65.07 Dectin-1 Signaling Promotes Pancreatic Cancer by Suppressing Adaptive Immunity

Posted on January 31, 2019

J. I. Kim1, D. Daley1, B. Aykut1, J. Gutierrez1, R. Abengozar1, P. Preiss1, R. Chen1, D. Wu1, S. Shadaloey1, A. Ochi1, G. Miller1  1New York University School Of Medicine,Surgery,New York, NY, USA

Introduction:

Pancreatic ductal adenocarcinoma (PDA) is a devastating disease with a high morality burden. Tumor progression in PDA is driven by activation of the innate and adaptive immune responses, and it is well established that innate immune cell subsets within the tumor microenvironment (TME) entrain adaptive immune effector cells towards a tumor-permissive phenotype. However, the key drivers of immune-suppressive monocytic cell differentiation have not been previously elucidated.

Dectin-1, a pattern recognition receptor expressed on the surface of dendritic cells, macrophages, and inflammatory monocytes, has been identified as a means by which the innate immune system recognizes fungal cell wall components. Our preliminary experiments showed high Dectin-1 expression as well as novel Dectin-1 agonists in the PDA TME. We therefore hypothesized that immune-suppressive cell differentiation and PDA infiltration is driven by Dectin-1 ligation in antigen-presenting cells (APCs), which in turn drives the degree of oncogenic progression in genetically susceptible individuals.

Methods:
We investigated the effects of Dectin-1 ligation using both a slowly progressive authochthonous PDA model (KC and KC;Dectin-1-/-) and an orthotopic PDA tumor model in wild-type versus Dectin-1-/- mice. During tumorigenesis, mice were serially treated with Dectin-1 specific ligand depleted Zymosan (d-Zymosan) or heat-killed candida albicans (HKCA).  Cellular characteristics of the pancreatic tumor microenvironment unique to Dectin-1 ligation or Dectin-1 absence were analyzed via various methods.

Results:

Dectin-1 was more highly expressed in tumor cells and tumor-infiltrating myeloid cells in the PDA TME of both KC mice and within orthotopic KPC tumors. Furthermore, ligation of Dectin-1 via serial d-Zyosan or HKCA treatments accelerated tumorigenesis in KC mice versus vehicle-treated controls, as well as in orthotopically implanted KPC-derived tumors.  Dectin-1 deletion in KC mice also delayed malignant progression, reduced pancreatic tumor weights, and extended median survival by ~5 months. At the cellular level, both KC;Dectin-1-/- pancreata and orthotopic KPC tumor-bearing Dectin-1-/- mice showed ~50% reduction in tumor-associated macrophages versus their respective controls. Additionally, cellular phenotyping of Dectin-1-/- TAMs expressed higher MHC-II and TNF-a, as well as decreased CD206 compared with their Dectin-1+/+ counterparts, suggestive of immunogenic reprogramming towards M1-like differentiation induced by Dectin-1 deletion.

Conclusion:

In PDA, Dectin-1 ligation and, conversely, the absence of Dectin-1 drive differential tumor progression.  Furthermore, in the PDA TME, Dectin-1 signaling pays a key role in reprogramming macrophages towards an immune-suppressive versus immunogenic phenotypes, which carries great promise for potential immunotherapies.

65.06 Neuroblastoma Tumor Growth Suppression with Locally Implanted Cisplatin-Loaded Reservoirs

Posted on January 31, 2019

J. S. Taylor1, B. Yavuz3, J. Zeki1, K. Harrington3, J. M. Coburn3, N. Ikegaki5, D. L. Kaplan3, B. Chiu1,5  1Stanford University,Pediatric Surgery / Surgery / Stanford School Of Medicine,Stanford, CA, USA 3Tufts University,Biomedical Engineering,Medford, MA, USA 4University Of Illinois At Chicago,Surgery,Chicago, IL, USA 5University Of Illinois At Chicago,Anatomy And Cell Biology,Chicago, IL, USA

Introduction:  Advanced stage neuroblastoma requires multi-agent chemotherapy but still portends a poor prognosis. Cisplatin is an effective systemic chemotherapy; however, treatment courses are often shortened because of side effects. We hypothesized that implanting a sustained release reservoir loaded with cisplatin can suppress tumor growth while limiting systemic toxicity.

Methods:  Specially designed silk protein reservoirs were prepared using previously described techniques and loaded with 0.2 mg or 0.5 mg cisplatin. In vitro testing was performed to evaluate the cisplatin release profile from the reservoirs, and dose-dependent toxicity was determined using human neuroblastoma KELLY cells. Subsequently, KELLY cells were injected into mouse adrenal glands to create orthotopic tumors. After the tumors reached 100 mm3 by ultrasound, a sustained release reservoir was implanted into the tumors; reservoirs were either empty controls or loaded with 0.2 mg or 0.5 mg cisplatin. Tumor growth was followed with serial ultrasounds and animals were euthanized when tumor volume exceeded 1,000 mm3. Paraffin-embedded tumor sections were stained with hematoxylin/eosin.

Results: Cisplatin killed 50% of the KELLY cells in vitro at approximately one μg/mL. In vitro, Cisplatin 0.2 mg-loaded reservoirs released 53.28 μg of the loaded drug in 24 hours and 53.31 μg in four days. Cisplatin 0.5 mg-loaded reservoirs released 114.96 μg of the loaded drug in 24 hours and 313.84 μg in 13 days (Fig 1A). In vivo, cisplatin-loaded reservoirs suppressed tumor growth in a dose-dependent manner compared to control treated tumors (p < 0.05). Tumors treated with 0.5 mg cisplatin reached 500 mm3 after 17.6 ±3 6.4 days; tumors treated with 0.2 mg cisplatin took 4.2 ± 1.8 days; tumors treated with control reservoirs took 1.2 ± 0.3 days (Fig 1B). Similar growth suppression was observed at 600, 700, and 800 mm3. No significant toxicity was observed in the cisplatin-treated animals. Hematoxylin/eosin staining demonstrated tumor necrosis adjacent to the cisplatin-loaded reservoir.

Figure 1: A) In vitro cisplatin release profile from silk protein reservoir. Error bars represent standard deviations. B)Tumor growth kinetics when treated with control, 0.2 mg, or 0.5 mg cisplatin-loaded reservoirs. Asterisk (*) indicated p-value < 0.05.

Conclusion: Sustained release cisplatin reservoirs implanted into orthotopic neuroblastoma xenografts were able to significantly suppress tumor growth. Silk reservoirs can be utilized to load a larger amount of cisplatin for tumor implantation.

 

65.05 Immune-Modulating Effects of SMAD4 Associated with Tumor Suppression in Colon Epithelium

Posted on January 31, 2019

P. M. Smith1, A. Means1, S. Novitskiy2, R. D. Beauchamp1  1Vanderbilt University Medical Center,Department Of Surgery,Nashville, TN, USA 2Vanderbilt University Medical Center,Department Of Medicine,Nashville, TN, USA

Introduction:  Evidence suggests that colorectal carcinogenesis is promoted by a combination of microbiota- and host-dependent mechanisms that trigger epithelial cell inflammatory signaling. This is particularly true in ulcerative colitis (UC)-associated cancer (UCAC) in which longstanding UC predisposes to colorectal cancer. Our lab has discovered that TGFβ  signaling via SMAD4 has a central inhibitory role in colon epithelial innate inflammatory signaling. Furthermore, by three months after DSS-induced colitis, 100% of mice with adult-onset deletion of the Smad4 gene in intestinal epithelium developed invasive carcinomas of the colon compared to 0% of control mice. Based on these preliminary observations, we hypothesize that SMAD4 functions as an immune-modulator in colonic epithelial cells through inhibition of pro-inflammatory cytokine production, and loss of SMAD4 results in altered immune cell infiltration.

