78.15 Downregulation of the Tumor Supressor RASSF2 in Colorectal Cancer Cell Lines

Posted on January 26, 2017

J. Carter1, J. Burton1, N. Galbraith1, M. R. Eichenberger1, S. Galandiuk1  1University Of Louisville,Department Of Surgery,Louisville, KY, USA

Introduction:
We have previously identified increased expression of all members of the miR-200 family in colorectal cancer (CRC) as compared to normal colon epithelium cell lines. The members of the miR-200 family are highly enriched in epithelial tissues and they have been linked to several cancers including CRC. Using our screening data, pathway analysis software identified all members of the miR-200 family to have a validated regulatory role upstream of Ras proteins in the MAPK canonical pathway. Further analysis of the miR-200 family confirmed validated target interaction with Ras associated domain-containing protein 2 (RASSF2). RASSF2 is one of six proteins in the RASSF family that is encoded by the RASSF2 gene. RASSF2 is a negative regulator of Ras and binds directly to K-Ras within the Ras effector domain in a GTP-dependent process. RASSF2 has previously been shown to promote apoptosis, cause cell cycle arrest and is regarded as a novel K-Ras-specific effector and potential tumor suppressor. Therefore, we hypothesize that miR-200 family and RASSF2 expression will differ between CRC cell lines and a normal colon epithelial cell line.

Methods:
Four CRC cell lines (SW116, SW480, HT29, T84) and one normal colon epithelial cell line (CCD841) were acquired for use (ATCC®, Manassas, VA). Total RNA was converted to cDNA. Specific TaqMan® miRNA primers and probes for the miR-200 family and RASSF2 mRNA were used to bind to complementary sequences on target cDNA during qRT-PCR (Life Technologies, Carlsbad, CA). All reactions were completed in duplicate. Statistical analysis was performed using a two sample t-test comparing each CRC cell line to the normal colon epithelial cell line. Western blots were performed on cell lysates to identify RASSF2 protein relative concentrations.

Results:
Data are shown in Figure 1. We observed up-regulation of all members of the miR-200 family in CRC cell lines as compared to the normal colon epithelial cell line (p<0.001). Conversely, RASSF2 mRNA was found to be down-regulated in CRC cell lines compared to the normal colon epithelial cell line (p<0.001). RASSF2 protein was absent in 3 of the 4 CRC cell lines and down-regulated in the remaining CRC cell line (Dukes' B, SW480) compared to the normal colon epithelial cell line.

Conclusions:
As hypothesized, miR-200 expression was increased and RASSF2 mRNA and protein was decreased in CRC cell lines, suggesting a potential association between the miR-200 family and RASSF2. Further study into the gain and loss of function of the miR-200 family should be investigated to determine whether the miR-200 directly targets RASSF2 and influences CRC tumorigenesis.

78.13 Characterization of MicroRNA Expression in Colorectal Cancer Cell Lines

Posted on January 26, 2017

J. Burton1, J. Carter1, N. Galbraith1, M. R. Eichenberger1, S. Galandiuk1  1University Of Louisville,Department Of Surgery,Louisville, KY, USA

Introduction:
Cell lines are a common experimental model used to study biological mechanisms involved in various diseases. In-vitro cell line studies allow for the investigation of signaling pathways, functional processes, identification of molecular markers of disease, and testing of cancer therapeutics. microRNAs (miRNAs) affect gene expression by negative regulation of target mRNA and are dysregulated in a variety of cancers including colorectal cancer (CRC). They exhibit a variety of regulatory functions related to cell growth, development, and differentiation. miRNAs are inherently stable accounting for their emerging use as biomarkers for human disease and as targets for disease intervention. We chose to characterize the differential expression of miRNAs in CRC as compared to a normal colon epithelial cell line in order to identify potential therapeutic targets for intervention.

Methods:
Sporadic CRC cell lines (SW116, SW480, HT29, T84)  representing locally confined to metastatic disease (modified Dukes’ A-D staging) and a normal colon epithelial cell line (CCD841) were acquired (ATCC®, Manassas, VA). Total RNA was extracted from cells and RNA quantity and purity determined. For each cell line, RT and qRT-PCR was performed. The expression levels of 380 miRNAs were examined using microfluidic array technology (Life Technologies, Carlsbad, CA). miRNA expression of each CRC cell line was compared to the miRNA expression of the normal colon epithelial cell line. Statistical analysis was performed using a two sample t-test and results were regarded significant when p<0.05.

Results:
Each cell line was screened 4 times. Comparison of miRNA expression in non-metastatic CRC cell lines (Dukes’ A, SW116, and Dukes’ B, SW480) compared to normal colon epithelium (CCD841) identified 190 significantly dysregulated miRNAs, 36 up-regulated and 139 down-regulated miRNA (p<0.05). Five of the 8 most significantly upregulated miRNAs were members of the miR-200 family (p<0.0001, fold-change 2688-18830). Comparison of metastatic CRC cell lines (Dukes’ C, HT29, and Dukes’ D, T84) as compared to normal colon epithelium identified 231 significantly dysregulated miRNAs, 39 up-regulated and 192 down-regulated miRNA (p<0.05). Five of the 6 most up-regulated miRNAs were all members of the miR-200 family (p<0.0001, fold-change 4171-35668). Single assay confirmation of individual miR-200 family members was performed in each cell line.

Conclusions:
We have characterized the miRNA expression in CRC cell lines as compared to a normal colon epithelial cell line. Identification of dysregulated miRNAs permits identification and investigation of clinically relevant miRNAs as potential targets for disease intervention in CRC. Members of the miR-200 family were highly expressed in all tested CRC cell lines. This provides an opportunity to study the miR-200 family already known to play a role in tumor progression and cancer metastasis.

78.12 High Levels of Ceramide in Breast Cancer and Its Microenvironment

Posted on January 26, 2017

K. Moro1, J. Tsuchida1, A. Ohtani1, M. Endo1, T. Niwano1, K. Tatsuda1, C. Toshikawa1, M. Hasegawa1, M. Ikarashi1, M. Nakajima1, Y. Koyama1, J. Sakata1, T. Kobayashi1, H. Kameyama1, K. Takabe2, T. Wakai1, M. Nagahashi1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, NIIGATA, Japan 2Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA

Introduction: Sphingolipids, including sphingosine-1-phosphate (S1P) and ceramide, have
emerged as key regulatory molecules that control various aspects of cell growth and survival in cancer. Despite their critical roles, the levels of sphingolipids have never been measured in patients due to lack of methods to precisely quantify them until recently. We have recently published high levels of S1P not only in breast tumor, but also in tumor microenvironment, such as tumor interstitial fluid (IF), and reported that S1P plays pivotal roles in breast cancer progression. On the other hand, the levels of ceramide, a bioactive metabolite of S1P, in breast cancer patients have not yet well investigated to date. The aim of this study is to determine the levels of ceramide in the tumor and its microenvironment by mass spectrometry utilizing surgical specimens obtained from breast cancer patients.

