60.05 Minnelide Synergizes with TRAIL Against Pancreatic Cancer

Posted on January 26, 2017

S. Modi1, B. Giri1, V. Sethi1, B. Garg1, J. George1, S. Banerjee1, V. Dudeja1, A. K. Saluja1  1Sylvester Cancer Center,Surgery,Miami, FL, USA

Introduction: Pancreatic cancer is an aggressive malignancy. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has been shown to cause cancer cell death in multiple cancers with minimal toxicity to un-transformed cells. Unfortunately, pancreatic cancer (PDAC) is resistant to TRAIL. We have previously shown that triptolide (TPL), a diterpene triepoxide isolated from a Chinese herb and its water soluble pro-drug Minnelide, is effective against pancreatic cancer. Minnelide is currently undergoing Phase-I trials against advanced GI malignancies. The aim of the current study is to evaluate the anti-tumor efficacy of lowered doses of Minnelide in combination with TRAIL in multiple models including an immunocompetent and stroma rich mouse model of pancreatic cancer. 

Methods: The effect of TRAIL (0-40ng/ml), low dose of TPL (50nM), or combination of TRAIL and TPL on the viability (WST-8 assay) and apoptosis (cleaved caspase-3 and cleaved PARP levels using flow cytometry) of human pancreatic cancer cell lines (S2-VP10) and human pancreatic stellate cells (hPSCs) was measured. To evaluate efficacy of combination therapy in animal models of pancreatic cancer, subcutaneous xenograft model in athymic nude mice, subcutaneous syngeneic model in C57BL/6J wild type mice and a novel tumor implantation model in which 3 mm3 pieces of KPC tumors were implanted in pancreas of C57BL/6J wild type mice. The animals were randomized into four groups and treated with Minnelide (0.21 mg/kg/day ip), TRAIL (20mg/kg 3Xweek ip) or combination of the two for 4-6 weeks. At the end of 4-6 weeks, tumors were harvested, and tissues were used for various experiments. The animal doses of Minnelide correspond to the clinically relevant and well tolerated doses from the Phase-I trial.

Results:Combination of TRAIL 1.25ng/ml and TPL 50nM decreased the viability of S2VP10 (viability, % of Control, 25±4.6% after 24h of treatment) and significantly increased cleaved caspase-3 and PARP levels suggesting the activation of apoptotic cell death, whereas TRAIL alone did not influence viability of these cancer cells (viability, % of Control, 96±8.3% at 24h). TRAIL alone caused a downregulation of DR5 receptors while addition of TPL abrogated this effect. In the xenograft SC model, Minnelide and TRAIL alone did not have any effect on tumor size while combination of the two resulted in marked decrease in tumor growth. In the tumor implantation model, combination of low doses of Minnelide and TRAIL markedly inhibited tumor burden when compared to Minnelide or TRAIL alone. Tumor weights (gm, mean±SEM) after 3 week of Rx: Saline-1.63±0.1; Minnelide-1.15±0.02; TRAIL-1.54±0.04; Minnelide+TRAIL-0.38±0.03. TUNEL staining showed significantly increased apoptosis in the combination arm.

Conclusion:Minnelide synergizes with TRAIL therapy and reverses the TRAIL resistance in multiple models of pancreatic cancer including immunocompetent stroma rich model. 

 

60.04 BIRC5 Is A Biomarker for Early Detection of PDAC in CRISPR-Cas9 Engineered Pancreatic Organoids

Posted on January 26, 2017

S. Liu1, J. Yu1, R. Sanchez1, E. Rozengurt2, F. Brunicardi1  1David Geffen School Of Medicine, University Of California At Los Angeles,Department Of Surgery,Los Angeles, CA, USA 2David Geffen School Of Medicine, University Of California At Los Angeles,Division Of Digestive Disease,Los Angeles, CA, USA

Introduction: Recent advances in CRISPR-Cas9 gene editing and organoid culturing are leading to pancreatic cancer (PDAC) tumor models with unprecedented speed and precision by introducing driver mutations such as KrasG12D, P53 mutation, which will hopefully identify early detection PDAC biomarkers. Over-expression of BIRC5 has been found in early stage of PDAC in human specimens and is a promising candidate for an early detection biomarker. The purpose of this study is to study BIRC5 as an early detection biomarker by taking the advantage of CRISPR-Cas9 and organoid techniques to develop an early onset pancreatic 3D tumor model.

Methods:  Three-dimensional culture conditions were optimized to maintain mouse pancreatic ductal progenitor organoids (ORGs). sgRNA was well designed and cloned into a GFP-tagged lentiviral vector to generate two vectors: Lenti-sgKrasG12D-GFP and Lenti-sgKrasG12D-sgp53KO-GFP. Co-transfection was performed using Lenti-sgRNA-GFP and Lenti-Cas9-2A-tdTomato (tdT) to infect ORG. KrasG12D and p53 mutations were confirmed by genome DNA sequencing. BIRC5 expression in ORGs and human specimens was measured using western blot and immunofluorescence assay. Knockdown of Kras and overexpression of wild type p53 was carried out. A reporter assay using BIRC5-GLuc was performed in these ORGs. 

Results:Immunofluorescence demonstrated that BIRC5 was expressed in PanIN3 and PDAC specimens.   ORGs were established in matrigel with additional growing factors in culture media. GFP (green), tdT (red) or GFP/tdT double staining was observed in ORGs following co-infection. Tumoroid ORGs were observed in CRISPR-Cas9 engineered ORGs displaying GFP/tdT double staining in 3-D culturing with either GFP or tdT only (Fig). The expression of GFP and tdT were further confirmed in the cryosection and paraffin sections. KrasG12D mutation only resulted in very limited expression of BIRC5 in ORGs, however KrasG12D/p53KO mutations markedly increased BIRC5 expression. Knockdown of KrasG12D expression did not alter BIRC5 levels, however, restoration of wild type P53 expression resulted in significant reduction of BIRC5 levels in ORGs (p<0.05). Furthermore, BIRC5-GLuc reporter assay revealed GLuc expression only in the ORGs with both KRAS and P53 mutations. 

Conclusion:CRISPR-Cas9 engineering of pancreatic ductal organoids with KRAS/P53 driver mutations resulted in a tumoroid ORGs associated with BIRC5 overexpression. BIRC5 was over-expressed in PanIN3 and PDAC. These data support the hypothesis that BIRC5 is biomarker for early detection of PDAC.

 

60.03 Enrichment of Polymerase Theta (POLQ) Single Nucleotide Polymorphisms in Papillary Thyroid Carcinoma

Posted on January 26, 2017

T. D. Murtha1, R. Korah1, T. Carling1  1Yale University School Of Medicine,New Haven, CT, USA

Introduction:  Recent comprehensive genetic analyses have demonstrated a high prevalence of gene fusions–suggestive of defective DNA break repair–in papillary thyroid carcinoma (PTC). Polymerase theta (POLQ) is an A-family mammalian polymerase that is unique in mediating replication-independent DNA repairs, including single and double strand DNA breaks. Differential expression of POLQ has been shown to correlate with poor outcomes in breast, ovarian, and lung cancer. Because of the high concentration of hydrogen peroxide in thyroid follicles and the consequent oxidation-induced single and double strand breaks in DNA, we hypothesized that genetic polymorphism of the DNA repair enzyme POLQ may play a role in predisposing follicular cells to de novo mutations and potentially tumorigenesis. To identify novel mutations and enrichment of SNPs, we performed classical Sanger sequencing of the exonic regions of POLQ in a large cohort of sporadic PTCs. 