Methods:  The Smad4 gene was conditionally and selectively knocked out (KO) of intestinal epithelium in adult mice using transgenic mice expressing intestinal epithelial cell selective and inducible Cre-recombinase (Cre) and crossed with mice with LoxP sites inserted into the Smad4 gene locus. Activation of Cre results in excision of the Smad4 gene selectively in the intestinal epithelial cells while retaining it elsewhere, including in leukocytes. The submucosal colonic stroma was isolated from KO and control mice and processed for either RNA-sequencing or for immunophenotyping by flow cytometry. KO and control mouse colon was also dissected and examined histologically by H&E staining for quantity and size of Gut-Associated Lymphoid Tissue aggregates (GALTs).

Results: Compared to control mice, KO mice exhibited a striking increase in stromal gene expression consistent with inflammatory cell signaling with increased expression of multiple pro-inflammatory genes including those related to T cell activation/differentiation, B cell development/signaling, myeloid cell differentiation/proliferation/chemotaxis, and communication between innate and adaptive immune cells (all p<0.0001). Correspondingly, KO of Smad4 in the colon epithelium resulted in a significant and robust increase in leukocyte (CD45+ cell) infiltration into the sub-epithelial compartment (34.0 vs 9.8%, p=0.03). GALT size and number did not vary significantly between KO and control mice (p=0.69 and p=0.91, respectively).

Conclusion: Epithelial SMAD4 modulates the immune system through inhibition of pro-inflammatory cytokines, and adult-onset loss of SMAD4 in intestinal epithelium is associated with increased immune cell infiltration. These results strongly suggest that the SMAD4 tumor suppressor role is associated with its inflammation inhibitory function.

 

65.04 Prenatal Stress Enhancement of NNK-Induced Lung Tumors in AJ Mouse Offspring

Posted on January 31, 2019

T. Ito1,3,4, H. Saeki2,3, X. Guo3, P. S. Shah6, J. Coulter8, K. Tamashiro7, H. Orita4, K. Sato4, A. Hulbert5, K. Rodgers1, B. Lee1, M. Garner1, N. Fackche1, Y. Mei1, M. V. Brock1, K. Gabrielson3  1The Johns Hopkins University School Of Medicine,Surgery,Baltimore, MD, USA 2The Johns Hopkins University School Of Medicine,Pathology,Baltimore, MD, USA 3The Johns Hopkins University School Of Medicine,Molecular And Comparative Pathobiology,Baltimore, MD, USA 4Juntendo University Shizuoka Hospital,Surgery,Izunokuni-shi, SHIZUOKA, Japan 5University Of Illinois At Chicago,Surgery,Chicago, IL, USA 6The Johns Hopkins University School Of Medicine,Radiology,Baltimore, MD, USA 7The Johns Hopkins University School Of Medicine,Psychiatry And Behavioral Sciences,Baltimore, MD, USA 8The Johns Hopkins University School Of Medicine,Radiation Oncology And Molecular Radiation Sciences,Baltimore, MD, USA

Introduction: Lung cancer is the most commonly diagnosed malignancy and the leading cause of cancer-related death worldwide. Maternal stress during pregnancy has both detrimental effects on both mother and fetus. Although in epidemiology studies, prenatal stress has been associated with an increased risk of cancers in later life, the causal relationship between prenatal stress and tumorigenesis has not been determined. To date, no animal studies linking prenatal stress to tumorigenesis have ever been reported. The purpose of this study is to investigate whether prenatal stress can increase tumor multiplicity in the well-established nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced lung tumor model in AJ mice.

Methods: Pregnant AJ mice were timed-bred and assigned randomly at gestation day (GD) 12.5 to either a prenatal stress (PS) group or a control group.  Mice in the PS group were restrained for 2 hours for 5 days through GD16.5. Pups from each group were weaned and housed until 11 weeks of age when all were administered 3 NNK injections of 50mg/kg every other day. All mice were euthanized at 27 weeks of age and examined for hyperplasia and lung tumors. Three different sections of each lung lobe were stained by H&E and examined. Categories of individual proliferation types were defined as follows: C1 for “single layer of hyperplastic alveolar Type II cells lined along alveoli”; C2 for “multiple layers of hyperplastic alveolar Type II cells lined along alveoli”; C3 for “adenoma within hyperplasia”; C4 for “adenoma” and analyzed.

Results: There were significantly more lung proliferative lesions in male and female offspring in the PS group compared to control group [n=16-22, control male group (CM) vs. PS male group (PSM), 9.6 ± 1.0 vs. 12.6 ± 1.0, P=0.021; control female group (CF) vs. PS female group (PSF), 10.3 ± 0.5 vs. 16.9 ± 1.0, P<0.001] (Fig. 1). In both genders, the control group had a significantly higher frequency of C1 lesions in 3 sections of lung compared to PS group. Yet for C2, the opposite was found as the control group had lower frequency of C2 lesions compared to the PS group (CM vs. PSM, C1, 67.9 ± 5.6 vs. 50.4 ± 5.2, %, P=0.041, C2, 28.6 ± 5.5 vs. 46.6 ± 4.6, %, P=0.028; CF vs. PSF, C1, 70.4 ± 5.4 vs. 50.4 ± 5.1, %, P=0.041; C2, 28.8 ± 5.3 vs. 43.5 ± 5.2, %, P=0.068). These results suggest that prenatal stress enhances NNK induced lung tumor pathogenesis.

Conclusion: This is the first report of an animal model that supports the findings in epidemiological studies that prenatal stress may influence lung tumor carcinogenesis. 

65.03 Repurposing of FDA Approved Drugs for Precision Targeted Therapy for Pancreatic Cancer

Posted on January 31, 2019

S. Liu1, J. Yu1, R. Damoiseaux2, R. Sanchez1, F. Brunicardi1  1University Of Toledo Medical Center,Department Of Surgery,Toledo, OH, USA 2David Geffen School Of Medicine, University Of California At Los Angeles,Molecular Screening Shared Resource,Los Angeles, CA, USA

Introduction: Precision cancer therapy requires matching of the tumor’s genomic profile to targeted therapy. Development of a novel targeted therapy takes years and is associated with tens of millions of dollars and low probability of success. In this study, we have tested the hypothesis that genomic profiling using RNA Seq and high throughput screening (HTS) of an FDA approved drug library using a target gene super-promoter can provide precision cancer therapy for pancreatic adenocarcinoma (PDAC) using repurposed FDA approved drugs.

Methods: RNA-Seq and Weighted Gene Coexpression Network Analysis (WGCNA) were performed on 45 human PDAC vs matched benign pancreatic specimens and networks of target genes (TG) were identified. BIRC5 was selected as the target gene for generation of a BIRC5 super promoter (BIRC5-SP). HTS of a 2000 FDA approved drug library was performed using PDAC cells stably transfected with BIRC5-SP-firefly luciferase reporter (Luc). Single vs combination of selected drugs that inhibited BIRC5-SP-Luc activity were further tested in vitro and in vivo using patient derived PDAC cell lines (PDCL 5 & 15) and commercial PDAC cell lines (PANC-1, Mia PaCa2, Capan-2, AsPC1). CMV-Gaussia luciferase stably transfected PDAC cells were used in vivo in xenograft mice to monitor tumor volume by bioluminescence imaging before and after one month of therapy ± standard chemotherapies. IHC, TUNEL assay and RNA-Seq were used for analysis of PDAC gene expression patterns before and after therapy.