Methods: Surgical specimens were obtained from 45 breast cancer patients who underwent mastectomy in Niigata University Medical and Dental Hospital. Tissue samples from 1) breast cancer (tumor), 2) peri-tumor normal breast defined as tissue within 1 cm from the gross edge of cancer and 3) normal breast distant from the cancer were collected from the surgical specimens. IF from the tumor, peri-tumor and normal tissue was also collected by a centrifugation method. Sphingolipids, including ceramides (C14:0, C16:0, C18:1, C18:0, C20:0, C22:0, C24:1, C24:0, C26:0) and their metabolites of monohexosylceramides and sphingomyelin, in the tissue samples and the IF were determined by mass spectrometry. Results were analyzed for statistical significance with the Kruskal-Wallis test.

Results:Levels of all species of the ceramides, except for C26:0-ceramide, were significantly higher in tumor tissue than those in peri-tumor and those in normal breast tissue. Similarly, all species of the monohexosylceramides, except for C26:0-monohexosylceramide, were significantly higher in tumor tissue than those in peri-tumor and those in normal breast tissue. All of the sphingomyelin were also significantly higher in tumor tissue than those in peri-tumor and those in normal breast tissue. Importantly, ceramide levels; C14:0, C16:0, C18:1, C18:0, C22:0, C24:1 and the total ceramides, in IF from tumor tissue were significantly higher than those in IF from peri-tumor and from normal breast tissue.

Conclusion:This is the first report that not only the levels of ceramides in tumor tissue, but also those in the IF from tumor tissue shows higher than those in the tissue and IF from peri-tumor and normal breast tissue. At this point, it is unclear whether ceramides in IF is from dead cancer cells, stromal cells, inflammatory cells or adipocytes. Given the fact that ceramides are known to play major role in cancer cell survival, further studies are warranted to elucidate the mechanism.

 

78.10 Inhibition of Cell Proliferation of Pancreatic Cancer Cells by Prostaglandins E1, A2, and B2

Posted on January 26, 2017

J. Yu1, R. Damoiseaux2, S. Liu1, R. Sanchez1, F. C. Brunicardi1  1University Of California – Los Angeles,Department Of Surgery,Los Angeles, CA, USA 2University Of California – Los Angeles,Department Of Molecular And Medical Pharmacology,Los Angeles, CA, USA

Introduction:
The prostaglandins are a family of lipid compounds, found in most human tissues with diverse hormone-like effects. We have recently identified prostaglandins E1, A2, and B2 (PGE1, PGA2, and PGB2) as potential inhibitors of pancreatic cancer proliferation via high-throughput drug screening of small molecules. However, the molecular mechanisms of how prostaglandins inhibit pancreatic cancer cell proliferation are poorly understood.

Methods:
A dilution series for a final concentration from 1mM to 1pM were used to determine the concentration required for 50% inhibition (IC50) for PGA2, PGB2 and PGE1 on human pancreatic cancer cell line MiaPaca2. To further determine whether PGA2, PGB2 and PGE1 inhibit pancreatic cancer cell growth via shared molecular targets or via independent mechanisms, RNA sequencing was applied to pancreatic cancer cell line MiaPaca2 with or without treatment of 5mM PGA2, PGB2 and PGE1. There were 3 replicates per each treatment group. Total RNAs were extracted 72 h post-treatment using Qiagen RNeasy columns. Sequencing reads were obtained using the Illumina HiSeq2000 platform. Differentially expressed genes from treated versus non-treated pancreatic cancer cells were calculated using “Cuffdiff” program from the Cufflinks suites and the top differentially expressed genes were then validated using QPCR.

Results:
The dose-response curve of PGE1 showed that IC50 of PGE1 on Mia Paca2 cell proliferation was 550nM, which was lower than the IC50 for PGA2 (1260nM) and PGB2 (1600nM). Giving a cut-off at log2 fold-change>0.5 and P<0.05, 205, 237 and 284 up-regulated genes and 289, 278 and 355 down-regulated genes were identified associated with PGA2, PGB2, and PGE1 treatment, respectively. Among these genes, only 31 genes in common were up regulated after treatments of all 3 prostaglandins. Gene ontology analysis revealed that these 31 up-regulated genes were enriched in genes of DNA damage response and apoptosis (P=0.05). In contrast, 93 genes were found significantly inhibited in all 3 prostaglandin-treated cells, including Tetraspanin 1 (TSPAN1), Sulforaphane (SFN) and Keratin 15 (KRT15). Gene ontology analysis revealed that these 93 down-regulated genes were enriched in genes involved in epidermal ectoderm development (P=2.9E-6) and cell proliferation (P=0.02). QPCR proved the significant reduction of gene expression for TSPAN1, SFN and KRT15, which have been associated with epidermal ectoderm development and oncogenesis.

Conclusion:
This analysis demonstrates the inhibitory effect of PGE1, PGA2, and PGB2 on pancreatic cancer cell proliferation in vitro. Moreover, by comparing the transcriptome of pancreatic cancer cells before and after prostaglandin treatments, the RNA Sequencing data revealed a list of shared gene targets including TSPAN1, SFN and KRT15, suggesting the mechanism of the inhibitory effect of PGE1, PGA2, and PGB2 on pancreatic cancer cells.
 

78.09 SphKs/S1P Activate Erk, p38 MAP Kinases, and PI3K/AKT/mTOR Pathways for Cancer Cell Survival

Posted on January 26, 2017

A. MAITI1, K. Takabe1, N. C. Hait1  1Roswell Park Cancer Institute,Division Of Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA

Introduction:   Cell proliferation and migration are essential components of cancer progression. Mechanism responsible for highly proliferative cancer cells can be specific targets for the treatments of cancer patients. PI3K/AKT/mTOR signaling pathways are involved in crosstalk with RAS/RAF/MEK/ERK and p38 MAPK pathways, which are considered the master controller of proliferation, migration and cell survival. A bioactive sphingolipid signaling mediator sphingosine-1-phosphate (S1P) has emerged as a key regulatory molecule in cancer progression. S1P is generated intracellularly by two isoenzymes, SphK1 and SphK2. Compared to cytosolic SphK1, SphK2 is mainly localized in the nucleus in many cells. S1P is critically involved in the major pathways in oncogenesis including promotion of cellular survival, proliferation and transformation, prevention of apoptosis, stimulation of angiogenesis and metastasis. Here we hypothesized that SphKs/S1P activate ERK, p38 MAP kinases, and PI3K/AKT/mTOR pathways to regulate cancer cell proliferation and migration.