Methods:  Genomic DNA was isolated from 51 histologically confirmed PTC specimens and matched adjacent non-tumor thyroid tissue. Genetic sequencing via capillary electrophoresis (Sanger method) was performed to detect novel variants and SNPs for all 30 exons and exon/intron splice sites of the POLQ gene. Discovered nonsynonymous and missense SNPs, and the corresponding amino acid substitutions, were analyzed using multiple bioinformatic algorithms to determine if alterations were deleterious to protein integrity or function. Novel variants were annotated and SNP frequency was compared to The Exome Aggregation Consortium (ExAC) database.

Results: Fourteen germline variants were confirmed in tumor and corresponding matched adjacent non-tumor thyroid tissue. Eight of those germline variants (57%) were determined to be disease-causing by mutation prediction algorithms. In the C-terminal polymerase domain (amino acids 2060–2590), there was an increased frequency of germline SNPs compared to the comprehensive population SNP database ExAC. The recurrent SNP rs532411, previously found to be significantly associated with breast cancer, was found in 22% (11/51) of PTC specimens while its frequency in the population is 6.5% (P < .0001, chi-squared test). We also identified 4 novel variants not previously reported.

Conclusion: The unique contribution of POLQ in DNA repair via non-homologous end-joining of double stand breaks makes it a critical regulator of genomic integrity in the hydrogen peroxide-rich thyroid gland. Sequencing demonstrates enrichment of multiple SNPs in the polymerase region of POLQ, potentially increasing the probability of carcinogenic point mutations and gene fusions. While expanded genome-wide association studies (GWAS) are essential for confirming the relationship, the current study offers preliminary information suggesting a role for POLQ SNPs in genetically predisposing individuals to PTC.

60.02 Utility of Circulating Tumor Cells for Preoperative Prediction of Micrometastatic Pancreatic Cancer

Posted on January 26, 2017

C. M. Court1,4, J. S. Ankeny1,4, S. Sho1, S. Hou1, P. Winograd1, Q. Li1, M. Song1, T. R. Donahue1,4, O. J. Hines1, H. A. Reber1, Z. A. Wainberg3, H. R. Tseng4, J. S. Tomlinson1,4  2University Of California – Los Angeles,Molecular And Medical Pharmacology,Los Angeles, CA, USA 3University Of California – Los Angeles,Hematology/Oncology,Los Angeles, CA, USA 4VA Greater Los Angeles,Surgery,Los Angeles, CA, USA 1University Of California – Los Angeles,Surgery,Los Angeles, CA, USA

Introduction: Current preoperative evaluation and staging of patients with pancreatic ductal adenocarcinoma (PDAC) is hampered by the limited sensitivity of cross-sectional imaging for micrometastatic disease. Furthermore, the majority of patients who undergo resection ultimately succumb to metastatic disease, suggesting that current staging systems are likely routinely understaging patients. Our goal was to investigate the utility of circulating tumor cells (CTCs) as a preoperative predictor of metastatic disease and postoperative outcomes.

Methods:  A total of 49 patients were taken to the operating room for attempted pancreatic resection and enrolled in the study. 15 (30.6%) had undergone neoadjuvant therapy but were deemed to be surgical candidates based on post-treatment imaging. Four milliliters of venous blood (VB) was evaluated for the presence and number of CTCs using the microfluidic NanoVelcro chip. CTCs were defined by immunocytochemical staining (CK+ or CEA +, CD45-, DAPI+). CTC number was then correlated with both surgical outcomes and standard clinicopathologic findings.

Results: Of the 49 PDAC patients taken to the operating room, 11 (22.4%) were found to have metastatic disease intraoperatively, 6 with liver metastases and 5 with peritoneal metastases. CTC presence and number per 4 mL VB were found to correlate with stage and distinguished patients with resectable disease from those with metastatic disease. CTCs were present in all (100%) of the 11 patients with metastatic disease versus only 63% of those with resectable PDAC and at significantly higher levels (Average CTCs/4mL VB- 11.5 vs. 1.7, p<0.01). Using a cutoff of ≥3 CTCs/4mL VB, CTCs were able to distinguish patients with metastatic disease from those with resectable cancer with a sensitivity of 90.9%, specificity of 84.2%, PPV of 62.5%, NPV of 97.0%, and AUROC of 0.898 (95% CI = 0.796 – 0.999, p < 0.001). Of note, CA19-9 levels did not statistically differ between the groups (p=0.88). Furthermore, higher CTC counts correlated with worse recurrence-free and overall survival for both the entire cohort as well as the group of patients who successfully underwent an operation.

Conclusion: In this small prospective study, preoperative CTC enumeration demonstrated a correlation with the presence micrometastatic disease not detected by preoperative cross sectional imaging. Larger studies with longer follow-up are needed to firmly establish CTCs as a predictive biomarker in PDAC. 

 

60.01 U1 adaptors suppress the KRAS-cMYC oncogenic axis in human pancreatic cancer xenografts

Posted on January 26, 2017

A. T. Tsang1,6, L. Yi1, C. Dudgeon1, X. Yu1, R. Goraczniak5, S. Gunderson3,5, D. R. Carpizo1,2,4  6Mount Sinai St. Luke’s Roosevelt General Surgery Residency Program,New York, NY, USA 1The Cancer Institute Of New Jersey,New Brunswick, NJ, USA 2Rutgers University Robert Wood Johnson Medical School,Surgery,New Brunswick, NEW JERSEY, USA 3Rutgers University,Department Of Molecular Biology And Biochemistry,PIscataway, NEW JERSEY, USA 4Rutgers University,Department Of Pharmacology,Piscataway, NEW JERSEY, USA 5Silagene, Inc.,Hillsborough, NEW JERSEY, USA

Introduction:
One of the most significant unmet needs in pancreatic cancer therapy is targeting the most commonly mutated gene, KRAS, and its downstream mediator MYC. Small interfering RNA targeting KRAS has produced potent anti-tumor activity in preclinical studies, but technical difficulties of in-vivo delivery have impeded clinical translation. U1 Adaptors are a novel technology for oligonucleotide-mediated gene silencing that acts by blocking polyadenylation of messenger RNA. They can accommodate extensive covalent modifications for nuclease resistance, targeted delivery and in-vivo imaging without loss of silencing activity, offering important advantages over siRNA and antisense oligos as therapeutic agents. The KRAS-cMYC oncogenic axis plays a key role in the generation of self-renewing metastatic cells, making cMYC an attractive target in KRAS-driven pancreatic cancers. Genetic studies in pancreatic cancer mouse models validate the therapeutic efficacy of silencing KRAS and MYC expression.