Results: 14 BIRC5-SPs were generated and the optimal BIRC5-SP was selected for further studies. BIRC5-SP-Luc HTS revealed panels of FDA-approved BIRC5 inhibitory drugs and three were selected for further dose response testing in PDAC cells. Based on extensive in vitro testing, and a list of criteria to enhance translational potential, simvastatin, metformin and digoxin, (C3), were determined to be the optimal combination and then tested in three in vivo studies using PDAC xenograft mouse models. Following therapy: 1) both PDCL and commercial PDAC tumor growth was significantly suppressed vs controls (p<0.05), 2) the effect of chemotherapy on tumor suppression was significantly enhanced (p<0.05), 3) tumors displayed complete suppression of BIRC5 expression and increased apoptosis, 4) there was no toxicity in any mice. RNA Seq of PDAC tumors revealed a) decreased cell proliferation gene expression, b) decreased ATP/energy gene expression and c) markedly increase cell death gene expression after C3 therapy.

Conclusions: RNA Seq and WGCNA identified BIRC5 as a PDAC target gene. BIRC5-SP HTS identified C3, which suppressed BIRC5 expression and PDAC tumor growth in mice, and enhanced the effect of chemotherapy. Our study demonstrates a feasible cost effective strategy of repurposing FDA-approved drugs for targeted precision PDAC therapy using a super-promoter of the target gene in a clinically relevant timeframe.

65.02 The Early Epigenomic Landscape of Oncogenic KRAS Signaling

Posted on January 31, 2019

R. Kerketta1, A. Mathison3, A. Zeighami3, J. Abrudan3, W. Demos3, M. Zimmermann2,4, G. Lomberk1, R. Urrutia1,3  1Medical College Of Wisconsin,Division Of Research, Department Of Surgery,Milwaukee, WI, USA 2Medical College Of Wisconsin,Bioinformatics Research And Development Laboratory, Genomic Sciences & Precision Medicine Center,Milwaukee, WI, USA 3Medical College Of Wisconsin,Genomic Sciences And Precision Medicine Center,Milwaukee, WI, USA 4Medical College Of Wisconsin,Clinical And Translational Sciences Institute,Milwaukee, WI, USA

Introduction: Pancreatic ductal adenocarcinoma (PDAC) develops through accumulation of genetic alterations, with the KRAS oncogene being the earliest genetic mutation found, which drives the progression of preneoplastic pancreatic intraepithelial neoplasia (PanIN) lesions into carcinoma. Direct targeting of the KRAS gene has been clinically unsuccessful and the downstream impact that the constitutive activation of KRAS has on chromatin remains unknown. Thus, in order to identify chromatin events downstream of oncogenic KRAS which can be clinically targeted, we investigated the earliest changes at the transcriptomic and epigenomic levels that occur following activation of this oncogene.

Methods: Our in vitro pancreatic cell model was derived from a genetically engineered mouse, carrying a doxycycline-inducible KRASG12Dtransgene. At different time points following doxycycline treatment, western blot was used to evaluate levels of oncogenic KRAS. Subsequently, mRNA, cross-linked chromatin and DNA were isolated for next generation sequencing (NGS). These NGS technologies included RNA sequencing (RNA-seq) for gene expression, chromatin immunoprecipitation sequencing (ChIP-seq) of a series of histone marks to assess active and silenced chromatin, and reduced representation bisulfite sequencing (RRBS) for DNA methylation. We used advanced bioinformatics tools to process, integrate and analyze changes in the gene pathways and epigenetic landscape.

Results:Induction of oncogenic KRAS was confirmed by western blot using a G12D specific antibody. RNA-seq data indicated that following KRAS induction, genes involved in the regulation of epithelial to mesenchymal transition (EMT) and metabolic pathways were downregulated, while genes involved in KRAS signaling and cellular proliferation were upregulated. ChIP-seq revealed an increase in the deposition of histone marks associated with enhancers/super-enhancers (H3K27ac and H3K4me1), activated promoters (H3K4me3), and regions silenced by polycomb (H3K27me3). Integration of RNA-seq and ChIP-seq data demonstrated that up- or down-regulated genes also had corresponding alterations of the H3K27ac and H3K4me3 activating histone marks near their promoters. DNA methylation levels of several CpG islands were also altered following KRAS induction.

Conclusion:Based on our results, exposure to oncogenic KRAS induced pancreatic cells to acquire a more epithelial-like phenotype with increased proliferation, which coincides with changes in the transcriptome and epigenome. RRBS indicated that KRAS induction resulted in differentially methylated regions across the genome. Through the analysis of histone marks, we observed a marked increase in active enhancers and super-enhancers, as measured by H3K27ac and H3K4me1 peaks, implicating the role of histone acetyltransferases as downstream epigenetic modulators of the KRAS signaling pathway. Thus, these enzymes may serve as potential drug therapy targets for mitigating the progression of PDAC.

65.01 PAI-1 Activates PSC to Increase Pancreatic cancer stiffness and Determine Early Relapse

Posted on January 31, 2019

Y. S. Shan1,2, Y. S. Shan1,2  1Institute of Clinical Medicine,College Of Medicine, National Cheng Kung University,Tainan, TAIWAN, Taiwan 2Department of Surgery,Division Of General Surgery, Department Of Surgery, National Cheng Kung University Hospital,Tainan, TAIWAN, Taiwan

Introduction:
Pancreatic stellate cells (PSCs) can be activated to induce intra-tumor fibrosis and influence patient survival; however, the molecular basics for the regulation of PSC activation remain unclear.

Methods:
The organotypic coculture system was used to study the interaction between pancreatic cancer cells and PSCs. Cytokine arrays, qPCR, and Western blotting were performed to identify the potential factors in PSC activation and elucidate the underlying pathway.

Results:
Activated PSCs was correlated with increased stiffness of pancreatic tumors, shorter disease-free survival and over-all survival of after resection. Organotypic coculture with cancer cells activated PSCs, which in turn increased the invasiveness of PANC-1 and Miacapa-2 cells and the stiffness of coculture gels. Cytokine array revealed elevated plasminogen activator inhibitor 1 (PAI-1) secretion after co-culture. Treatment with PAI-1 activated PSCs to secrete fibronectin and collagen type 1 and consequently increased gel stiffness. Knockdown of PAI-1 in tumor cells or knockdown of PAI-1 receptor LRP-1 prevented PSC activation after coculture. KRAS mutation in pancreatic cancer cells was associated with increased PAI-1 expression. Pharmacological inhibition of KRAS downstream signaling molecule ERK decreased PAI-1 expression. In addition, in PSCs, PAI-1 triggered ERK phosphorylation and its downstream target c-JUN expression. Inhibition of ERK activation or c-JUN expression blocked PAI-1-induced PSC activation and expression of fibronectin and collagen type 1.

Conclusion:
KRAS-mutant pancreatic cancer cells can activate PSCs through PAI-1/LRP-1 signaling to increase fibrosis and cause early relapse.
 

64.10 Platelet Rich Plasma Induces M2 Macrophage Polarization and Improves Metrics of Mesh Incorporation

Posted on January 31, 2019

J. Van Eps2,3, X. Wang2, S. Minardi2, E. Tasciotti2, J. Fernandez-Moure1,2  1Hospital Of The University Of Pennsylvania,Traumatology, Surgical Critical Care, And Emergency Surgery,Philadelphia, PA, USA 2Houston Methodist Research Institute,Houston, TX, USA 3Houston Methodist Hospital,Surgery,Houston, TX, USA

Introduction:  Platelet rich plasma (PRP), a growth factor rich form of superconcentrated platelets, has been shown to improve metrics of incorporation (MOI), improve acellular dermal matrix (ADM) strength, and reduce recurrence in ventral hernia repair (VHR) with ADM. Macrophage phenotypic differentiation to the M2 phenotype has been shown to promote ADM incorporation. We hypothesized that PRP would induce a differential pattern of macrophage polarization towards a regenerative phenotype and improve early MOI.