Methods:  Experiments were done in human breast cancer cells, MCF7 and MDA-MB-453 cells. Serum starved cell were treated with epidermal growth factor (EGF) and S1P. Endogenous activities of SphKs was inhibited with N,N-Dimethylsphingosine (DMS) and either of SphK1 or SphK2 were ectopically over expressed and treated with EGF. Western blots analysis were done to examine protein expression. Cell migration assay was done by DMS pretreatment followed by EGF and S1P treatment. In order to downregulate one particular SphK, we used specific siRNAs in breast cancer cells and treated with chemotherapeutic drug, doxorubicin to measure cell proliferation, apoptosis, and ceramide levels.

Results:  Treatment of cells with S1P from outside activates S1PRs, enhances phosphorylation of ERK, p38, AKT and p70 S6 Kinase1 (SK6). Interestingly, ectopic expression of SphK1 drastically enhanced basal as well as EGF-mediated phosphorylation of those signaling molecules in cultured cells.  Down regulation of SphK2 with siRNA, reduced EGF-mediated SphK2 activity and drastically reduced basal as well as EGF-mediated cell proliferation. Further, inhibiting SphKs activities with DMS reduced S1P mediated P-ERK, P-AKT levels. In addition, DMS significantly reduced basal as well as EGF and S1P-mediated cell migration. Downregulation of individual SphK with specific siRNA, enhanced ceramide levels and sensitizes breast cancer cells to doxorubicin mediated apoptosis.

Conclusion:  Our data strongly suggested that SphK1, SphK2 and generated S1P are playing critical roles to regulate AKT, ERK and p38 signaling pathways to regulate cancer cell proliferation and migration by using different mechanisms based on their cellular location. This study warrants immediate attention to develop specific inhibitors of each SphK for therapy.

 

78.08 Precision Oncology At a Single-Cell Level: Quality Control for Single-cell Whole-Genome Amplification

Posted on January 26, 2017

S. Sho1, C. M. Court1, P. Winograd1, J. S. Tomlinson1  1University Of California – Los Angeles,General Surgery,Los Angeles, CA, USA

Introduction:

Obtaining circulating tumor cells from a peripheral blood draw opens the door to the possibility of applying precision oncology strategies from a “liquid biopsy.” Realization of this potential requires whole genome amplification (WGA) for genomic analysis of single cells. However, WGA remains prone to non-linear and uneven amplifications, resulting in inaccurate representation of the starting genome. WGA reactions are also known to be stoichastic, resulting in highly varied amplified DNA quality between samples. Thus, appropriate selection of the high-quality WGA product is needed to ensure the accuracy and reliability of downstream analysis. Currently, quality analysis methods for WGA products is not well defined, and no clear workflow exists for obtaining and selecting optimal WGA products for downstream molecular assay. 

Methods:
Laser microdissection (LMD) was used isolate pancreatic cancer cell line (HPAF-II) cells. Nine single-cells, six 5-cell groups and six 10-cell groups were obtained for WGA using multiple displacement amplification (MDA) method. Quality of WGA products was assessed by performing real-time quantitative (RT-qPCR) PCR for 8 cancer related genes. Quality score (QS) of 0-8 was assigned based on the ability of RT-qPCR to detect these cancer genes in the WGA DNA product (0: absence, 1: presence). Single-cell WGA with perfect QS of 8 and low QS of 1 were subjected to array comparative genomic hybridization (aCGH) and next generation sequencing (NGS), in order to assess if QS could predict the success of downstream analysis. WGA products of 5-cells and 10-cells with QS of 8 were also subjected to aCGH and NGS, in order to determine the impact of increasing number of starting cells on the quality of downstream analysis. 

Results:

Successful DNA amplification was noted in all samples undergoing WGA. However, only 22% (2 of 9) single-cell WGAs had the perfect QS of 8, with the rest of ranging between 1-3. For both 5- and 10-cell WGA, 50% (3 of 6) of the WGA products had QS of 8. When applied to aCGH and NGS, the single-cell WGA product with low QS of 1 resulted in poor quality data (Table 1). On the other hand, the single-cell WGA with the perfect QS of 8 resulted in a significantly higher quality aCGH and NGS data. When the WGA products of 5-/10-cells with perfect QS of 8 were subjected to aCGH and NGS, results were even better, approximating that of nonamplified DNA.

 

Conclusion:
WGA quality control by RT-qPCR offers a simple, inexpensive way of selecting optimal WGA products for downstream analysis. Furthermore, aCGH/NGS results approximating nonamplified DNA can be obtained by using high quality WGA product (QS of 8) obtained from using 5-10 cells as starting template. 

71.07 Amitriptyline Treatment Improves Survival in a Murine Model of Sepsis

Posted on January 26, 2017

B. T. Xia1, Y. Kim1, E. Gulbins1,2, C. C. Caldwell1  1University Of Cincinnati College Of Medicine,Department Of Surgery,Cincinnati, OH, USA 2Universität Duisburg-Essen,Essen, NORTH RHINE-WESTPHALIA, Germany

Introduction:  During traumatic stress and sepsis, ceramide accumulates within the lipid bilayer, due to increased acid sphingomyelinase (Asm) and decreased acid ceramidase activities.  It has been demonstrated that ceramide augments the innate immune response, such as increased superoxide formation, which may damage tight junction proteins and disrupt epithelial barriers.  Disruption of epithelial barriers in the intestines and lungs has been shown to lead to edema, ischemia, and organ dysfunction.  We hypothesized that Asm inhibition will blunt the innate response and improve survival in a murine model of polymicrobial sepsis.

Methods:  Cecal ligation and puncture (CLP) was used to induce polymicrobial sepsis.  Mice were randomized to intraperitoneal injection of saline vehicle or amitriptyline (16 mg/kg) at the time of CLP.  Systemic and peritoneal bacterial load, immune response, and survival were the primary endpoints.

Results:  Septic mice treated with amitriptyline demonstrated improved survival compared to vehicle (p=0.03). Amitriptyline treatment resulted in decreased peritoneal neutrophil accumulation 16 hours post-CLP compared to vehicle (3.79 x106 cells/ml vs. 6.14 x106 cells/ml, p=0.03), and no changes in systemic and peritoneal bacterial burden. Of note, we did not observe changes in peritoneal neutrophil accumulation, oxidative burst, and systemic and peritoneal bacterial burden 6 hours after sepsis.

Conclusion:  Amitriptyline treatment improved survival in polymicrobial sepsis and decreased neutrophil accumulation after 16 hours.  We postulate that Asm inhibition may reduce epithelial exposure to activated neutrophils such that organ integrity and functionality are maintained.  Future experiments will be conducted to determine neutrophil infiltration, edema, and permeability in the lung and small bowel.  Altogether, we propose that ASM inhibition, in conjunction with antibiotics, is a promising therapeutic approach in the treatment of polymicrobial sepsis.
 