Methods:
Candidate U1 Adaptors targeting KRAS were screened in vitro using the human pancreatic cancer cell line MIAPaCa2 (KRAS G12D mutant). The best Adaptors were applied in cell growth inhibition assays over 10 days in multiple pancreatic cancer cell lines. They were then tested for efficacy in mice bearing subcutaneous MIAPaCa2 xenograft tumors. For in-vivo delivery, Adaptors were covalently linked to a cyclic RGD-motif peptide (cRGD), a targeting ligand for integrin receptors expressed on tumor cells and endothelia, or alternately, internalizing RGD (iRGD), a variant peptide that triggers endothelial permeabilization and internalization by cells through neuropilin-1 binding. The cRGD- and iRGD-conjugated KRAS Adaptors were administered by tail vein injections twice weekly for 3 to 4 weeks once tumors reached 20mm3.. In parallel, U1 Adaptors targeting cMYC were screened in B-cell lymphoma lines and tested for efficacy in mice bearing MIA-PaCa-2 xenograft tumors using the same method.

Results:
The best KRAS Adaptors reduced KRAS mRNA expression by up to 76% – as effectively as an siRNA control. Knockdown of KRAS protein expression and its downstream effectors was confirmed by western blot. Cell growth inhibition was demonstrated for MIAPaCa2 and other established human pancreatic cancer cell lines in vitro. The potency of inhibition was dependent on mutant KRAS. Over a series of in vivo mice experiments, MIAPaCa2 xenograft tumor growth was inhibited by averages of 68% to 93% by cRGD- and iRGD-conjugated KRAS Adaptors as compared to vehicle-only controls. Tumor stasis or regression occurred in some treated mice. Remarkably, cMYC Adaptors were similarly effective in suppressing xenograft tumor growth.

Conclusion:

U1 Adaptors can successfully target human KRAS and cMYC in vivo. These results support the continued development of U1 Adaptor technology as a strategy for therapeutic suppression of KRAS, cMYC and possibly other oncogenes in pancreatic cancer.

 

59.10 IκK-16 decreases miRNA-155 expression and attenuates the human monocyte inflammatory response

Posted on January 26, 2017

N. J. Galbraith1, S. P. Walker1, C. Bishop1, J. V. Carter1, M. Cahill1, S. A. Gardner1, H. C. Polk1  1University Of Louisville,Department Of Surgery,Louisville, KY, USA

Introduction:

The magnitude of the immune response following major trauma is predictive of mortality. IκK-16, a selective inhibitor of IκB kinase (IκK), has shown promising results by limiting end-organ damage in experimental models of sepsis and hemorrhagic shock. The purpose of this study was to determine the influence of IκK-16 on miRNA-155 expression and the human monocyte inflammatory response. 

Methods:

Primary human monocytes were freshly isolated from healthy donors using CD14 magnetic beads and cultured in standard medium. Purity of ≥95% was confirmed by flow cytometry. Cells were treated for 1h with either IκK-16 (100nM unless otherwise specified) or diluted DMSO as a control. Monocytes were then stimulated with lipopolysaccharide (LPS) 100ng/mL for 16 h. Cell viability was determined by Trypan Blue staining and manual counting by standard microscopy. The influence of IκK-16 on IκK phosphorylation was assessed by Western Blot. Intracellular expression of miRNA-155 was measured at qRT-PCR. TNF-α and IL-10 supernatant protein concentrations were measured by ELISA. Cell surface protein expression of CD14 and HLA-DR were measured by flow cytometry. Paired T-test or Wilcoxon Rank Sign Test was used where appropriate, with signficance set at 0.05. 

Results:

IκK-16 did not influence monocyte cell viability. IκK-16 treatment suppressed IκK phosphorylation. IκK-16 treatment lead to a downregulation of miRNA-155 expression compared with control (p<0.05). Levels of both TNF-α and IL-10 were suppressed in a dose-dependent fashion with IκK-16 treatment (p<0.05). However, levels of monocyte HLA-DR expression were not significantly altered by IκK-16 treatment, and monocyte surface CD14 expression was augmented by IκK-16 treatment. 

Conclusion:

IκK-16 treatment suppresses activation of the IκK signaling pathway in the human monocyte, and does not appear to be toxic to cells. Both pro-inflammatory and anti-inflammatory cytokine responses are also suppressed, which may occur due to modulation of MiRNA-155 expression. The antigen presenting capacity of monocytes was not impaired by IκK-16 treatment. These results are promising for future studies examining the role of IκK-16 as a potential immunomodulatory therapy.  
 

59.09 TH17 Cells Mediate Cancer Progression and Development Due to Neutrophil Extracellular Traps in Liver

Posted on January 26, 2017

V. Sud1, S. Tohme1, H. Huang1,2, D. J. Van Der Windt1, A. Tsung1  1University Of Pittsburg,General Surgery,Pittsburgh, PA, USA 2Huazhong University Of Science And Technology,Surgery,Wuhan, HUBEI, China

Introduction:
Colorectal cancer is the third leading cause of cancer in the United States, a majority of which is due to liver metastasis. In liver metastasis of colorectal cancer, surgical resection is superior to chemotherapy alone. However, the rate of recurrence of metastasis after surgical resection is 50-60%, a major cause of treatment failure. It has been shown that the Neutrophil Extracellular Traps (NETs), a unique method of neutrophil apoptosis, contribute significantly to this process of cancer growth and development. We hypothesized that NETs influence the growth of metastasis by altering the tumor immune environment to a more pro-tumorigenic state.

Methods:
C57BL/6J wild-type mice (WT) and global genetic knockouts for peptidylarginine deaminase 4 (PAD4) gene (PAD4 KO mice), a gene which codes for an integral enzyme for NET formation, hence preventing the formation of NETs in the mice, were used. At 12wks of age both groups were injected through the portal vein with 0.5 million cells of Murine colorectal cancer cell line (MC-38). After 3 weeks, livers from both groups were harvested and underwent flow cytometry to analyze the innate and adaptive cell populations. 

Results:
Flow cytometry analysis was run on mice from both groups and the samples from the background liver and tumor of the mice were independently assessed. On examination, there were far fewer tumors in the PAD4KO mice than the WT. Flow analysis on the liver demonstrated a 5.6 fold decrease in the TH17 cell population in the PAD4KO mice as compared to the WT (0.082% PAD4KO; 0.448% WT). Which was accompanied by an increase in the CD8+ population (66.1% WT v 33.4% PAD4KO of total T cells) and a small decrease in the CD4+ population (18.9% WT v 20.6% PAD4KO of total T cells). 

Conclusion:

We found that absence of NETs leads to an anti-tumorigenic environment by a decrease in TH17 cells and increase in CD8 cells. Further studies are underway to further characterize the crosstalk between NET’s and immune cells in the tumor microenvironment.

 

59.08 Monocyte Depletion Protects Against Murine Neurogenic Pulmonary Edema

Posted on January 26, 2017

S. Chiu1, H. Makinde1, M. Akbarpour1, H. Perlman2, J. I. Sznajder2, G. S. Budinger2, S. J. Schwulst1, A. Bharat1  1Northwestern University,Surgery,Chicago, IL, USA 2Northwestern University,Medicine,Chicago, IL, USA

Introduction:  Up to 75% of patients who suffer from traumatic brain injury (TBI) or intracranial hemorrhage develop life-threatening neurogenic pulmonary edema (NPE). Catecholamine surge resulting from TBI has been traditionally postulated to cause pulmonary vasodilation and capillary leak, resulting in NPE. However, cathecholamine antagonists do not confer protection against TBI-associated NPE, suggesting an alternate pathogenesis. Here, we demonstrate that intravascular non-classical monocytes (NCM) play a crucial role in the development of NPE which is ameliorated when NCM are pharmacologically depleted.