Methods:  Lewis rats underwent chronic hernia creation and subsequent repair with non- crosslinked porcine ADM after 30 days(d). A standardized dose (200μl)  of PRP with a concentration of 1×106 platelets/μl  was applied prior to skin closure. The abdominal wall including mesh was harvested at 3d and 10d. To determine macrophage phenotype, the excised mesh and 2 mm  of surrounding tissue was decellularized and flow cytometry performed at 3d using CD3, CD45, CD206, and antimacrophage marker. Expression of genes associated with an inflammatory (M1) phenotype (IL-1b, iNOS, TNFα) and a regenerative (M2) phenotype (ARG-1, IL-10) were then quantified with RT-PCR. To assess MOI, tissues were fluorescently stained for αSMA and CD31 and the expression of Col1, Col3, VEGF, and vWF at 10d quantified by RTPCR .

Results: PRP treated ADMs had increased percentage of M2 (antimacrophage+CD206+) macrophages at 3d (p<0.05) (Figure 1a). This correlated with a significant increase in expression of M2 associated genes (Figure 1 b-c) and decreased expression of M1 associated genes at 3d (Figure 1d-f). PRP treated ADMs showed increased αSMA and CD31 intensity in histology. This finding correlated with increased Col1, Col3, VEGF, and vWF expression at 10d.

Conclusion: PRP, a growth factor rich blood product, improves metrics of wound healing. We demonstrate that M2 polarization is a responsible mechanism for the clinically relevant findings of improved ADM performance seen in our previous studies. PRP induced a differential pattern of macrophage polarization to the M2 phenotype and was associated with improved MOI. These findings suggest that PRP is a useful adjunct with ADM and may have a putative role in other wound healing scenarios, especially those in which graft incorporation is essential.

 

64.09 Improved Pedicle Skin Flap Survival with Hypoxia-Inducible Factor (HIF) DNA Plasmid

Posted on January 31, 2019

P. Shoureshi1, K. Chang1, F. Lay1, Z. Alikhassy Habibabady2, J. M. Abraham1, A. K. Ahmed1, R. Sebastian3, J. W. Harmon1  1Johns Hopkins University School Of Medicine,Dept Of Surgery,Baltimore, MD, USA 2Massachusetts General Hospital,Cardiac Surgey,Boston, MA, USA 3George Washington University School Of Medicine And Health Sciences,General Surgery,Washington, DC, USA

Introduction:

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator that orchestrates the cellular response to hypoxia. Its actions in dermal wounds promote angiogenesis and improve healing. In plastic and reconstructive surgery, surgical skin flaps are frequently used to repair acquired and congenital disabilities. Necrosis of the distal flap is a common complication resulting from insufficient blood supply to the distal flap. Improving blood flow to such flaps utilizing HIF-1 is an attractive strategy for improving flap survival. To explore the feasibility of a local injection of 3194bp plasmid-encoded to express a human degradation resistant HIF-1 (Nature Technology Company, Lincoln, NE) to improve the viability of dermal pedicle flaps by increasing angiogenesis in aged rats 

Methods:

One-year-old Sprague Dawley rats were prophylactically injected intradermally in the marked region of dermis where the flaps would be made. The treatment group received 50μl of 1μg/μl HIF-1α DNA plasmid while the control group received a 50μl saline sham injection. Seven days after treatment, each rat had two 1.6×8 cm isolated pedicle flaps raised with a silicone implanted in between the skin flap and muscle layer. Surviving flap area and blood perfusion measured by laser Doppler imaging were compared for the treatment and control groups. Immunohistochemistry was assessed on tissues stained with CD 31. Levels of human HIF mRNA were measured with qRT-PCR.

Results:

Necrotic area of the skin flap assessed photographically was significantly less in the treatment compared to the control group (the Necrotic area on day 1: 0.4 ±1.2 vs. 2.3±2.5, day 7: 1.0±2.3 vs. 4.0±4.6, day 14: 1.0±2.0 vs. 2.5±2.8, Tukey test, p<0.001). CD31 showed angiogenesis with a significant increase in the number of vessels present per high power field (t-Test, p=0.027). We confirmed that the DNA plasmid was being transcribed using qRT-PCR in skin tissues 24 hours after treatment was administered.  Human HIF mRNA from the plasmid was expressed at 25X the level of endogenous rat HIF.

Conclusion:

Local injection of HIF-1α DNA plasmid promotes angiogenesis and viability of dermal pedicle flaps significantly. This approach has a potential for clinical application in different fields of surgery

 

64.08 Penicillin Induces Colonic H,KATPase via Nitric Oxide Precursors: Novel Target for Diarrheal Control

Posted on January 31, 2019

V. Norz1,2, T. Spingler1,2, T. M. Gisinger1,2, V. M. Baratta2, M. J. Barahona2, J. Ollodart2, D. Mulligan2, J. P. Geibel2  1Paracelsus Medical University,Medicine,Salzburg, SALZBURG, Austria 2Yale University School Of Medicine,Surgery,New Haven, CT, USA

Introduction: Antibiotic-associated diarrhea (AAD) is a well-known complication of antibiotic administration, although the pathogenesis is not clearly elucidated. Symptoms range from mild gastrointestinal disturbances to fulminant gastroenteritis. Surgical patients who receive perioperative antibiotics are at higher risk. Antibiotic-associated diarrhea is thought to be due to an imbalance in the host microbiome, leading to pathogen overgrowth and increased intestinal secretion. We propose a novel pathway of AAD, whereby Penicillin G stimulates intestinal H, KATPase. Activation of H,KATPase leads to intraluminal fluid loss. Here, we demonstrate that L-arginine, a nitric oxide (NO) precursor, works synergistically with Penicillin G to activate H,KATPase. We corroborate this by showing how an inhibitor of the NO pathway, L-NAME, N (ω)-nitro-L-arginine methyl ester, eliminates the secretory effect of L-arginine and Penicillin G.

Methods: Rat distal colons were harvested and placed in solution for crypt isolation. Glands were maintained in a thermostatically controlled perfusion chamber and loaded with a pH indicator dye 2',7'-Bis(2-carboxyethyl)-5(6)- carboxyfluorescein,acetoxymethyl ester (BCECF) to measure intracellular pH in real time. Proton extrusion, a measure of acid secretion of the individual cells, was monitored by observing the recovery of pH, as previously described. A higher pH recovery rate indicates a higher rate of cellular secretion, i.e. diarrhea. Rat colonic crypts cells were perfused with the following solutions: 1.) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES + 5 mM Penicillin G sodium salt (Control) 2.) HEPES + 10 mM L-arginine 2.) HEPES + 5 mM Penicillin G + 10 mM L-arginine 3.) HEPES + 5 mM Penicillin G + 30 μM L-NAME. Statistical analysis of data was carried out with Graphpad Prism 7.0 software.