71.05 Inflammatory Regulation Promotes Angiogenesis in Chronic Renal Disease Model

Posted on January 26, 2017

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

Introduction:
Tubulointerstitial fibrosis, compounded with peritubular capillary loss, is a common finding in progressive renal disorders. Treating progressive renal disorders patients’ costs over $40 billion annually in the US alone. Beyond dialysis and transplantation, novel therapies are needed. Previous reports suggest that there is a role for the anti-inflammatory cytokine, interleukin-10 (IL-10), in attenuating renal fibrosis in a disease murine model. We and others also showed that IL-10 can regulate angiogenesis and endothelial progenitor recruitment during dermal and ischemic cardiac tissue repair.  We hypothesize that IL-10 can promote angiogenesis and regulate TGFβ isoforms in unilateral urethral obstruction (UUO) murine model. 

Methods:
Primary fibroblasts (FB) were isolated from 8-10 week-old male C57BL/6J (WT) mice. IL-10 (50 ng/ml or 200 ng/ml) was added to cultures. VEGF and TGFβ-1 gene expressions were assessed by qPCR at 1, 2, 3 and 6h. Levels of TGFβ-1 and TGFβ-3 were determined at 48h by ELISA. Eight week-old WT and IL-10 null male mice were injected with lenti-IL-10/lenti-GFP (1×1010 IU) under kidney capsule. Three days after the injection, unilateral ureteral obstruction (UUO) was performed. UUO/sham kidneys and serum were collected at 14 days after UUO for RNA, ELISA and immunohistochemical analysis. Data presented as mean ± SD, n=3/treatment group. P value by ANOVA.

Results:

In primary FB culture, IL-10 treatment increased VEGF expression and altered the differential expression of TGFβ isoforms, with three-fold increment of ratio between TGFβ-3 to TGFβ-1. The role of IL-10 in regulatory angiogenesis was further validated in IL10-null mouse with UUO.  Lenti-Il10 treatment reduced intertubular fibrotic change (45±7%, p<0.05) and attenuated tubular dilatation in UUO (p<0.05, n=30/group). The CD31, an established endothelium marker, was essential to preserve tubular integrity, normally expressed in healthy tubules and abrogated after UUO. IL-10 null mice revealed a lower basal level of CD31 compared to WT mice (Fig.1). In both WT and IL-10 null mice, IL-10 treatment preserved CD31, suggested a potent capability to rescue peritubular capillary (Fig.1). 

Conclusion:

Our results indicate that IL-10 can effectively promote angiogenesis in vitro and prevent microvascular rarefaction in vivo. Taken together, our study might lead to a novel therapeutic for the treatment of CKD and associated angiogenic morbidity.

71.03 Diabetic MSCs Promote Endothelial Cell Invasion through Unique Angiogen Pathways

Posted on January 26, 2017

A. D. Morris1, H. Li1, L. Brewster1,2  1Emory University Hospital,Division of Vascular Surgery,Atlanta, GA, USA 2Atlanta VA Medical Center,Division of Vascular Surgery,Atlanta, GA, USA

Introduction: Diabetes increases the risk of cardiovascular disease and major amputation. Mesenchymal Stem Cells(MSCs) have robust regenerative potential, but concerns remain that MSCs from diabetic patients or under diabetic conditions may limit therapeutic effect. The objective of this project was to compare healthy MSCs(hMSCs) and diabetic MSCs(dMSCs) angiogenic activity under standard and high glucose conditions.

Methods: MSCs were harvested from 4 patients (2 healthy and 2 diabetic). Human aortic endothelial cells (HAECs) (20,000 cells), HAECs with hMSCs(10,000/10,000 cells) or HAEC with dMSC(10,000/10,000 cells) were formed into pellets, suspended in fibrin gel, and cultured in 5mM, 20mM, or 40mM glucose media. Cell invasion was captured by daily microscopy for 3 days. To obtain secretome, hMSCs and dMSCs were cultured under 5mM glucose, 20mM glucose, or 40mM glucose in 10% FBS media for 24 hours. Secretome analysis was performed using a commercial microarray for 20 individual angiogenic factors. Densitometry using positive control as reference allowed comparison of relative secretion.

Results: High glucose environments did not significantly inhibit HAEC invasion at any time point. At day 3, co-culture with hMSCs improved HAEC invasion under high glucose conditions(20mM and 40mM, p= 0.004 and p=0.02) but not in 5mM glucose media(p=.24). dMSC co-culture also improved HAEC invasion at day 3 (p<0.001 for all conditions). On day 3, dMSCs encouraged HAEC invasion more than hMSCs in 5mM glucose and 40mM glucose, and equivalently in 20mM glucose (p<0.01,p<0.01,p=0.14). hMSC co-culture improved HAEC invasion in 20mM glucose compared to 5mM and 40mM glucose conditions(Day 2:p=0.034,p<0.001, Day 3:p=0.029,p=0.013). On day 1, 40mM glucose inhibited HAEC invasion in dMSC co-culture compared to 5mM glucose(p=0.002), but that difference did not persist on day 2 or day 3(p=0.366, p=0.176).

Compared to hMSCs, dMSCs secreted increased levels of CXCL-1(p=0.002). Secretion of CXCL-1 was an average of 6.5 times greater by dMSCs versus hMSCs. Additionally, 20mM glucose environments stimulated secretion of CXCL-1 compared to 5mM and 40mM glucose in dMSCs(p=0.043, p=0.021).

Conclusions: We have shown that addition of hMSCs and dMSCs are capable of improving cell invasion. Diabetic MSCs are not inferior to hMSCs and may have superior function in certain instances. While dMSC and hMSC are functionally similar, dMSCs secrete the unusual angiogen CXCL-1 at higher levels. We theorize that CXCL-1 may serve as an alternate pathway for encouraging endothelial function under diabetic conditions. With future in vivo experiments, we hope to show dMSCs retain their function by alternate mechanisms and dMSCs can be used in clinical trials with equal efficacy.

71.01 Integrin αVβ3 as a Target to Increase Endothelial Cell Recruitment Under Shear Stress Conditions

Posted on January 26, 2017

E. S. Lee1, K. Samadzadeh1, A. Afkhami1, A. Rona1, D. Hao2, A. Wang2  1Sacramento Veterans Affairs Medical Center,Research Service,Mather, CA, USA 2University Of California – Davis,UC Davis Medical Center,Sacramento, CA, USA

Introduction:  Endothelial cell seeding has been shown to improve long term vascular graft patency and minimize intimal hyperplasia. Previous work has shown endothelial cells wash away after exposure to shear stress conditions. We hypothesize that the use of a novel ligand targeting αVβ3 can improve upon endothelial cell recruitment and adhesion under shear stress conditions.  The purpose of this study is to compare the effects of shear stress in vitro on cell recruitment and adhesion between an αVβ3 Ligand and other conventional ligand substrates.