Methods:  C57BL/6J mice were subject to either sham craniotomy or controlled cortical impact with severe TBI. The neurologic severity score (NSS) was used to evaluate motor and behavioral function following TBI. 24 hours prior to TBI, mice were treated with intravenous clodronate liposomes to deplete pulmonary non-classical monocytes using our recently described protocols. Flow cytometry was used to characterize lung myeloid cell populations at 24 hours after TBI. The ratio of wet- to dry-weight of lungs was used to measure pulmonary edema. 

Results: Controlled cortical impact resulted in severe TBI as measured by NSS (Sham: 1.3 vs. TBI: 7.0, p<0.001, Figure A).  Controlled cortical impact resulted in NPE, as indicated by a 30% increase in the wet-to-dry ratio of lungs versus controls (Sham: 4.7 vs. TBI: 6.0, **p=0.03, Figure B). NPE was associated with an influx of CD11b+ cells, including NCM and NK Cells (**p=0.03, ***p=0.01, Figure C). Treatment with intravenous clodronate liposomes resulted in selective depletion of intravascular NCM, while preserving interstitial classical monocytes in the lung. Depletion of NCM resulted in protection against NPE (Wet-to-dry ratio 6.0 vs. 4.5, *p<0.001, Figure B), but did not protect against TBI severity (NSS 7.0 vs. 6.5, p=n.s., Figure A). Sham craniotomy and liposome treatment alone did not alter the lung wet-to-dry ratio. 

Conclusion: NPE is associated with an influx of non-classical monocytes and NK cells into the lung. Pharmacologic depletion of non-classical monocytes in the lung protected against NPE in this murine model. Ongoing studies will determine whether NCM mediate NPE through the recruitment of NK cells.

59.07 Integrin VLA3 Mediates Endothelial Barrier Damage by Human Sepsis Patient Neutrophils in vitro

Posted on January 26, 2017

C. M. Dickinson1, X. O’Brien1, D. S. Heffernan1, M. Kim2, W. G. Cioffi1, J. Reichner1  1Brown University School Of Medicine,Division Of Surgical Research, Rhode Island Hospital, Affiliated With Warren Alpert Medical School,Providence, RI, USA 2University Of Rochester,Department Of Microbiology And Immunology, Center For Vaccine Biology And Immunology,Rochester, NY, USA

Introduction: Critical illness induces endothelial dysfunction mediated in part by neutrophil-to-endothelial interactions. Integrins play a major role in the neutrophil-endothelial interface. It was recently reported that the integrin VLA3 (α 3β 1, CD49c/CD29) is significantly up-regulated on the surface of human neutrophils during an infectious cause of inflammation (sepsis), but not in sterile inflammation (SIRS only). Here we sought to determine the role of VLA3 in an in vitro model of neutrophil-induced damage of endothelial barrier function.

Methods:  We used TNFα-activated human umbilical vein endothelial cell (HUVEC) monolayers and Electrical Cell-substrate Impedance Sensing (ECIS) to quantify in real-time barrier disruption after neutrophil adhesion. Blood was obtained from healthy volunteers and patients with either sepsis (Surgical ICU) or sterile trauma inflammation (TICU). APACHE II scores were calculated on patients at the time of blood draw. We used HUVEC monolayers grown on fibronectin (a VLA3 ligand) to determine the effect of a function blocking anti-VLA3 antibody (MAb 1992, clone MKID-2) on endothelial barrier dysfunction.  Neutrophils from all subjects were isolated by dextran sedimentation, pretreated with anti-VLA3 antibody or isotype control, and allowed adhere to monolayers with and without fMLP stimulation at 30 mins.

Results:Blood was obtained from critically ill TICU patients (n=6) and SICU septic patients (n=5). Healthy donors (n=8) served as controls. Comparing TICU to Septic patients, there was no difference in age (73+/-5.6 vs 56+/-6;p=0.07) or WBC (16.3+/-2.5 vs 16.5+/-1.4;p=0.9). Septic patients had higher APACHEII scores (17.4+/-2.1 vs 5.8+/-0.6;p<0.001). Neutrophils from healthy donors and TICU patients did not induce significant differences in barrier function, measured as a decrease in normalized resistance(nR). Neutrophils from sepsis patients, however, induced a significantly exaggerated loss of barrier function across conditions when compared to neutrophils from healthy donors (nR= 0.67+/-0.08 vs 0.87+/-0.05 at 120min; p<0.05) or TICU patients (nR= 0.67+/-0.08 vs. 0.83+/-0.06 at 120 min;p<0.05). Neutrophils from sepsis patients that were pre-treated with the VLA3 blocking antibody showed significantly less damage than isotype control (nR= 0.88+/-0.05 vs. 0.68+/-0.07 at 120min; p<0.05), with barrier function comparable to healthy donors (nR= 0.88+/-0.05 vs. 0.88+/-0.05 at 120min).

Conclusion: The integrin VLA3 is specifically up-regulated on neutrophils in septic patients.Pretreatment with a function blocking anti-VLA3 antibody was able to attenuate the loss of barrier function to levels indistinguishable from that induced by healthy donors, consistent with the hypothesis that VLA3 mediates the rapid and more pronounced loss of barrier function caused by neutrophils from septic patients. Therefore, VLA3 is a therapeutic target in the treatment of endothelial dysfunction in sepsis warranting further investigation. 

59.06 Strategies to Induce Anti-Tumor Immunity in Colorectal Liver Metastases

Posted on January 26, 2017

N. M. Kunda1, J. Qin1, G. Qiao1, B. S. Prabhakar2, A. V. Maker1,2  1University Of Illinois At Chicago,Division Of Surgical Oncology, Department Of Surgery, College Of Medicine,Chicago, IL, USA 2University Of Illinois At Chicago,Department Of Microbiology & Immunology, College Of Medicine,Chicago, IL, USA

Introduction:
Anti-tumor immune responses have been shown to improve outcomes in patients with advanced stage colon cancer.  Immunogenic cell death (ICD) is a specific mechanism of drug-induced apoptosis that can stimulate an anti-tumor immune response through activation of specific T-cell responses.  Mitoxantrone (MTX) is an anthracenedione antineoplastic agent known to trigger ICD, however, it has not been evaluated as a potential immunotherapeutic potentiator in colon cancer or its metastases.  

Methods:
Murine colon cancer CT-26 cells were treated with 1 μM MTX. For cell cycle studies, cells were fixed with 70% ethanol and DNA staining was performed using DAPI. DNA content was measured with FACS and cell cycle distribution was analyzed.  Treated colon cancer cells were then injected subcutaneously into the right flank of Balb/c mice on days 0 and 7. On day 14, isolated colorectal liver metastases were induced using our well-established model of intrasplenic tumor cell injection of untreated CT-26 cells. All animals were sacrificed on day 28 after establishment of liver metastases.