Results: The mean rate of pH recovery in the control was 0.00623 ± 0.00026 ΔpHi/min, p 0.0004. L-arginine administration increased H,KATPase activity 0.00097 ± 0.00011 Δ  pHi/min, p 0.0002. L-arginine + Penicillin G administration led to a greater, statistically significant increase in H-KATPase activity (Figure 1, 0.00883 ± 0.00051 ΔpHi/min, p 0.0004). Exposing crypts to a combination of L-NAME and Penicillin G provoked a significant reduction in H,KATPase activity (0.00292 ± 0.0001 ΔpHi/min, p 0.0022).          

Conclusion: It is well-known that antibiotic exposure disrupts the microbiome, leading to diarrhea. We propose a novel mechanism of antibiotic-associated diarrhea through Penicillin G’s activation of the H,KATPase. Maximum fluid secretion was found with Penicillin G and L-arginine, a nitric oxide precursor. In contrast, fluid secretion was significantly decreased when tissues were exposed to L-NAME, a known NO pathway inhibitor. This study may provide new therapeutic opportunities to address AAD in clinical settings by modulation of the colonic H,KATPase.
 

64.07 "Natural Brush Border Enzyme IAP Blocks Gut Inflammatory State after Severe Burn Injury"

Posted on January 31, 2019

F. Adiliaghdam1, P. M. Cavallaro1, M. Najibi1, Y. Liu1, L. Rahme1, R. A. Hodin1  1Massachusetts General Hospital,Department Of Surgery/ Harvard Medical School,Boston, MASSACHUSETTS, USA

Introduction: Our previous data has shown that severe burn injury induces gut barrier dysfunction and leads to a gut-derived systemic inflammatory response.We hypothesized that there is a vicious cycle occurring in the gut after acute burn injury which progressively damages the gut barrier and transforms the gut into an inflammatory organ.Furthermore, we investigated how targeting this pro-inflammatory state with supplemental intestinal alkaline phosphatase (IAP) can prevent this vicious cycle.

Methods: Mice were subjected to a 30% total body surface area burn +/- burn site super-infection induced by injection of Pseudomonas aeruginosa into the dermis. IAP was given by gastric gavage.(2000unit)The pro-inflammatory characteristics of intestinal contents was measured in vitro by Caco2 trans-well epithelial electrical resistance (TEER).Primary mouse macrophages were incubated with ileocecal contents to evaluate the immune response.Peritoneal macrophages were evaluated for inflammatory gene expression.

Results: Our data suggest that burn injury leads to an increased expression of pro-inflammatory cytokines such as TNF, IL-6, IL-1B,and lipocalin in the intestine(P<0.01).Burn injury increased peritoneal macrophage pro-inflammatory cytokine expression compared to sham treatment(TNF-a and IL-6, 3-fold increase,P<0.01).Incubation of ileocecal contents of burned mice with Caco2 monolayer negatively affected the monolayer integrity and downregulated the tight junction protein(TJP)expression.There was a significant higher inflammatory response to the ileocecal content of burn-injured mice in both epithelial cells and macrophage cells compared to sham-treated ones(P<0.01).IAP supplementation after burn decreased the intestine inflammation(3-fold decrease,P<0.01).Peritoneal macrophages isolated from IAP-treated mice showed less inflammatory gene expression(P<0.01).Also, IAP treatment was associated with an improved monolayer integrity(3-fold improvement in TEER drop,P<0.05).IAP-treated ileocecal content caused a less inflammatory response in both epithelial cells and primary macrophages(P<0.01).Ex-vivo incubation of burn-injured ileocecal content with IAP decreased the pro-inflammatory characteristics of the contents(3-fold with primary macrophages,4-fold with epithelial cells,P<0.05).Furthermore, IAP treatment improved TJPs expression in in-vitro and in vivo burn models(ZO-1,P<0.05 and Occludin,P<0.01).Interestingly,the Pseudomonas strain(PA14) which was injected subcutaneously into the burn site was detectable in the ileum and stool of the burn infected mice, confirming the existence of a vicious inflammatory cycle in the gut after burn.

Conclusion:Acute burn injury causes an inflammatory response in the gut.IAP supplementation as a natural,anti-inflammatory enzyme significantly decreases gut inflammation and improves gut barrier function,and can represent a novel therapy to prevent a gut-induced systemic inflammation.

64.06 The Potential Role of B Cells in Mitigating Metabolic Disease After Sleeve Gastrectomy

Posted on January 31, 2019

D. A. Harris1, R. Subramaniam1, T. Brenner1, A. Tavakkoli1, E. G. Sheu1  1Brigham And Women’s Hospital,General Surgery,Boston, MA, USA

Introduction:  Immune dysregulation in obesity causes insulin resistance. Sleeve Gastrectomy (SG) leads to diabetes remission through weight-loss dependent and independent mechanisms. We developed a model of SG in both lean and obese mice to characterize the weight-loss dependent and independent, global and organ-specific, immune cell changes following SG.

Methods:  C57Bl/6J mice were divided into 4 groups: lean sham and SG (n=10,9); diet induced obese sham and SG (n=7,11). Weights and food intake were measured daily. Oral glucose tolerance testing (OGTT) was performed at 2 weeks. B, T, myeloid, NK, NKT, innate lymphoid cell (ILC) populations from jejunal, ileal, cecal, liver, and splenic tissues were profiled at 4 weeks using a 31-antibody panel and time-of-flight mass cytometry (CyTOF). ViSNE analysis and t-tests were used for comparisons.

Results: SG enhanced glucose tolerance in obese and lean mice (OGTT: p<0.01, 30/60 min), but only led to weight loss in obese mice. CyTOF analysis demonstrated a 15% and 10% reduction in splenic CD19+CD11BnegCD21+CD23+ B cells in obese (p=0.002) and lean (p=0.05) SG mice, respectively, compared to shams (figure 1). Further, there was a 14% reduction in mature IGM+IGD+ B cells in both obese (p=0.03) and lean (p=0.02) SG animals. Interestingly in the jejunum of obese, SG animals, there is a conserved, 63% reduction in the same CD21+CD23+ B cell population (p=0.009).

Further, there were population level changes that were unique to the obese phenotype. SG led to an increase in multiple 11B+ B cell subsets including a 2-fold increase in CD21-CD23+ (p=0.022) and a 4-fold increase in CD21+CD23- (p=0.01) B cells compared to shams.

Changes were also seen across myeloid populations. There was a 5.6- and 4.8-fold increase in splenic neutrophils in obese (p=0.06) and lean (p=0.025) SG compared to shams. In obese SG there was a 4.3-fold increase in cecal neutrophils as well. Finally, there was an increase in splenic, M2 (arginase 1+) macrophage polarization in all tested macrophage populations in obese SG animals (p<0.05).

There were no sustained T, NK, NKT, or ILC population changes in spleen, intestine, or liver in either lean and obese SG groups compared to their respective shams.

Conclusion: SG induces both weight-loss dependent and independent immune cell changes. There is a conserved reduction in total and follicular B cells among these changes, which are associated with improved glucose handling and are independent of weight-loss. B cells have been implicated in diet-induced obesity and diabetes. Thus, their downregulation may play a central role in mediating metabolic improvements following surgery.
 

64.05 Sleeve Gastrectomy with Ileal Transposition Increases Peptide YY and Improves Weight Loss in Mice

Posted on January 31, 2019

L. Ying1, G. Breuer1, M. Hubbard1, J. Hwa1, G. Nadzam1, K. Martin1  1Yale University School Of Medicine,New Haven, CT, USA

Introduction: Sleeve gastrectomy with ileal transposition (SGIT) is superior to sleeve gastrectomy (SG) for promoting weight loss in rat and porcine models, and for preventing weight gain in a mouse model of diet-induced obesity. However, it is unknown if SGIT is superior to SG for promoting weight loss and lowering blood glucose in obese mice, and its weight loss mechanism is unclear.

