47.10 Resistin Promotes Inflammation by Upregulating Expression of Inflammatory Markers in Macrophages

Posted on January 18, 2016

M. C. Zuniga1,2, G. Raghuraman1,2, E. Hitchner1,2, W. Zhou1,2 1VA Palo Alto Healthcare Systems,Vascular Surgery,Palo Alto, CA, USA 2Stanford University,Vascular Surgery,Palo Alto, CA, USA

Introduction: Monocytes and macrophages (MΦ) are key players in atherosclerotic plaque inflammation. Resistin has been associated with increased risk for cardiovascular complications but its mechanisms of action remain to be elucidated. We have previously observed higher levels of inflammatory cytokines in patients with ‘high’ resistin levels (4.97 – 13.3 ng/mL, AVE = 7.29), implicating that resistin may act through inflammatory pathways. In this study, we evaluated the direct effects of high levels of resistin on the monocyte/macrophage inflammatory state.

Methods: Monocytes were isolated from buffy coats of healthy controls, and some monocytes were differentiated into MΦ. Monocytes and MΦ were treated with resistin at a ‘high’ physiological level (10 ng/mL) with or without a selective PKC-ε inhibitor (1 µM). Monocytes/MΦ profiles were evaluated using immunocytochemistry (ICC) and verified with Western blot. CD80 and CD40 were used as markers for the M1 (pro-inflammatory) phenotype, and CD206 (mannose receptor) and CD163 were used for the M2 (anti-inflammatory) macrophage phenotype; CD14 and CD16 were used to assess monocyte states. Levels of resistin post-treatment were measured with ELISA. Data was analyzed with ANOVA, and a p-value of <0.05 was considered significant.

Results: Despite a very low basal expression of resistin (0.15 ng/mL), MΦ treated with resistin (10 ng/mL) showed significantly higher than expected production of resistin up to 18.2 ng/mL ± 1.17 (AVE ± SEM), suggesting a potential positive feedback mechanism in MΦ. This phenomenon was not observed in monocytes. ICC revealed that monocytes treated with resistin did not exhibit a change in CD14 or CD16 expression compared to the controls. Similarly, resistin-treated MΦ did not show significant changes in CD80 or CD163 expression. However, decreased CD206 and a markedly increased CD40 expression were observed (p<0.01). Western blot results confirmed these observations. Moreover, inhibition of PKC-ε mitigated resistin-induced CD40 upregulation (Figure 1 a, b, c).

Conclusion: Our study suggests that resistin stimulates MΦ toward a pro-inflammatory subtype through a positive feedback mechanism. PKCε-specific inhibition decreased CD40 up-regulation, highlighting a possible approach to prevent resistin-mediated M1 polarization and associated cardiovascular complications.

47.05 CD44 Increases Matrix Production By Promoting Inflammatory Responses During Fistula Maturation

Posted on January 18, 2016

J. J. Hanisch1,2, G. Kuwahara1, K. Yamamoto1, T. Hashimoto1, C. D. Protack1,2, T. Foster1,2, H. Bai1, S. M. Jay3, A. Dardik1,2 1Yale University School Of Medicine,Department Of Surgery,New Haven, CT, USA 2VA Connecticut Healthcare System,Department Of Surgery,West Haven, CT, USA 3University Of Maryland,Department Of Bioengineering,College Park, MD, USA

Introduction:
Arteriovenous fistulae (AVF) remain the optimal conduit for hemodialysis access but continue to demonstrate poor patency and with poor rates of primary maturation. We have previously shown that there is a distinct temporal regulation of extracellular matrix (ECM) components during AVF maturation. CD44 is a widely expressed cellular adhesion molecule that serves as a major receptor for ECM components such as hyaluronic acid as well as promoting adhesion of leukocytes to endothelial cells and stimulating smooth muscle cell proliferation and migration. We hypothesized that CD44 promotes wall thickening and ECM deposition during AVF maturation by promoting inflammation in the maturing vein.

Methods:
Aortocaval fistulae were performed by needle puncture in wild-type (WT) C57BL/6J and CD44 knockout (KO) mice. AVF diameter was serially assessed weekly by duplex ultrasound. AVF were harvested at days 7 or 21 and histology analyzed using computerized morphometry, as well as qRT-PCR and Western blot. In both WT and CD44 KO mice, after AVF formation, microspheres containing MCP-1 were placed over the adventitia using a pluronic gel.