Results:
At clinical concentrations of MTX, the fraction of cells in the G2-M phase increased from 15.6%-38.8%, and the fraction of cells in the G1 phase decreased correspondingly from 62.6% to 32.8%, consistent with cell cycle growth arrest. The fraction of cells in the sub-G1 phase increased from 0.8% to 11.2%, consistent with DNA degradation and apoptosis.  Liver metastatic tumor burden and average liver weights were significantly decreased in the MTX-treated group compared to control vaccinated animals (4.6g vs 1.8g, p<0.05). The percentages of infiltrating NK cells and activated T-cells in metastatic liver tumors were significantly higher in the MTX-treated group compared to controls (p<0.05). Splenocytes from treated animals also trended towards increased populations of CD3 cells (7.9% vs 17.9%), NK cells (3.6% vs 6.3%), & CD8 cells (24.1% vs 28.6%) compared to control-treated animals. 

Conclusion:
Treatment of colon cancer cells with MTX induced cell cycle arrest, and vaccination of animals with MTX-treated colon cancer cells induced significantly increased immune cell infiltration and decreased tumor burden in distant colorectal liver metastases.  Evaluation of MTX as a stimulator of immunogenic cell death and as a strategy to induce anti-tumor immunity for the treatment of advanced stage colorectal cancer is warranted. 
 

59.05 Interleukin-22 Promotes Chemoresistance in Pancreas Cancer by Enhancing DNA Damage Repair

Posted on January 26, 2017

J. Lazarus1, M. Perusina Lanfranca1, W. Wang1, T. Maj1, W. Zou1, T. Frankel1  1University Of Michigan,Ann Arbor, MI, USA

Introduction:
Pancreatic cancer (PDAC) is the 3rd leading cause of cancer death in the United States.  Patients diagnosed with PDAC have a poor 5-year survival which has not significantly improved in decades. Chemotherapy, including platinum based agents, is often ineffective and the development of chemo-resistance is common.  Recent data has shown that interleukin-22 (IL-22), a cytokine produced by immune cells which acts primarily on epithelial cells, is associated with a worse prognosis in PDAC. We hypothesize that IL-22 enhances chemoresistance leading to the observed impairment in survival.

Methods:
PDAC cells were incubated with and without IL-22 for 3 days followed by cisplatin treatment for 24 hours.  Cell death was determined by propidium iodide (PI) staining.  Apoptosis was assessed by annexin V staining by flow cytometry and immunoblotting for cleaved caspase 3 (cCASP3). Cisplatin mediated DNA damage was evaluated by western blot for the DNA damage marker γH2AX. Genes and proteins involved in apoptosis, DNA damage recognition and repair were assessed by rtPCR and western blotting, respectively.

Results:
Cells pretreated with IL-22 exhibited less platinum chemotherapy-mediated cell death compared to controls as determined by PI staining. In particular, IL-22 decreased the number of cisplatin-induced apoptotic cells and inhibited cCASP3. To investigate the mechanism of IL-22 protection, we first examined differences in apoptotic related gene expression and determined no appreciable differences in Bcl-2, Bcl-xL, caspase 3, and BAX. However, we found cisplatin-induced γH2AX was decreased significantly following IL-22 pre-treatment, suggesting IL-22 controls cisplatin resistance at the DNA damage response level. The role of IL-22 in DNA damage response was investigated by examining expression of genes involved in homologous recombination, non-homologous end-joining, base excision repair and nucleotide excision repair. Data revealed that IL-22 increased gene expression of BRCA2 and PARP in cells also treated with cisplatin.

Conclusion:
Elevated IL-22 is associated with a poor prognosis in pancreas cancer. We determined that IL-22 protected PDAC cells from platinum induced cell death by enhancing DNA damage repair by homologous recombination and base excision repair as evidenced by decreased γH2AX and up-regulation of BRCA2 and PARP.  Strategies to derail DNA damage repair in tumors with a high level of IL-22 may improve sensitivity to chemotherapy and subsequently survival.
 

59.04 Importance of Lymphodepletion prior to Cancer Immunotherapy with Specific T Cell Subsets

Posted on January 26, 2017

M. V. Beems1,2, A. Contreras1, T. K. Luther2, A. Tatar1, P. Srinand1, S. Sen1, C. S. Cho2  1University Of Wisconsin,Department Of Surgery,Madison, WI, USA 2University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA

Introduction:
Adoptive cell transfer (ACT) immunotherapy traditionally involves the infusion of tumor-specific T cells into cancer patients after a lymphodepletion regimen designed to enhance T cell engraftment. Lymphodepletion is a potential source of patient morbidity. Our laboratory and others have shown that ACT using memory T cells is superior to traditional effector T cell-based ACT. Given the innate ability of memory T cells to persist well after antigen encounter, we hypothesized that ACT with memory cells would be less dependent on pre-transfer lymphodepletion.

Methods:
C57BL/6 mice were inoculated with B16GP33 melanoma flank tumors. Half of the mice underwent pre-transfer lymphodepletion using 5Gy total body irradiation. Equal numbers of tumor-specific effector and memory T cells were adoptively transferred. Serial tumor measurements and flow cytometric analyses of tumors, tumor draining lymph nodes (TDLNs), and circulating blood were performed.

Results:
Lymphodepletion significantly reduced tumor growth in all groups. Tumor control was strongest in mice receiving memory T cell ACT with lymphodepletion, with minimal tumor growth seen in this group. Flow cytometric analyses revealed a significantly higher proportion of adoptively transferred cells in the tumors, TDLNs, and circulating blood in mice treated with pre-transfer lymphodepletion. Lymphodepletion benefited memory ACT more than effector ACT in terms of increasing adoptively transferred T cell numbers in all lymphoid compartments.

Conclusion:
Contrary to our hypothesis, lymphodepletion with total body irradiation improved the efficacy of memory ACT to a greater extent than effector ACT. ACT of tumor-specific memory T cells following pre-transfer lymphodepletion may represent a novel modality of immunotherapy for melanoma cancer patients.

59.03 Immunothrombosis and organ injury after liver ischemia/reperfusion

Posted on January 26, 2017

J. Goswami1, P. Varley1, D. Van Der Windt1, H. Waring1, P. Loughran3, D. Geller1, H. Huang1,2, A. Tsung1  1University Of Pittsburgh Medical Center,Surgery,Pittsburgh, PA, USA 2Huazhong University Of Science And Technology,Surgery,Wuhan, Hubei, China 3Center For Biologic Imaging,Cell Biology,Pittsburgh, PA, USA

Introduction:  We have recently shown that liver ischemia/reperfusion (I/R), an unavoidable consequence of liver resection, releases neutrophil extracellular traps (NETs). However, the mechanisms by which liver I/R leads to remote organ injury are as yet unknown. We now hypothesize that NET formation after liver I/R stimulates a pro-coagulant state with resultant distal organ injury.

Methods:  Wild-type and wild-type treated with DNase (to inhibit NETs) were subjected to 70% one-hour hepatic ischemia with 1-24 hours reperfusion. Immunofluorescence and hematoxylin & eosin staining of liver, lung, and kidney were performed to assess for platelet microthrombi. Platelet aggregometry was assessed with Chronolog-Lumi aggregometer. Flow cytometry for platelet activation (CD62P, P-selectin) and circulating platelet neutrophil aggregates (CD41, Ly6G) was performed. 