Methods: SGIT was performed on a Pilot Cohort of 5 C57Bl/6J mice (7-8 weeks old), which were followed for 10 weeks to test viability. Whole transcriptome sequencing (RNAseq) was performed on the transposed ileal segments of these mice to identify highly expressed genes potentially responsible for weight loss. Next, SGIT, SG, or sham surgery (SH) was performed on a Study Cohort of 16-week old obese C57Bl/6J mice (40-45 grams, n=12 each). Prior to surgery, mice were grouped to match initial weight and fasting blood glucose. After surgery, mice were fed a low-fat diet and weighed weekly. 4 weeks after surgery, food intake was measured by weighing food over 4 days. 6 weeks after surgery, fasting blood glucose was re-measured. Additionally, after administering a liquid diet bolus via oral gavage, postprandial serum Peptide YY was measured after 15-minutes, 30-minutes, 1-hour, and 2-hours with competitive enzyme immunoassay.

Results: The overall mortality in the Study Cohort was 0%. In the Study Cohort, SGIT mice lost significantly more weight than SG or SH mice (6-week weight in grams±standard error of mean (sem): SH: 35.7±1.1, SG: 31.9±0.7, SGIT: 25.2±0.9). 4 weeks after surgery, SGIT mice consumed significantly less food than SG or SH mice (daily food intake in grams±sem: SH: 3.8±0.3, SG: 2.4±0.4, SGIT: 1.9±0.5). Fasting blood glucose (mg/dl±sem) was not statistically different between SG (77.8±8.6) and SGIT (87.7±7.7) mice 6 weeks after surgery, but both were significantly lower compared to SH mice (134.8±3.7). RNAseq of the transposed ileum from the Pilot Cohort revealed high expression of the ileal brake Peptide YY (PYY). Consequently, postprandial serum PYY was measured 6 weeks after surgery in the Study Cohort. Fasting serum PYY was not significantly different between the three groups. However, after administering a bolus of liquid diet, serum PYY rose more rapidly in SGIT mice than in SG or SH mice (graphic).

Conclusion: In this study, we show that SGIT is superior to SG for promoting weight loss, and similarly effective for lowering fasting blood glucose. SGIT mice also consume less food than SG or SH mice. An early release of Peptide YY, confirmed directly via serum measurement, provides a potential mechanistic explanation for the enhanced weight loss observed in SGIT mice.

64.04 Anti-IL-6 receptor Monoclonal Antibody Robustly Ameliorated Postoperative Adhesion Formation in Mice.

Posted on January 31, 2019

J. Fujimoto1, N. Uyama1, H. Tsutsui1, W. Songtao1, M. Sudo1, E. Hatano1, J. Fujimoto1  1Hyogo College of Medicine,Surgery,Nishinomiya, HYOGO, Japan

Introduction: After abdominal surgeries more than 90% of patients reported to have adhesion formation.  It often results in significant morbidity, and treatment of these morbidity costs approximately $1.3 billion per year.  We have developed an experimental mouse model of abdominal adhesion, and revealed that IFN-g and PAI-1 played a pivotal role in adhesion formation by regulating the coagulation fibrinolysis system (Nat Med 2018).  Recently, we have performed RNA profiling and pathway analysis in this mouse model and found that IL-6 is the key molecule for adhesion formation.  In response to IL-6, IFN-g, PAI-1, TGF-b were up-regulated which stimulated the peritoneal mesothelial cells to produce collagen fibers.  Thus, we administrated anti-IL-6 receptor monoclonal antibody (IL-6R Ab) to the mouse to examine its preventive effect on the adhesion formation.

Methods: We induced intestinal adhesion using BALB/c mouse by cecal cauterization as previously reported.  Adhesions strongly connected the cecum to the large bowel, the abdominal wall or both at day 7. We used (1) IL-6 receptor antibody: IL-6R Ab (MR16-1) or (2) IL-6 ligand antibody: anti-IL-6 monoclonal antibody (MP5) to the adhesion mouse models.  PBS or rat IgG was injected to the adhesion mouse models as control groups.  These antibodies or PBS/rat IgG was injected intraperitoneally 24 hours before the cauterization.  Adhesion score (0: no adhesion to 5: very thick vascularized adhesion) was examined at day 7, and immuno- histopathological study and molecular analysis were performed between day 0 and day 7.  Moreover, mice treated with PBS or MR16-1 were injured by skin biopsy to examine wound healing.  The repair rate of the wound was monitored every day until day 7.

Results:A single injection of MR16-1 significantly reduced intestinal adhesion (N=11, score: 1.9±0.49) compared to PBS group (N=10, score: 4.9±0.1, p=0.003)  and rat IgG group (N=4, score: 5.0, p=0.00002).  A single injection of MP5 did not reduce the adhesion score (N=4, score: 5.0).  MR16-1 treated mice had significantly lower expression of IFN-g, IL-6, CXCL2, collagen 1a1, and TGF-b1 mRNA in their ceca.  Histopathological analysis revealed that MR16-1 treated mice had significantly reduced fibrotic change and the neutrophil infiltration.  The skin wound of the first biopsy day were 19.68 mm2 and 19.2 mm2 in rat IgG treated and MR16-1 treated mice, respectively.  They were reduced to 1.6mm2 and 1.07mm2 at day 7, respectively.  Thus, wound healing progressed normally in MR16-1 treated mice.

Conclusion:We revealed that IL-6R Ab strongly prevents intestinal adhesion after surgery in mice.  Human IL-6R Ab, Tocilizmab/ Actemra R is available,
IL-6R Ab treatment presented here may eventually be translated into a useful clinical regimen for the preventing adhesion formation in patients undergoing surgery.

 

64.03 Cerium Oxide Nanoparticle with MircoRNA 146a Delivered via Zwitterionic Gel Improves Skin Strength

Posted on January 31, 2019

S. A. Hilton1, C. Zgheib1, L. C. Dewberry1, M. M. Hodges1, S. Singh3, S. Seal3, G. Sener2, M. Krebs2, K. W. Liechty1  1University of Colorado,Laboratory For Fetal And Regenerative Biology, Department Of Surgery,Aurora, CO, USA 2Colorado School of Mines,Department Of Chemical And Biological Engineering,Golden, CO, USA 3University of Central Florida,Department Of Material Science Engineering, AMPAC And NSTC Center,Orlando, FL, USA

Introduction

Impairments in wound healing and wound strength are a significant clinical problem in diabetic wounds.  We have previously shown that local injection of cerium oxide nanoparticles conjugated to MicroRNA-146a (CNP-miR146a) improves diabetic wound healing in a murine model through decreased inflammation and improved angiogenesis. We hypothesized that topical delivery of CNP-miR146a in a zwitterionic gel would be more clinically relevant, result in improved healing and improved wound strength.

 

Methods

12 week old female mice that are breed homozygous diabetic (Db/Db) were used. A single 8mm full thickness punch wound was made on the dorsal neck skin of each mouse. Zwitterionic gels were prepared by dissolving [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA) and 2-hydroxyethyl methacrylate (HEMA) in water. To load CNPs into the zwitterionic cryogels, CNP-miR146a was added in the gelation solution. Polymerization was initiated using APS and TEMED, then the reaction mixtures were poured into a plastic mold with inner diameter of 0.5 cm and polymerized at -20 ºC for 24 hr. Cryogels were thawed at room temperature. To remove unreacted monomers and other unbound ingredients, the cryogels were washed with PBS several times.