Methods:  Channels in a BioFlux200 48 well plate were treated with Avidin (20 μg/mL) + Biotin (2 μM) and at various concentrations of the αVβ3 ligand or a fibronectin analog arginylglycylaspartic acid (RGD) (0.05 μM, 0.2 μM, 0.5 μM, 2.0 μM, and 5.0 μM) to determine optimal standard concentration. All channels were treated with phosphate-buffered saline (PBS), Avidin (20 μg/mL), RGD (2 μM) or the αVβ3 ligand (2 μM). Human umbilical vein endothelial cells (HUVECs) from commercial stock of low passage were suspended in media and seeded into channels at a concentration of 1×106 cells/mL under 2 dyn/cm2 shear stress for 2 hours at 37 C. Following cell seeding, the channels were fixed using 3.7% PFA and imaged using an Olympus IX81 fluorescent microscope to determine recruited cell count.

Results: Under shear stress, greater cell recruitment was seen from the αVβ3 ligand treatment (2.62×105 ± 1.02×105) when compared to RGD treatment (1.13 x105 ± 1.03×104) in static concentrations (Figure 1, p = 0.03). Cell adhesion and recruitment were directly correlated with increasing concentrations of both the αVβ3 ligand and RGD treatments.  Cell recruitment between 2 μM and 5 μM αVβ3 ligand was not significantly different. In concentrations above 2 μM, greater adhesion overall was seen from the αVβ3 ligand, which influenced more cell-cell adhesion and clustering resulting in a vastly greater number of recruited HUVECs (4.16×105 vs. 2.57×106).

Conclusion: Targeting for the integrin αVβ3 increases endothelial cell recruitment and adhesion under shear stress conditions much greater than other accepted standard ligand substrates. Clinical application in targeting for the αVβ3 ligand may assist in improving endothelial cell seeding and thereby decreasing vascular bypass graft complications. 

 

63.10 The Role of the Etoposide Induced 2.4 Gene Ei24 in Regulating Pancreatic β Cell Function

Posted on January 26, 2017

S. R. Hamarneh1, J. M. Ramirez Decrescenzo1, F. M. Kuehn1, A. R. Munoz1, S. Morrison1, F. Adiliaghdam1, R. A. Hodin1  1Massachusetts General Hospital,Department Of Surgery, General & Gastrointestinal Surgery,Boston, MA, USA

Introduction:  Type 2 diabetes is characterized by insulin resistance, inadequate insulin secretion and declined pancreatic β-cell mass. EI24 is a tumor suppressor gene and has emerged as a regulator of autophagy and inflammatory pathways. We sought to explore the role that EI24 plays in insulin production and β-cell proliferation.

Methods:  We performed expression analysis of the EI24 gene using Gene-ontology data from patients with or without diabetes. To study the role of Ei24 in β-cell function, rat insulinoma cell line INS-1 was incubated with different inflammatory mediators and under nutrient deprivation conditions to study the effect of these stressors on Ei24 levels. Additionally, we studied the effect of Ei24 deletion or activation on inflammation levels, autophagy influx and insulin production in β-cells in vitro. Furthermore, functional analysis was performed after immunoprecipitation of Ei24 protein complexes to elucidate the plausible cellular pathways affected by Ei24 in pancreatic β-cells.

Results: Ei24 expression levels were lower in human β-cells from diabetic compared to non-diabetic patients (-1.27 vs. 1, p=0.0004). In INS-1 cells, inflammatory mediators such as TNF-α, LPS and bacterial contents suppressed Ei24 levels. Furthermore, the Ei24 expression was induced by nutrient availability. Overexpression of Ei24 increased insulin levels (Ei24 WT vs. Ei24-overexpressing Cells, 1.0 ± 0.3 Vs. 2.8 ± 0.45 Relative Expression, p< 0.01) in INS-1 cells in vitro.  Ei24 deletion in β-cells altered the expression of genes involved in β-cell activity such as Ppar-α (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.24 vs. 0.4 ± 0.056 Relative Expression, p< 0.05), Ppar-γ (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.16 Vs. 7.2 ± 1.0 Relative Expression, p< 0.01) and Pgc1-α (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.18 Vs. 3.7 ± 0.68 Relative Expression, p< 0.01). Also, EI24 deletion increased inflammatory cytokine levels: TNF-α (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.08 vs. 63 ± 7.8 Relative Expression, p< 0.001), IL-1β (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.23 Vs. 57 ± 5.5 Relative Expression, p< 0.001) and IL-6 (Ei24 WT vs. Ei24-KO Cells, 1.0 ± 0.1 Vs. 42 ± 3.8 Relative Expression, p< 0.001) and impaired autophagic flux in β cells in vitro. Functional analysis demonstrated an extended role for Ei24 in β-cell function.

Conclusion: EI24 plays a major role in β-cell function and homeostasis. The EI24 pathway in pancreatic β-cells may represent an important therapeutic target to prevent or treat diabetes in humans.

 

63.08 Menin/MicroRNA-24 Regulatory Axis Drives Hepatic Fibrosis in Mdr2-/- Mice

Posted on January 26, 2017

C. Hall1, L. Ehrlich2, T. Shepperd2, A. O’Brien2, G. Alpini2, S. Glaser2, T. C. Lairmore1  1Scott & White Healthcare,Surgery,Temple, Texas, USA 2Texas A & M Health Science Center College Of Medicine,Bryan, TX, USA

Introduction:
Liver transplantation remains the primary treatment for Primary Sclerosing Cholangitis (PSC), one of several cholangiopathies that result in cholestatic liver disease. Mdr2-/- mice provide an in vivo model of PSC with characteristic biliary inflammation and fibrosis that subsequently develop cirrhosis and hepatic malignancies. Since cholangiocytes express a neuroendocrine phenotype within the liver, we tested the hypothesis that the tumor suppressor protein menin is implicated in the progression of liver fibrosis and that menin expression can be regulated in the liver via MicroRNA-24. 

Methods:
Menin expression was measured in human PSC and Mdr2-/- mice. Twelve-week old Mdr2-/- mice were treated with MicroRNA-24 Vivo Morpholino (miR-24 VM) to knockdown microRNA-24 expression levels. Liver fibrosis was evaluated using sirius red staining, hydroxyproline assay, and qPCR for genes associated with liver fibrosis (fibronectin 1 (FN1), collagen type 1 alpha1 (Col1α1), transforming growth factor-Beta1 (TGF-β1), and alpha-smooth muscle actin (α-SMA)). Intrahepatic bile duct mass was visualized using immunohistochemistry for CK-19, a cholangiocyte specific protein. Studies were replicated in vitro using mouse cholangiocytes and human hepatic stellate cells treated with microRNA-24 hairpin inhibitor and mimic. Menin and microRNA-24 gene expression measured via qPCR.

