,Translational Research Core,Orlando, FLORIDA, USA

Introduction:
Mutations in the KRAS oncogene (K-RASmut) have been associated with pancreatic ductal adenocarcinoma (PDAC) progression and aggressive behavior. We have previously reported the presence of K-RASmut transcriptionally active circulating tumor cells (CTC) in the portal circulation of PDAC patients. We hypothesized that active chromatin K-RASmut DNA from tumor cells may be detected in pancreatic juice following pancreatico-duodenectomy and indicative of risk for progression among surgically treated patients with peri-ampullary neoplasms.

Methods:
We collected portal venous blood and pancreatic juice samples from 17 patients who underwent pancreatico-duodenectomy for surgical treatment of various peri-ampullary neoplasms (PDAC=6, ampullary cancer=5, neuroendocrine tumor=3, cholangiocarcinoma=1, IPMN=1), and chronic pancreatitis (n=1). Portal vein circulating tumor cells (CTC) were isolated by high-speed FACS and analyzed by quantitative RT-PCR for K-RAS exon 12 mutant gene expression (K-RASmut). DNA, chromatin, and histone acetylated active chromatin were isolated from pancreatic juice samples (collected post-operatively from temporary external pancreatic duct stents) by chromatin immunoprecipitation (ChIP) and analyzed for the presence K-RASmut and other cancer related gene sequences by quantitative PCR and ChIP SEQ.

Results:
DNA, chromatin, and acetylated chromatin containing mutated K-RAS gene were detectable in pancreatic juice secreted after surgical resection of pancreatic, ampullary and bile duct carcinomas. The detection of pancreatic juice K-RASmut in chromatin and acetylated chromatin directly correlated with the number of CTC found in the portal venous blood (p=0.0108, p=0.0405, respectively). ChIP SEQ analyses indicated the presence of acetylated chromatin in pancreatic juice from PDAC patients, including RET and two other candidate chromatin loci, not found in similar analysis of non-malignant pancreatic juice from pancreatitis. PDAC patients with K-RASmut+ DNA in their portal blood CTC exhibited K-RASmut mRNA expression, indicative of transcriptionally active CTC surviving after primary tumor resection. K-RASmut gene activation in CTC correlated positively with progression free survival (p=0.0267, r2=0.4827, n=13, median follow-up of 4.5 months). 

Conclusion:
Detectable K-RASmut chromatin in pancreatic juice suggests that viable cancer cells remain in the pancreatic duct following surgical resection. Analysis of pancreatic juice and portal venous blood CTC may be useful for stratification of tumor recurrence potential and identification of molecular therapeutic targets in peri-ampullary neoplasms.
 

23.08 Priming with IL-7/15 to Generate Metabolically Fit CD8+ T Cells in the Tumor Microenvironment

Posted on January 18, 2018

S. Patel1, T. Hoki1, T. Yamauchi1, K. A. Collins1, C. A. Eppolito1, A. J. Francois1, J. V. Welch1, J. A. DiTursi1, K. Odunsi1,2,3, F. Ito1,4,5  1Roswell Park Cancer Institute,Center For Immunotherapy,Buffalo, NY, USA 2Roswell Park Cancer Institute,Department Of Gynecologic Oncology,Buffalo, NY, USA 3Roswell Park Cancer Institute,Department Of Immunology,Buffalo, NY, USA 4State University Of New York At Buffalo,Department Of Surgery, University At Buffalo Jacobs School Of Medicine And Biomedical Sciences,Buffalo, NY, USA 5Roswell Park Cancer Institute,Department Of Surgical Oncology,Buffalo, NY, USA

Introduction:
Current approaches to adoptive cell therapy (ACT) with antigen-specific T cells are limited by the difficulty of obtaining sufficient numbers of T cells against targeted antigens with effective in vivo characteristics. Whereas interleukin (IL)-2 has been widely used for generation of antitumor T cells in vitro clinically, dose-dependent effects of IL-2 on differentiation of T cells are associated with decreased proliferative and self-renewal capacity in vivo. IL-7 and IL-15 are also common γ  chain cytokines that play pivotal roles in homeostasis, proliferation, and maintenance of memory CD8+ T cells. Accumulating evidence largely from examining hematological malignancies indicates that the combined use of IL-7 and IL-15 (IL-7/15) can produce T cells that confer superior antitumor immunity in vivo. However, antitumor efficacy of IL-7/15-primed T cells in an orthotopic tumor model has not been rigorously evaluated.

Methods:
Pmel-1 T-cell receptor transgenic CD8+ T cells were activated with the cognate antigen gp100 expressed on B16 melanoma in IL-7/15 for 6 days. IL-2 was used for a control. Phenotype and function as well as metabolic profile of IL-2- and IL-7/15-primed T cells were evaluated. To determine in vivo antitumor efficacy, C57BL/6 mice bearing subcutaneous B16F10 melanoma were treated with adoptive transfer of IL-2- or IL-7/15-primed Pmel-1 T cells, followed by systemic administration of IL-2, and vaccination with gp100, anti-CD40 antibody, and toll-like receptor (TLR) agonist to augment antitumor efficacy of transferred T cells.

Results:
Cell expansion was significantly higher when T cells were activated in IL-7/15 at day 6 compared with ones in IL-2. IL-7/15-primed T cells consisted of a higher proportion of less-differentiated CD44+CD62L+ T cells, and secreted significantly more IL-2 against the target antigen compared to IL-2-primed T cells while both had comparable effector function such as specific lysis of targets and IFNγ  production in vitro. Furthermore, IL-7/15-primed T cells had higher mitochondrial spare respiratory capacity than IL-2-primed T cells, suggesting that IL-7/15-primed T cells have capacity to produce more ATP in case of a sudden increase in energy demand. In line with this, adoptively-transferred IL-7/15-primed T cells expressed significantly higher Ki67 than IL-2-primed T cells in the tumor microenvironment (TME). Significantly delayed tumor growth and improved survival were observed in mice treated with IL-7/15-primed T cells compared to IL-2-primed T cells.

Conclusion:
Taken together, our studies suggest that IL-7/15 modulates the metabolic programming of T cells to promote more robust and efficient CD8+ T cells that can proliferate in the TME. In particular, IL-7/15-primed T cells have higher self-renewal and spare respiratory capacity with potent effector function that correspond to significantly improved survival in an orthotopic tumor model.
 

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