 

Wounds were treated with one time administration of zwitterionic gel only (n=5) or gel impregnated with CNP-miR146a (n=5, ~10ng  Wounds were photographed over time to closure, and animals were euthanized 4 weeks after wound closure for biomechanical testing. A dumbbell shaped sample was taken from cranial to caudal on each mouse with the healed wound in the center. The Instron 5942 testing unit with Bluehill 3 Software was used for examining maximum load, extension, tensile strain.

 

Results

Mice wounds treated with CNP-miR146a gel demonstrated a significant improvement in time to complete wound healing. Untreated diabetic mouse wounds typically heal at day 22-24 post healing. Wounds treated with control gel healed at day 20 and wounds treated with CNP-miR146a impregnated gel healed at day 14 (P-value=0.002). Wounds also showed improved strength after healing with increased maximum load of 3.24N compared to 2.04N (P-value = 0.03). Elastic modulus measures resistance to being deformed when stress is applied. We see improved modulus with CNP-146a gel compared to control gel – 22.26MPa compared to 14.68MPa (P-value = 0.02). Tensile stress at maximum load is also improved – 1.63MPa in control gel compared to 2.59 in treatment gel (P-value = 0.03).

 

Conclusions

Diabetic mice wounds treated with zwitterionic gel impregnated with CNP-miR146a demonstrated improved time to complete wound healing, strength, elasticity, and resistance to stress after healing. This study shows feasibility of topical delivery of a therapeutic for diabetic wound healing via zwitterionic gel, as well as no adverse effects on wound strength.

64.02 Post-operative Ileus is Reduced by Pharmacologic and Genetic Inhibition of Toll-like Receptor 4

Posted on January 31, 2019

M. L. Kovler1, C. P. Sodhi1, M. R. Ladd1, A. Werts1, W. B. Fulton1, T. Prindle1, S. Wang1, Y. Yamaguchi1, D. J. Hackam1  1Johns Hopkins University School Of Medicine,Baltimore, MD, USA

Introduction:

Post-operative ileus (POI) occurs frequently after abdominal surgery and leads to increased morbidity and prolonged hospitalization. Inflammation within the gut wall triggered by intestinal manipulation is recognized as an inciting event in post-operative ileus, suggesting that the innate immune receptor toll-like receptor 4 (TLR4) may be involved. We have recently identified a novel TLR4 inhibitor, C34, which is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which is safe and effective in a variety of pre-clinical studies of gut inflammation. We now hypothesize that TLR4 activation mediates POI, and that pharmacologic TLR4 inhibition with C34 will reduced this postoperative condition.

Methods:

Post-operative ileus was induced by standardized small bowel manipulation in adult wild-type (Tlr4WT) and TLR4 knockout (Tlr4-/-) mice, which we previously generated. Twenty-four hours after manipulation, GI transit was measured by treating mice with fluorescent dye by oral gavage. After 60 minutes, mice were sacrificed, and gastrointestinal motility was expressed as small intestinal transit (%) = the fluorescent pass distance/the total length of small intestine x 100. GI mucosal inflammation was characterized by quantitative RT-PCR measuring expression of TNFa, IL-1b, Lipocalin-2, iNOS, RORC, and FOXP3 from ileal segments. The mice in the control group did not undergo any operation. Motility and inflammatory indices were measured in mice treated with lipopolysaccharide (LPS), the main TLR4 ligand. To evaluate the effects of pharmaceutical inhibition of TLR4, the same experiments were conducted in Tlr4WT mice after intraperitoneal injection with C34. Comparisons were by student’s t-test with p<0.05.

Results:

In Tlr4WT and Tlr4-/- control mice, the degree of GI motility was similar (Tlr4WT: 100% vs. Tlr4-/-: 95.7% p=.41), and the addition of C34 to unperturbed Tlr4WT mice had no effect on motility (saline: 90% vs C34: 93%, NS). By contrast, Tlr4WT mice treated with the TLR4 agonist LPS (dose 3mg/kg) showed decreased gut motility (saline: 87.5% vs. LPS 60.4%, p<0.05) and also significantly increased gut inflammation as represented by TNFa level (saline: 1.39 vs LPS 10.31, p<0.05). The induction of post-operative ileus resulted in significantly reduced transit (Tlr4WT: 44.6%, Tlr4-/-: 57.9% p<0.05) and increased inflammation (Tlr4WT: 21.3, Tlr4-/-: 9.91 p<0.05), which were milder in Tlr4-/- mice. Strikingly, treatment of mice with the TLR4 inhibitor C34 significantly reduced inflammation and improved motility after intestinal manipulation, consistent with reversal of this post-operative complication.

Conclusion:
 

These results prove that experimental post-operative ileus is mediated through a TLR4-dependent inflammatory pathway and reveal that a novel TLR4 inhibitor can attenuate inflammation and dysmotility.

64.01 Remote Ischemic Conditioning to Decrease Postoperative Complications After Major Abdominal Surgery

Posted on January 31, 2019

J. Rosado1, A. Alvarez1, K. P. Oberoi1, G. Dikdan1, S. R. Pentakota1, S. Husain2, B. Koneru1  1Rutgers New Jersey Medical School,Surgery,Newark, NEW JERSEY, USA 2Rutgers New Jersey Medical School,Genomics Center,Newark, NJ, USA

Introduction:  Remote ischemic conditioning (RIC) modulates inflammation after ischemia reperfusion, and may decrease postsurgical inflammation and complications. Here we present preliminary data regarding peripheral blood leukocyte gene expression in subjects from a prospective randomized a phase II clinical trial of RIC in progress (NCT03234543).

Methods: Adults undergoing abdominal surgery (duration > 2 hours, hospital stay > 2 days) are randomized (1:1) to receive either RIC or sham intervention immediately before surgery and on postoperative days 1 and 2. Each RIC intervention comprises 3 cycles of 5/5 minutes of inflation/deflation of mid-thigh cuff in one lower extremity. Inflation pressures were 250 mmHg for the first and systolic pressures plus 50 mmHg for the others. Sham interventions comprise cuff inflation pressure of 20 mmHg.  Primary outcome is 30-day complications measured as comprehensive complications index. Peripheral blood was collected for plasma and RNA before (R0) and 1hr post surgery (R1), and 1 hour after 2nd and 3rd interventions (R2 and R3). Leukocyte mRNA transcripts were examined in R0 and R3 samples of both groups using RNA sequencing (30 million reads) for global expression profiles (n=6 each; fold change >1.5 and false discovery rate p<0.05) and RT-PCR for cytokine transcripts (IL-1β, IL-6, IL-8, IL-10, and TNF-α; n=8 each; Wilcoxon p< 0.05).

Results: 45 of planned 100 subjects (23/22 RIC/No RIC groups) were enrolled. At baseline (R0), mRNA profiles significantly differed between two groups in only 14 transcripts, and none were related to inflammation. 122 transcripts had significant differential expression between two groups at R3. 39/122 were transcripts of immunoglobulins, and expression levels of all were increased in No RIC. Importantly, TMED7, LGALS2, GZMH, and IL32 transcripts encoding proteins, which promote inflammation via IL-1 signaling, macrophage M1 phenotype, granzyme, and p38 kinase and NF-kb pathways, respectively were increased in No RIC. Transcripts of RELL1, IL1RAP, FKBP5, which encode member of TNF-a receptor family, IL-1 receptor accessory protein, and a cellular glucocorticoid responsiveness protein, respectively were decreased in No RIC.  RT-RCR data showed a trend towards increased expression of IL-10 in No RIC that did not reach significance (p=0.10).