Results:
Menin gene expression was increased in Mdr2-/- mice and advanced stage human PSC samples. Treatment of Mdr2-/- mice with miR-24 VM increased menin expression, which correlated with increased expression of fibrotic genes (Figure 1). Mice treated with miR-24 VM showed significant increase in peri-ductular fibrosis and bile duct mass. In vitro, inhibition of microRNA-24 significantly increased cholangiocyte expression of FN1, Col1α1, TGF-β1, and α-SMA. 

Conclusion:
The menin/miR-24 regulatory system is implicated in cholestatic liver fibrosis. Inhibition of microRNA-24 increases menin and TGF-β1 expression, subsequently driving hepatic fibrosis and bile duct mass in Mdr2-/- mice, a murine model of PSC. Previous studies in other models of cholestatic liver disease have shown that TGF-β1 drives hepatic fibrosis through increased expression of phosopho-Smads. Modulation of the menin/microRNA-24 axis may provide novel targeted therapies to slow the progression of hepatic fibrosis into cirrhosis by altering TGF-β1 and phospho-Smad expression. 
 

63.05 Endogenous Intestinal Alkaline Phosphatase Modulates Inflammatory Pathways and Homeostasis in the Gut

Posted on January 26, 2017

F. M. Kuehn1, S. R. Hamarneh1, J. M. Ramirez1, A. R. Munoz1, F. Adiliaghdam1, S. A. Morrison1, R. A. Hodin1  1Massachusetts General Hospital,Department Of Surgery,Boston, MA, USA

Introduction:  The inability of epithelial cells to cope with various stresses and cellular damage plays a crucial role in the development of many inflammatory diseases in the gut. The exact functions of the brush border enzyme intestinal alkaline phosphatases (IAP) in various cellular pathways are not well understood. We propose that IAP functions as an important modulator of intracellular homeostasis and stress responses in the gut.

Methods: Functional analysis and identification of IAP-binding proteins were performed using Liquid chromatography-tandem mass spectrometry (LC-MS) after immunoprecipitation of IAP complexes from Caco-2 cells in vitro. Furthermore, IAP knockout and overexpressing Caco-2 cells were developed using CRISPR/Cas9 gene editing technique and cell transfection, respectively. Subsequently, the effect of IAP activation or inhibition on inflammatory cytokine levels was measured using qPCR in the IAP-knockout and overexpressing Caco-2 cells. Additionally, the role of IAP in Caco-2 cell survival was assayed after incubation with high doses of TNF-α.

Results: Analysis of IAP protein complexes showed that IAP binds to key modulators of the NFκB, TNF-α and TLR-4 pathways. Overexpression of human IAP significantly reduced mRNA-levels of inflammatory cytokines in Caco-2 cells: TNF-α (IAP WT vs. IAP-overexpressing Cells, 1.0 ± 0.08 Vs. 0.19 ± 0.1 Relative Expression, p= 0.01), IL-1β (IAP WT vs. IAP-overexpressing Cells, 1.0 ± 0.4 Vs. 0.47 ± 0.06 Relative Expression, p= 0.001) and IL-8 (IAP WT vs. IAP-overexpressing Cells, 1.0 ± 0.26 vs. 0.46 ± 0.16 Relative Expression, p= 0.046). IAP deletion increased the expression of TNF-α (IAP WT vs. IAP-KO Cells, 1.0 ± 0.2 Vs. 7.9 ± 0.7 Relative Expression, p= 0.007) and IL-1β (IAP WT vs. IAP-KO Cells, 1.0 ± 0.6 Vs. 11.4 ± 0.1.1 Relative Expression, p= 0.004) and IL-8 (IAP WT vs. IAP-KO Cells, 1.0 ± 0.12 Vs. 15.9  ± 0.8 Relative Expression, p= 0.0013). Furthermore, overexpression of IAP resulted in significantly less inflammation and cytokine production in Caco-2 cells when incubated with inflammatory mediators such as TNF-α (IAP WT vs. IAP-overexpressing Cells, 9.7 ± 1.8 Vs. 1.2 ± 0.46 Relative Expression, p= 0.004), LPS and bacterial contents. Additionally, higher IAP levels significantly increased Caco-2 survival after incubation with TNF-α 20ng/mL for 24 hours (IAP WT vs. IAP-overexpressing Cells, 30.5 ± 7.2 Vs. 90.3 ± 11.1 % Survival, p= 0.018). 

Conclusion: Endogenous IAP functions as a modulator of the stress response and inflammatory pathways in intestinal epithelial cells. Intracellular IAP pathways in the gut may represent an important therapeutic target to prevent or treat a variety of gut inflammatory conditions.

 

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

Posted on January 26, 2017

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

Introduction:

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

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

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

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

63.02 Del1 Knockout Affects Bone Cartilage Stroma Progenitor Cells Following Femur Fracture in Mice

Posted on January 26, 2017

T. V. Boyko1,2, O. Marecic1, E. Y. Seo1, C. K. Chan1, T. Leavitt1, M. T. Longaker1, G. P. Yang1,3  1Stanford University,Surgery,Palo Alto, CA, USA 2State University Of New York At Buffalo,Surgery,Buffalo, NY, USA 3VA Palo Alto Healthcare Systems,Surgery,Palo Alto, CA, USA

Introduction:  DEL1 is a secreted protein, which has been shown to be involved in bone fracture healing. In previous experiments we have demonstrated that Del1 gene knockout (KO) mice healed fractures with 15% less bone when compared to wildtype (WT) mice. Increased apoptosis was also seen in fracture calluses of KO mice. Separately, we identified the mouse skeletal stem cell (mSSC) and 7 other unique subpopulations of skeletal progenitor cells that are capable of self-renewal and giving rise to all three components of the skeleton: bone, cartilage and stroma. One subpopulation, the Bone Cartilage Stroma Progenitor Cells (BCSPs), are the primary skeletal stem cell population involved in fracture repair. Following fracture, BCSPs transition to another sub-type, f-BCSPs, that has greater osteogenic potential. We hypothesized that Del1 deletion leads to decreased fracture callus due to an effect on BCSP biology. 

Methods:  Femurs were fractured in KO and WT mice. Fracture calluses were harvested on post-operative day 7. BCSPs, mSSCs and f-BCSPs were isolated by Fluorescent Activated Cell Sorting (FACS) following staining for signature cell surface markers. Antibody staining for Annexin V was used to determine extent of apoptosis. KO and WT BCSPs were cultured in vitro and colony-forming units (CFUs) were counted 14 days after plating. 