Results: In wild-type mice, platelet-rich microthrombi and neutrophil infiltration were identified in liver, lung, and kidney microvasculature within 6 hours after reperfusion. This correlated with elevated sALT and serum cystatin C (a marker of kidney injury) levels at 6 hours. Platelet aggregation increased significantly within 1 hour after reperfusion and returned to baseline by 24 hours. Platelet surface CD62P and circulating platelet-neutrophil aggregates were increased, most prominently at 6 hours, both indicative of increased platelet activation. In DNase-treated mice, platelet aggregates were not seen in remote organ microcirculation, and platelet function studies did not reveal significant changes. DNase treatment was protective against both local and distal organ injury as measured by sALT and cystatin C levels, suggesting that NET formation is necessary for induction of systemic hypercoagulability and organ injury. 

Conclusion: NET formation after liver I/R induces a systemic pro-coagulant state with resultant remote organ injury.

 

59.02 CaMKIV Activation Promotes Pathogenic Th17 T-lymphocyte Differentiation During Experimental Colitis

Posted on January 26, 2017

K. E. Cunningham1,2, E. A. Novak2, G. Vincent2, J. D. Piganelli3, M. R. Rosengart1, K. P. Mollen2  1University Of Pittsburgh Medical Center,Surgery,Pittsburgh, PA, USA 2University Of Pittsburgh Children’s Hospital,Pediatric Surgery,Pittsburgh, PA, USA 3University Of Pittsburgh Children’s Hospital,Immunogenetics,Pittsburgh, PA, USA

Introduction:  The incidence and prevalence of inflammatory bowel disease (IBD) is increasing worldwide. Chronic intestinal inflammation, as seen in IBD, is largely mediated by T-lymphocytes. Ca2+/Calmodulin-dependent kinase IV (CaMKIV) is a multifunctional serine/threonine specific protein kinase predominantly found in lymphocytes and is known to play pivotal roles in immune activation and inflammation by regulating T-lymphocyte differentiation. CaMKIV has been extensively studied in diseases of autoimmunity, but its role in the intestine remains unclear. We have previously shown that activation of CaMKIV contributes to inflammation during murine experimental colitis. However, it is unknown whether CaMKIV activity in immune or non-immune cells primarily contributes to intestinal inflammation. We now hypothesize that CaMKIV upregulation in intestinal T-lymphocytes plays a key role in the pathogenesis of colitis and absence of CaMKIV will confer protection against the development of intestinal inflammation.

Methods:  Male CaMKIV KO mice (Camk4tm1Tch/Camk4tm1Tch) and wild-type (WT) counterparts (C57BL/6J) were subjected to dextran sodium sulfate (DSS) colitis for 7 days. Animal weights and Disease Activity Index (DAI) scores were recorded daily. Colonic lamina propria lymphocytes (LPLs) were isolated, stimulated with PMA/Ionomycin, and analyzed by flow cytometry. Intestinal tissue was analyzed for expression of inflammatory cytokines. H&E staining was used to evaluate histological changes. Immunofluorescence was used to observe differences in the quantity and localization of key proteins as well as to identify infiltrating bacteria within the intestinal mucosa.

Results: CaMKIV KO mice subjected to DSS were protected from intestinal inflammation relative to their WT counterparts. Clinical disease severity correlated with CaMKIV activation, as did inflammatory changes demonstrated by H&E staining, PCR, and western blot. Flow cytometry analysis of colon LPLs after DSS treatment verified a population of pathogenic IFNγ+/IL-17+ Th17 T-lymphocytes in WT mice which was absent in CaMKIV KO mice.

Conclusion: We investigate for the first time the role of CaMKIV in the pathogenesis of intestinal inflammation. We demonstrate the presence of a pathogenic Th17 T-lymphocyte population within the colons of WT mice subjected to DSS colitis, but not CaMKIV KO mice, suggesting that CaMKIV activation contributes to the differentiation of IFNγ+/IL-17+ Th17 T-lymphocytes during experimental colitis. Interestingly, this highly inflammatory T-lymphocyte population has been demonstrated to play a key role in the initiation and propagation of inflammation in human IBD. Strategies aimed at suppressing CaMKIV activity may lead to novel immune-based strategies to treat IBD.

 

59.01 Humanized Immune System with Multi-Organ Engraftment in Mice Generated by In Utero Transplantation

Posted on January 26, 2017

R. G. Witt1, E. Kreger1, P. Moradi1, L. Buckman2, S. C. Derderian1, C. Baker1, J. V. Garcia2, T. MacKenzie1  1University Of California – San Francisco,Pediatric Surgery,San Francisco, CA, USA 2University Of North Carolina At Chapel Hill,Chapel Hill, NC, USA

Introduction:  ‘Humanized’ mouse strains are immunodeficient mice that allow the engraftment of human hematopoietic stem cells (HSCs) and they can be used to study the human immune system in a murine model. Despite continued advances in these mouse models, several barriers such as low levels of engraftment in lymphoid organs, poor lymphoid tissue development, low levels of T cell engraftment, and graft-versus-host disease (GVHD) continue to prevent perfect recapitulation of a functional human immune system. In our study, we hypothesized in utero hematopoietic stem cell transplantation (IUHCT) would yield a more tractable system to study the human immune response.

Methods:  25,000 – 50,000 human cord blood-derived CD34+ HSCs were transplanted into the livers of fetal NSG mice on E14.5. One group of mice was treated with ACK2, an antibody against the murine c-Kit receptor, to deplete fetal host hematopoietic stem cells and increase space within the hematopoietic niche prior to transplantation. The levels of human cell chimerism and lineage differentiation including T cell, B cell, granulocyte, and monocyte differentiation were measured in the peripheral blood every 4 weeks, and in all lymphoid tissues and other organs at the time of harvest (24-26 weeks). Weight and coat appearance were assessed for evidence of GVHD.

Results: 29/30 of harvested mice were chimeric (defined as >1% human CD45+ cells in at least one tissue). Tissue-specific chimerism levels were determined in the peripheral blood (11.61±1.85%), crushed bone (10.00±2.21%), bone marrow (8.12±1.79%), and spleen (24.69±3.29%) in all mice. A subset of mice with greater than 5% chimerism in peripheral blood underwent further examination of chimerism levels in their lungs (26.94±5.28%), liver (43.19±7.58%), lymph nodes (83.48±5.95%) female reproductive tract (24.13±9.34%), and brain (4.20±2.08%). Immunohistochemistry confirmed the presence of human leukocytes in the intestines. Chimerism was T cell predominant; however, treatment with ACK2 yielded higher levels of B cells chimerism within the spleen (p=0.01). No mice displayed evidence of GVHD.

Conclusion: In utero transplantation in NSG mice more closely recapitulates a functional human immune system than traditional murine models as engraftment of human HSCs in multiple tissues remains robust without evidence of GVHD. B cell engraftment can be enhanced with ACK2 treatment. This new model of human HSC engrafted mice overcomes the limitation of poor engraftment and demonstrates human leukocytes in multiple mucosal tissues, with important implications for future experiments to understand organ-specific immune responses.