Conclusion: Preliminary results from this novel trial suggest that remote ischemic conditioning in the perioperative period down regulates expression of leukocyte proinflammatory genes. Further studies of gene expression in additional subjects and other time points and studies of plasma acute phase proteins and complement components are in progress. Also, the associations between the molecular data and the primary outcome will be examined. 

 

46.18 Evaluation of AIM2 Inflammasome Expression in Sporadic Ascending Aortic Dissection.

Posted on January 31, 2019

W. Ageedi1, P. Ren1, Y. Wang1, J. Guo1, J. Coselli1,2,3, Y. Shen1,2,3, S. LeMaire1,2,3  1Baylor College Of Medicine,Division Of Cardiothoracic Surgery, Michael E. DeBakey Department Of Surgery,Houston, TX, USA 2Texas Heart Institute,Cardiovascular Surgery,Houston, TX, USA 3Baylor College Of Medicine,Department Of Molecular Physiology And Biophysics,Houston, TX, USA

Introduction: Ascending thoracic aortic aneurysms and dissections (AAD) are extremely lethal conditions. No effective medical treatment to prevent AAD currently exists. Identifying the biological pathways responsible for aortic destruction is critical for developing effective treatment. We have recently shown that NLRP3 (nucleotide oligomerization domain–like receptor family, pyrin domain containing 3)–caspase-1 inflammasome cascade degrades smooth muscle cell contractile proteins, leading to aortic biomechanical dysfunction and AAD development. Increasing evidence suggests that Absent In Melanoma 2 (AIM2), another member of the inflammasome family with cytosolic DNA sensing ability, is critically involved in tissue inflammation and destruction.We hypothesized that patients with ascending AAD have elevated aortic tissue levels of AIM2 

Methods: Ascending aortic tissues were obtained from patients with ascending aortic dissection (n=6), patients with ascending aortic aneurysm without dissection (n=6) and organ-donor controls without aortic disease (n=6). We excluded patients with heritable aortic diseases, such as Marfan syndrome. AIM2 expression levels were determined by western blot and immunofluorescence analyses and compared between the three groups. 

Results: Western blot analysis showed that while AIM2 was barely detectable in control and non-dissection aortic aneurysm tissues, AIM2 was markedly increased in aortic dissection samples. Double immunofluorescence analysis showed a significant amount of AIM2 in the aortic media and adventitia of aortic dissection tissue, particularly in smooth muscle cells.

Conclusions: The expression of AIM2 inflammasome is increased in sporadic ascending aortic dissection. Further studies are needed to confirm this finding in larger samples, and to determine the role of AIM2 in aortic inflammation, extracellular matrix destruction, smooth muscle cell dysfunction, and AAD development.

46.17 Cardioscope: A New Innovation for Visualization of Intracardiac Pathology

Posted on January 31, 2019

A. Alotaibi1, G. A. Al-Dossari1, P. T. Roughneen1  1University Of Texas Medical Branch,Cardiovascular And Thoracic Surgery,Galveston, TX, USA

Introduction: Currently, cardiologists and cardiac surgeons visualize intracardiac anatomy through the echocardiography and cardiac catheterization or by open-heart surgery. The concept of cardioscopy, or endoscopy of the heart, dates back to the early 20th century with the first cardioscope developed by Drs. Rhea and Walker in 1913. The first published article was in 1922 by Drs. Allen and Graham. Since then, several attempts have been made to design the ideal cardioscope. Cardioscopy, however, has not advanced as rapidly as other forms of endoscopic surgery because of problems with visualization through blood within the beating heart. We present a novel endoscopic technique in performing a direct visualization of intracardiac anatomy in a porcine heart utilizing carbon dioxide (CO2) and normal saline (NS), and we describe its use as a diagnostic and therapeutic treatment to advance the future of the cardiovascular disease.

Methods: Our model involves cardioscope access in a porcine heart and great vessels with the use of CO2 and NS. During the first trial in 2016, a flexible endoscopy machine was used to visualize intracardiac anatomy. We expanded our work in 2017 and present the data herein. Purse strings were applied on both the right heart side (right atrium and pulmonary artery) and left heart side (aorta and left atrium). The pulmonary veins, the superior vena cava, and the inferior vena cava were closed with 3-0 Prolene sutures to allow the heart to fill with the NS and CO2. A flexible Olympus bronchoscope was used. This endoscope has outside diameters of approximately 5-6 mm, with an ability to flex 180 degrees and extend 120 degrees. The endoscope was inserted through the harvested porcine aortic artery, and CO2 was utilized to inflate the heart.

Results: We were able to view intracardiac structure through different heart axis views (the left and right heart axes). However, despite rotation and flexion of the 180-degree endoscope, it was not possible to see the mitral valve; although, other areas in the left ventricle were visualized.

Conclusion: Cardioscopy has potential as a diagnostic and therapeutic technique. However, the design of a cardioscope should include 360 degrees of rotational capacity and a side arm, the capability for therapeutic intervention, and improved optic visualization through blood utilizing digital subtraction technology.

 

46.16 The Molecular Signature of Deep Partial and Full Thickness Burns

Posted on January 31, 2019

A. S. Karim1, Z. Wang2, C. Kendziorski2, A. L. Gibson1  1University Of Wisconsin,Department Of Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Biostatistics & Medical Informatics,Madison, WI, USA

Introduction: Thermal injury can result in substantial morbidity and mortality. The most important determinant of burn wound healing and the need for surgical intervention is burn depth. There is a gap in our knowledge of how burns progress and if they possess the regenerative capacity needed at various depths of injury to heal the wounds without skin grafting. This is in part due to our incomplete understanding of the interplay between inflammation and the regenerative response. In this study, we sought to characterize the wound microenvironment in deep partial (DPT) and full thickness (FT) burns using high throughput RNA sequencing in order to identify pathways for therapeutic interventions.

Methods: Non-burned (NB; n=6), DPT (n=5), and FT (n=5) tissue biopsies were obtained at the time of burn excision on days 5-10 (median: 6 days) after burn injury then submitted for RNA sequencing. After mapping and normalization, probabilities with a false discovery rate of 5% were calculated to determine differentially expressed genes (DEG). Gene sets of genes that are involved in inflammation, reactive oxygen species (ROS), regeneration, apoptosis and necroptosis were created from the list of DEG and compared in DPT and FT samples normalized to NB. One-sided, Paired t-tests of log2 transformed fold changes were used to compare the two conditions.

Results: 5416 DEG in FT and 4131 DEG in DPT tissues were identified. 1442 of the DEG were different in DPT versus FT. There were no significant differences between DPT and FT with regards to apoptosis and necroptosis (p=0.0763), but there were significant changes with regards to regenerative potential, inflammation and ROS. DPT had greater regenerative potential than FT (p<0.0001), while FT had greater inflammatory response and ROS than DPT (p<0.0001).

Conclusion: In this study we present findings suggesting that there are differences in gene expression profiles between DPT and FT burn tissues that were operatively managed. Genes that are involved in inflammation, ROS, apoptosis and necroptosis were upregulated in FT and DPT relative to NB. Regenerative capacity was significantly lower in FT compared with DPT burns. Overall, this points to an imbalance between the inflammatory and regenerative capacity which is leading to apoptosis, necroptosis and cellular senescence. Gene set enrichment analysis of the mechanisms leading to this imbalance suggests mitochondrial stress and ROS (specifically the NAPDH oxidase system) as some of the biggest mediators. Targeting ROS and preventing mitochondrial depletion may be a viable therapeutic target aimed at preventing burn wound progression and accelerating healing of burn wounds. This will ultimately reduce the need for skin grafting in these patients.

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