Results: FACS analysis revealed that the BCSP populations in KO and WT femurs were equivalent prior to fracture (p=0.498, n=3 each). There is an increase of skeletal progenitors in the callus following fracture, but fewer BCSPs were found in KO mice compared to WT (11,700 cells/1 million events vs 77,706 cells/1 million events, p<0.01, n=4 each). Additionally, mSSCs showed a similar pattern in the KO mice with an attenuated increase after fracture (12,452 cells/1 million events in KO vs 57,451 cells /1 million events in WT, p<0.02, n=4 each). The percentage of apoptotic cells was found to be higher in both BCSPs (23.98% KO vs. 6.56% WT, p<0.001, n=4 each) as well as for mSSCs (14.83% KO vs. 4.74% WT, p<0.002, n=4 each). Following culture in vitro, KO BCSPs showed no difference in proliferation compared to WT, but did form significantly fewer CFUs (16.3 vs. 24 CFUs, p<0.05, n=6 each).  Examining f-BCSPs showed an equivalent percentage in KO compared to WT.

Conclusion: Following fracture, skeletal progenitors expand to create bone. In KO mice, this expansion is attenuated leading to decreased bone formation. BCSPs still transition to a more osteogenic phenotype, but there are just fewer of them. These data suggest DEL1 may have a therapeutic role in promoting fracture healing or regenerating bone.

 

62.03 Enhanced serotonin signaling stimulates mucosal growth along the entire small intestine

Posted on January 26, 2017

C. J. Greig1, R. A. Cowles1  1Yale School Of Medicine,Department Of Surgery,New Haven, CT, USA

Introduction:

Intestinal adaptation is regulated by a variety of factors and is thought to be most robust in the ileum. In patients with short bowel syndrome (SBS), resection of the distal intestine is associated with more severe disease and worse outcomes which may be explained by the regional differences in adaptive potential. Enteric serotonin (5-HT) signaling is known to induce mucosal growth in the distal small bowel but its actions at other sites have not been characterized. We hypothesized that enhanced 5-HT signaling would stimulate mucosal growth in all areas of the small intestine, potentially allowing for the development of novel therapies for SBS patients with distal resections.

Methods:

The serotonin reuptake transporter (SERT) inactivates 5-HT. Knockout or pharmacologic inhibition of SERT results in enhanced 5-HT signaling. P-chlorophenylalanine (PCPA) is an inhibitor of 5-HT biosynthesis. With institutional approval, C57Bl/6 wild-type (WT), SERT knockout (SERTKO), and selective serotonin reuptake inhibitor (SSRI)-treated WT mice were used for experiments. Four small bowel segments were harvested from proximal jejunum to distal ileum. Histologic sections were H&E-stained and villus height (VH), crypt depth (CD) and crypt proliferation index (CPI) were measured (n=40 per parameter). Values were compared using Student’s t-test and significance assumed when p≤0.05.

Results:

At baseline, morphometric (VH/CD) and proliferative (CPI) parameters varied from jejunum to ileum. Enhanced 5-HT signaling resulted in significant increases in morphometric and proliferative parameters for nearly every region of the small intestine (see table). Inhibition of 5-HT synthesis with PCPA resulted in reversal of the 5-HT-mediated effects, confirming the role of 5-HT in stimulating mucosal growth.

Conclusion:

Morphometric and proliferative parameters vary throughout the small intestine of WT mice. Enhanced 5-HT signaling resulted in taller villi, deeper crypts and increased crypt proliferation throughout the small bowel. The ability of this model to stimulate mucosal growth from the jejunum to the ileum suggests serotonergic signaling is a viable target for therapies aimed at increasing mucosal surface area in the residual bowel of SBS patients.

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.

 

58.04 Physiological Levels of Resistin Stimulate Inflammation in Macrophage and VSMC Co-culture via PKC-ε

Posted on January 26, 2017

M. C. Zuniga1,2, G. Raghuraman1,2, W. Zhou1,2  1VA Palo Alto Healthcare Systems,Vascular Surgery,Palo Alto, CA, USA 2Stanford University,Vascular Surgery,Palo Alto, CA, USA

Introduction:  Resistin, an adipokine with inflammatory properties, has been associated with atherosclerosis and cardiovascular disease. However, its specific mechanisms of action in key cells involved in atherosclerotic plaque formation are not well defined. We have shown that resistin promotes vascular smooth muscle cell (VSMC) dysfunction at a pathological concentration. The purpose of this study was to examine the direct effect of resistin on macrophage-related inflammation and its indirect effects on VSMCs in the presence of macrophages.

Methods:  Human monocytes were isolated from healthy controls and differentiated into macrophages (MΦ). MΦ were treated with resistin at a “high” physiological level (10 ng/ml) for 18 h, with or without selective PKC-ε inhibitor, εV1-2, at 1 µM. MΦ supernatants (conditioned media) were analyzed with ELISA for pro-inflammatory cytokines. MΦ were assessed for gene and protein expression of inflammatory markers using RT-PCR, immunocytochemistry (ICC) and Western blot, respectively. Conditioned media (CM) generated from MΦ cultures were used to treat human coronary artery smooth muscle cells (HCASMCs) for 24 h.  HCASMCs were evaluated for changes in proliferation using the MTT assay and flow cytometry, as well as gene expression changes of nuclear factor-kappa-B (NF-kB) subunits and of proliferation-associated genes.

Results: Physiological levels of resistin triggered increased gene expression and protein production of the inflammatory cytokines TNF-α, CD40L, IL-6, and resistin in MΦ (Figure 1 A-D). MΦ showed significantly upregulated expression of membrane receptor CD40 and downregulated CD206 (mannose receptor) after resistin treatment. Inhibition of PKC-ε reduced gene expression of inflammatory cytokines and protein expression of CD40. HCASMCs exposed to CM from resistin-treated MΦ showed significantly increased proliferation, while blocking PKC-ε in MΦ during resistin treatment reduced CM-induced proliferation in HCASMCs (Figure 1 E). Furthermore, HCASMCs upregulated gene expression of NF-kB1 (p50), NF-kB2 (p52), cyclin-D1 and proliferating cell nuclear antigen, whereas this effect was not observed in HCASMCs treated with CM from control MΦ.

Conclusion: Resistin at a high physiologic concentration promotes pro-inflammatory MΦ transformation via PKC-ε, and it induces VSMC dysfunction indirectly through MΦ. Targeting resistin may represent a therapeutic strategy in atherosclerosis-associated complications. Further in vivo investigations are warranted.