 

58.11 Ameliorating Hind Limb Ischemia-Reperfusion Injury in Mice with a Mitochondrial Targeting Peptide

Posted on January 26, 2017

H. Albadawi1, A. A. Tzika3, J. D. Long1, G. M. Cach1, H. Yoo1, W. G. Austen2, M. T. Watkins1  1Massachusetts General Hospital,Department Of Surgery, Division Of Vascular And Endovascular Surgery,Boston, MA, USA 2Massachusetts General Hospital,Plastic And Reconstructive Surgery,Boston, MA, USA 3Massachusetts General Hospital And Shriners Burns Institute,NMR Surgical Laboratory,Boston, MA, USA

Introduction: Skeletal muscle necrosis following an acute ischemic event may lead to a limb threatening complication in the lower extremity. Mitochondrial dysfunction is a component of ischemic muscle necrosis, which leads to metabolic deficit and a subsequent inflammatory response during reperfusion. Recent studies have reported that targeting mitochondria with d-Arg-2′, 6′-dimethyltyrosine-Lys-Phe-NH2 peptide (SS-31) resets mitochondrial function, reduces apoptosis, and protects against tissue injury in models of acute renal, cardiac, and cerebral ischemia. Experiments were undertaken to assess whether SS-31 treatment can ameliorate myofiber injury and reduce metabolic deficit in a mouse model of acute hind limb ischemia reperfusion injury (IR).  

Methods: Two groups of C57BL6 mice were subjected to 1.5 hours of hind limb tourniquet ischemia followed by 72 hours of reperfusion. Mice received intraperitoneal injections of either 3 mg/kg SS-31 (n=8) or normal saline (n=8) at 5 minutes before ischemia, 5 minutes before reperfusion, and at 2, 4, 6, 24, 48 and 70 hours following reperfusion.  Assessment of ischemic limb function was serially performed using a rodent scoring system. After 72 hours of reperfusion, hind limb muscles were harvested for quantitative analysis of myofiber injury (histologic assessment), infiltrating leukocytes (Ly6b immunohistochemistry), ATP levels (chemiluminescent assay), pro-apoptotic marker (cleaved caspase-3 by western blot), and expression of the proinflammatory chemokine-keratinocyte chemoattractant (KC) protein (ELISA). Data were analyzed using a Student t-test.

Results: At 72 hours following IR, SS-31 treated mice had a greater recovery of limb function when compared to the saline group (p=.008), which corresponded with reduced myofiber injury (47±12 vs. 85±5 percent injured fibers per field, p=.049) and enhanced steady state levels of ATP (12±2.4 vs. 3±1 pmol/µg protein, p=.001). Furthermore, SS-31 treatment correlated with diminished inflammatory cell infiltration (35±23 vs. 115±13, average counted Ly6B+ leukocytes per field, p=.006), lower KC protein content (6.6±0.8 vs. 13.5±2 pg/mg protein, p=.001) and decreased caspase-3 activation (1.7±0.3 vs. 4.8±1 AU, p=.002) in skeletal muscle tissues.

Conclusion: Our results demonstrated that SS-31 treatment is protective against myofiber necrosis following acute IR. This effect may be attributed to the preservation of mitochondrial function and the modulation of apoptotic and pro-inflammatory responses. SS-31 may be useful as a therapeutic adjunct for limb salvage in patients following revascularization. 

 

58.10 Importance of c-kit Signaling in Arteriogenesis.

Posted on January 26, 2017

R. M. Lassance-Soares1, D. R. Hernandez1, M. T. Pinto3, C. D. Rodrigues2,3, Z. Liu1, R. I. Vazquez-Padron1, O. C. Velazquez1  1University Of Miami,DeWitt Daughtry Family Department Of Surgery,Miami, FL, USA 2University Of Miami,Molecular And Cellular Pharmacology,Miami, FL, USA 3University Of Miami,Interdisciplinary Stem Cell Institute,Miami, FL, USA

Introduction:  Chronic limb ischemia is associated with high morbidity with approximately 200,000 major lower limb amputations annually in the USA. Despite the progress in bypass or endovascular procedures, 50% of affected patients are not eligible for these treatments. The spontaneous enlargement of native collaterals, called arteriogenesis, functions as a natural bypassing of a main occluded artery, recovering the blood flow distal to the occlusion. Despite significant efforts to understand the vascular process during arteriogenesis, the molecular mechanisms are not fully understood. c-kit is a tyrosine receptor and its pathway plays an important role during angiogenesis; however its role in arteriogenesis (collaterals remodeling) is poorly investigated. Hypothesis: c-kit signaling dictates proper arteriogenesis in the murine ischemic hindlimb.

Methods:  c-kit mutant mice (W/Wv) and their littermates (controls) were subjected to femoral artery ligation. Laser Doppler assessed blood flow after hindlimb ischemia with time (days: 3, 7, 14, 21 and 28). Ischemic tissue and function of the foot were quantified postoperatively (days: 1, 3, 7, 14). Pressure myograph measured vascular function of mesenteric arteries in dose response to acetylcholine and sodium nitroprusside (NO donor). 

Results: Blood flow recovery was impaired in c-kit mutant mice on days 7, 14, 21 and 28 (p<0.05; n=10). This data suggests dysfunction in collaterals remodeling and not anatomical differences (number and/or diameter of native collaterals), since blood flow was similar between groups immediately post and on day3. Foot ischemic damage was greater (on days 3, 7 and 14 p<0.05; n=10) and foot function was impaired (on days 1 and 14 p<0.05; n=11) in c-kit mutant mice compared to controls. These data support the blood flow recovery finding confirming the importance of c-kit in arteriogenesis. Vasodilatation of mesenteric arteries subjected to different doses of acetylcholine was not different between the groups (p>0.05; n=5). However, when subjected to different doses of sodium nitroprusside, c-kit mutant mesenteric arteries showed significant impairment in vasodilation (p<0.05; n=5). Because sodium nitroprusside acts in smooth muscle cells (SMCs) as an NO donor, these findings suggest SMCs dysfunction on c-kit mutant mice.

Conclusion: c-kit signaling is required to an appropriate blood flow recovery after hindlimb ischemia. The lack of c-kit impairs arteriogenesis and it is associated with SMCs dysfunction.

 

58.09 Specific Gut Microbe-Derived Metabolite Signatures Associated with Severe Human Atherosclerosis

Posted on January 26, 2017

C. A. Cason1, K. Dolan5, G. Sharma3, M. Tao3, A. Longchamp3, R. Kulkarni2, L. Xiong2, E. B. Chang5, M. M. McDermott4, C. K. Ozaki3, K. J. Ho2  1Northwestern University,General Surgery,Chicago, IL, USA 2Northwestern University,Vascular Surgery,Chicago, IL, USA 3Brigham And Women’s Hospital,Vascular Surgery,Boston, MA, USA 4Northwestern University,Medicine,Chicago, IL, USA 5University Of Chicago,Medicine,Chicago, IL, USA

Introduction:  Altered gut microbial composition and gut microbe-dependent metabolism of dietary components has been associated with atherogenesis. To expand our understanding of the role of gut microbe-dependent metabolites in atherosclerosis, we selected a panel of 5 metabolites that are known to be differentially produced in germ-free versus conventional mice. To test the hypothesis that specific gut microbe-derived metabolite signatures are associated with severe human atherosclerosis, we determined circulating plasma levels of these metabolites in cohorts of patients with surgical peripheral arterial disease (PAD) and non-PAD controls.  