 

41.20 Smooth Muscle Cell LRP1 Deficiency Affects Genes Regulating Extracellular Matrix Composition

Posted on January 25, 2017

S. Tsai1,2, D. Ramnarain1, M. Kanchwala4, C. Xing4, J. Herz3  1University Of Texas Southwestern Medical Center,Vascular Surgery,Dallas, TX, USA 2Dallas Veterans Affairs Medical Center,Vascular Surgery,Dallas, TX, USA 3University Of Texas Southwestern Medical Center,Molecular Genetics,Dallas, TX, USA 4University Of Texas Southwestern Medical Center,Clinical Sciences,Dallas, TX, USA

Introduction: LDL receptor related protein 1 (LRP1) is an integral regulator of vascular wall structural stability.  A mouse model generated by targeted deletion of LRP1 in smooth muscle cells (smLRP1-/-) in the context of hyperlipidemia is characterized by aggressive aortic atherosclerosis and aneurysmal degeneration.  Even under normolipidemic conditions, smLRP1-/- mice have evidence of structural abnormalities in the aortic wall.  The objective of this study was to understand the enhanced gene expression patterns and signaling pathways driving the changes in aortic wall structure using whole transcriptome sequencing.

Methods:  The aortas of 6 smLRP1-/- mice and their wild type littermate controls were harvested into RNAlater.   The adventitia and peri-adventitial tissues were stripped using micro-dissection techniques.  RNA was extracted and pooled RNA from each genotype was submitted for whole transcriptome analysis, which was performed in duplicate.  Whole transcriptome data was mapped with TopHat and differential expression analysis was carried out using EdgeR.  Genes with false detection rate (FDR) <0.05 (999 genes) were used for pathway/network analysis using Ingenuity Pathway Analysis (IPA).   

Results: LRP1 deficiency in aortic smooth muscle cells (SMCs) was confirmed by immunohistochemistry of descending thoracic aortic cross sections from smLRP1-/- and wild type mice.  Elastin staining revealed diffuse elastin breaks in the smLRP1-/- aortas, accompanied by thickened medial layers of the aortic wall (Figure).  Among the genes that were significantly upregulated in the smLRP1-/-  aorta were 15 collagen genes, including the fibrillar collagens Col1a1 (14.92 fold change, FDR<0.01), Col1a2 (6.39 fold change, FDR<0.01), and Col3a1 (5.82 fold change, FDR<0.01), as well as collagens associated with the basement membrane and regulation of cell migration, including Col4a1, Col4a2, Col4a4, and Col15a1. Elastin gene expression was also upregulated (4.01 fold change, FDR<0.01), in addition to expression of MMP14 (34.19 fold change, FDR=0.01), an activator for MMP2, consistent with observed elastin degradation and partial compensatory upregulation of elastin.  Pathway analysis using IPA software demonstrated upregulation of genes involved in cell migration and cell proliferation pathways, converging on erk1/2, which is known to undergo enhanced phosphorylation/activation in the absence of LRP1.  

Conclusion: Our findings demonstrate that the major aortic wall structural changes associated with LRP1 deficiency in SMCs are driven by enhanced expression of pro-fibrotic genes, in addition to genes involved in cell migration and cell proliferation, as well as a shift towards elastin degradation.

 

41.19 Endogenously Expressed IL-10 Contributes to Wound Healing and Regulates Tissue Repair Response

Posted on January 25, 2017

M. M. Rae1, T. Lu1, C. M. Moles1, X. Wang1, M. Fahrenholtz1, H. Li1, P. Duann1, P. Bollylky2, S. Balaji1, S. G. Keswani1  1Texas Children’s Hospital And Baylor College Of Medicine,Division Of Pediatric Surgery,Houston, TX, USA 2Stanford University School Of Medicine,Infectious Diseases And Microbiology And Immunology,Stanford, CA, USA

Introduction:
Our lab has shown a significant role for the anti-inflammatory cytokine IL-10 in regulating inflammation and ECM production, thereby attenuating fibrosis in skin wounds. Previous reports have shown, paradoxically, that wounds in IL-10-/- mice heal faster, with increased rates of re-epithelialization as compared to wildtype mice. However, these wounds were not controlled for contraction and wound environment. Therefore, we sought to determine the role of contraction on IL-10’s wound healing and anti-fibrotic effects in a controlled moist wound environment.

Methods:
Full thickness excisional 6mm wounds were made in IL-10+/+ and IL-10-/- mice that were controlled for contraction using a silicone stent. A consistent and moist wound environment was provided by semi-occlusive dressing Tegaderm. Wounds were serially photographed at 3, 5 and 7d and harvested at 7d and 14d post wounding, then examined for epithelial gap, granulation tissue (H&E), myofibroblasts (a-SMA staining) and leucocyte infiltration (CD45). Data is presented as mean+/-SD, n=5 wounds/group/time point; p-value by ANOVA.

Results:
Macroscopic appearance of unstented wounds with no dressing showed accelerated wound closure in IL-10-/- mice by day 7 compared to controls(IL-10+/+). This effect was lost when a semi-occlusive wound dressing was applied to unstented wounds to create a consistent moist wound environment, with no significant differences observed in re-epithelialization(IL-10-/- 2380±508.4 vs IL-10+/+ 2480.4.7±824.6, p=ns), epithelial gap (IL-10-/- 1433.7±558.1 vs IL-10+/+ 1436.2±527.7, p=ns), granulation tissue (IL-10-/- 1.65±0.5 vs IL-10+/+ 1.21±0.4, p=ns), or CD45 positive cells (IL-10-/- 10.1%±6.2 vs IL-10+/+ 8.9%±4.2, p=ns). In unstented wounds, a-SMA was abundantly expressed at the wound margins, but in IL-10-/- wounds, a-SMA was present throughout the granulation tissue and extended into deep dermal layers, whereas a-SMA expression was less pronounced in the IL-10+/+ wound bed. Stenting of wounds, which controlled for the contractility of mouse skin, significantly delayed wound healing. However, there was no statistical difference in either epithelial gap (IL-10-/- 4883.5±610.8 vs. IL-10+/+  4152.2.7±480.6, p=ns) or granulation tissue (IL-10-/- 0.33±0.1 vs. IL-10+/+  0.55±0.2, p=ns) at day 7, although stented IL-10-/- wounds exhibited increased a-SMA density. Interestingly, the CD45+ cellular infiltrate significantly increased in stented IL-10-/- mice as compared to unstented (IL-10-/- 10.1%±6.2 vs. IL-10-/- with stent 32.8%±15.6, p<0.01), but did not change in IL-10+/+ mice. Finally, wounds in IL-10-/- mice also developed significantly more scar tissue and much thicker epidermis on day 14 compared to IL-10+/+ mice. 

Conclusion:
IL-10 expression does not delay normal wound healing of skin wounds when wounds are controlled for contraction and moist environment. However, the loss of IL-10 leads to increased fibrosis. This data signifies a previously unrecognized role for endogenously expressed IL-10 contributing to the tissue repair response.  
 

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