Methods:  After informed consent, plasma and medical information were collected from patients undergoing carotid endarterectomy (N=48) and lower extremity major amputation (N=12) or revascularization (femoral endarterectomy or bypass surgery; N=39). Control non-PAD plasma specimens (N=22) were obtained from consented age- and gender-matched patients with normal lower extremity ankle-brachial indices. Patients with serum creatinine ≥ 2 mg/dL were excluded. Metabolites and internal standards were extracted in methanol, separated by reverse-phase HPLC, and measured by tandem mass spectrometry on a triple quadrupole instrument operating in multiple reaction monitoring mode. Normalized peak areas (NPA) were calculated using internal standards and converted to concentration using standard curves. Median plasma metabolite concentrations were compared between groups using Wilcoxon signed rank tests.

Results: There were statistically significant differences in 2 of the 5 gut microbe-dependent metabolites between the patients and controls (see Table). In PAD patients, tryptophan was significantly lower (P < 0.001), indole-3-propionic acid was significantly lower (P<.0001), and there was a trend toward a lower kynurenine (P=.15) than in controls. The kynurenine/tryptophan ratio was higher (P = .0005) in PAD patients than in controls.

Conclusion: Specific gut microbe-derived metabolite signatures associate with severe human atherosclerosis, including differences in tryptophan, kynurenine, and indole-3-propionic acid levels in severe atherosclerosis patients. Beyond potential roles as powerful quantitative biomarkers for atherosclerotic disease burden, these findings suggest mechanistic links between defined microbial metabolic pathways and atherogenesis. ?
 

58.07 ATP, a Molecular Mediator of Surgical Trauma to Blood Vessels

Posted on January 26, 2017

C. M. Guth1, W. Luo1, O. Jolayemi1, J. Cheung-Flynn1, C. M. Brophy1,2  1Vanderbilt University Medical Center,Nashville, TN, USA 2VA TN Valley Healthcare System,Nashville, TN, USA

Introduction: Surgical traction injury occurs to human saphenous veins (HSV) at the time of harvest. This injury is associated with vasomotor dysfunction and is reversible by treatment after injury with FCF, a purinergic (P2X7R) inhibitor. Thus, we hypothesized that stretch-induced ATP release acts as a mediator of injury by activating P2X7R, propagating further ATP release, and impairing vascular smooth muscle function.  

Methods: A model of stretch injury was developed using rat aorta (RA).  Tissue was stretched to twice the resting ex vivo length, which represented the haptic endpoint (subfailure overstretch). ATP release was measured using an ATP Bioluminescent Assay Kit. Contractile responses to phenylephrine were determined in a muscle bath. To verify ATP was the mediator of stretch-induced injury, non-stretched RA was treated with exogenous ATP. Both subfailure overstretched- and exogenous ATP-injured RA were treated with A438079 and oATP, antagonists to P2X7R, to examine the role of P2X7R activation in vascular injury.

Results: Subfailure overstretch injury of RA led to ATP release and decreased contractile function as compared to non-stretched controls (p < 0.05). Contractile function was partially restored in subfailure overstretch RA when treated with A438079 after injury (p < 0.05). RA subjected to exogenous ATP treatment had decreased contractile function (p < 0.05) that was mitigated by co-treatment with oATP (p < 0.05).

Conclusion: Data using a subfailure overstretch RA model suggests that vascular dysfunction after surgical traction stretch injury is associated with ATP release, induced by exogenous ATP, and partially restored by treatment with P2X7R antagonists. These data suggest that ATP represents a molecular mediator of surgical traction injury to blood vessels and that P2X7R represents a novel target for drug therapy for reducing pathologic vascular response to injury.

 

58.06 Sca-1 Differentially Affects Atherogenesis in Male and Female LDL Receptor Knockout Mice

Posted on January 26, 2017

N. A. Mansukhani1, Z. Wang1, V. P. Shively1, M. Kelly1, M. R. Kibbe2  1Northwestern University,Surgery,Chicago, IL, USA 2University Of North Carolina At Chapel Hill,Surgery,Chapel Hill, NC, USA

Introduction:

Atherosclerosis is the leading cause of death and disability in the United States.  The manifestations of atherosclerotic occlusive disease affect men and women differently.  Beyond estrogen, the etiologies for these differences are not well characterized.  Stem cell antigen 1 (Sca-1) positive cells are multipotent progenitor cells that reside in the adventitia of arteries, just external to the external elastic lamina.  Their role in atherosclerosis has not been defined.  Thus, the purpose of this study was to characterize the expression of atherosclerosis over time in male and female low density lipoprotein receptor knockout (LDLR KO) mice, and to identify the role of Sca-1 progenitor cells in the development of atherosclerosis.  We hypothesize that Sca-1 progenitor cells impact the development of atherosclerosis.  

Methods:
Male and female LDLR KO, Sca-1 KO, and Sca-1/LDLR double KO mice were bred, genetically confirmed, and colonies maintained.  To assess the development of atherosclerosis in male and female LDLR KO mice, mice were fed a high-fat diet for 8, 14, 18, and 22 weeks prior to sacrifice.  To assess the role of Sca-1 progenitor cells in the development of atherosclerosis, male and female Sca-1/LDLR double KO mice were fed a high-fat diet for 14 weeks prior to sacrifice. Control mice consisted of LDLR KO and Sca-1/LDLR double KO mice fed regular chow. At the time of sacrifice, aortic roots were harvested and oil-red-O staining of the atherosclerotic area was quantified.  n=4-6/treatment group.   

Results:
Time-course analysis of the development of atherosclerosis in the aortic root of LDLR KO mice fed the high-fat diet for 8, 14, 18, and 22 weeks revealed 18.9%, 35.8%, 43.5%, and 47.4% atherosclerosis, respectively (P<0.001).  Unexpectedly, female LDLR KO mice fed the high fat diet developed more severe atherosclerosis compared to males after 14 weeks (females 41.9% vs. males 32.3%), 18 weeks (females 48.3% vs. males 41.6%), and 22 weeks (females 48.8% vs. males 45.4%) (P<0.001).  To determine the role of Sca-1 progenitor cells in this process, Sca-1/LDLR double KO mice fed the high-fat diet for 14 weeks were analyzed.  Interestingly, female Sca-1/LDLR KO mice developed only 36.4% atherosclerosis, which was significantly less compared to the female LDLR KO mice (41.9%, p<0.05).  Male Sca-1/LDLR KO mice developed 32.7% atherosclerosis, which was similar to male LDLR KO mice (32.3%, p=NS). 

Conclusion:
Our results demonstrate that LDLR KO mice fed a high-fat diet develop progressive atherosclerosis in the aortic root over time and that female LDLR KO mice develop more atherosclerosis compared to male LDLR-KO mice.  Additionally, Sca-1 progenitor cells appear to play an important role in the differential development of atherosclerosis between male and female mice and warrants further study.
 

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