Results:
CD44 expression was increased 2.1-fold in AVF (day 7). CD44 KO mice showed reduced AVF wall thickness (8.9 μm vs. 26.8 μm; p = 0.0114, ANOVA), collagen density (1.7 fold; p = 0.0186, ANOVA), and hyaluronic acid density (2.6 fold; p = 0.0004, ANOVA), but similar elastin density (p = 0.9315) when compared to control AVF (day 21). CD44 KO mice also showed reduced VCAM-1 expression (12.0 fold; p = 0.0092, ANOVA), ICAM-1 expression (30.5 fold; p = 0.0002, ANOVA ), and MCP-1 expression (11.4 fold; p = 0.0006, ANOVA) in the AVF compared to control AVF; there were also reduced M2 macrophage markers (TGM2: 81.5 fold; p = 0.0001, ANOVA and IL-10: 7.6 fold; p = 0.0228, ANOVA) in CD44 KO mice. Delivery of MCP-1 to the AVF in WT mice resulted in thicker AVF walls (32.0 μm vs. 24.5 μm; p = 0.0383, t-test), increased collagen density (1.6 fold; p = 0.0184, t-test), and increased number of M2 macrophages (1.5 fold; p = 0.0241, t-test).

Conclusion:
CD44 promotes accumulation of M2 macrophages, ECM deposition and inflammation, enhancing AVF maturation. These data suggest that promoting CD44 activity may be a strategy to enhance AVF maturation, and also show the importance of inflammation to enable wall thickening during AVF maturation.

47.06 Eph-B4 Mediates Arteriovenous Fistula Maturation via Akt-1

Posted on January 18, 2016

T. Foster1,2, C. Protack1,2, T. Hashimoto1, K. Yamamoto1, H. Bai1, J. Hanisch1,2, A. Dardik1,2 1Yale University School Of Medicine,New Haven, CT, USA 2VA Connecticut Healthcare System,West Haven, CT, USA

Introduction:
Although the arteriovenous fistula (AVF) is the gold standard for dialysis access, low rates of fistula maturation prevent optimal fistula use. We have previously shown that stimulation of Eph-B4 in vein grafts prevents wall thickening during venous remodeling. Since Akt-1 is a downstream mediator of Eph-B4 signaling in endothelial cells, we hypothesized that Akt-1 mediates Eph-B4 function during AVF maturation.

Methods:
The infrarenal aorto-caval AVF model was created in wild type C57BL/6 mice and Akt-1 knockout mice as previously described. To stimulate Eph-B4 activity, mice were treated with either EphrinB2/Fc (20 μg IP) or a control vehicle at 48 hr intervals over a 21 day study period. Fistula maturation was monitored with weekly ultrasound measurements; AVF were harvested at day 21 and infrarenal IVC wall thickness was measured using computerized morphometry. Eph-B4 and Akt-1 expression were measured with immunofluorescence.

Results:
In WT mice, Eph-B4 expression increased 4.6-fold in the vein after AVF surgery compared to veins that had a sham operation (p=0.007, t-test). Stimulation of Eph-B4 in WT mice resulted in increased merged Eph-B4 and phospho-tyrosine signal (60% vs 92%; p=0.04, t-test) as well as an 8.2-fold increase in phospho-Akt-1 signal (p=0.04, t-test). Eph-B4 stimulation in WT mice was associated with reduced fistula diameter (45% vs. 98.6%; p<0.05, ANOVA) and reduced fistula wall thickness (8.9μm vs. 20.0μm; p=0.0261, t-test). However, in Akt-1 KO mice, Eph-B4 stimulation resulted in no change in fistula diameter (38% vs 22%; p=0.09, ANOVA) and no change in fistula wall thickness (15.3 vs 17μm; p=0.46, t-test).

Conclusion:
Although Eph-B4 expression is increased in an AVF, Eph-B4 in the AVF is not phosphorylated. However, stimulation of Eph-B4 increases the percentage of phosphorylated Eph-B4 that appears to be active with reduced fistula diameter and wall thickness. Akt-1 knockout abolishes the effects of Eph-B4 stimulation on wall thickness and diameter suggesting that Akt-1 mediates the effect of Eph-B4 on venous adaptation to the arterial environment.

47.07 Limb Demand Ischemia Modulates Revascularization and Adipocyte Differentiation in Obese Mice

Posted on January 18, 2016

H. Albadawi1,2, L. M. Crowley1, M. W. Koulopoulos1, H. Yoo1, M. T. Watkins1,2 2Harvard School Of Medicine,Brookline, MA, USA 1Massachusetts General Hospital,Department Of Surgery, Division Of Vascular And Endovascular Surgery,Boston, MA, USA

Introduction: Claudication, a form of demand ischemia (DI) in patients with peripheral arterial disease (PAD) has been associated with altered skeletal muscle fiber morphology and fatty degeneration. Metabolic syndrome exacerbates exercise intolerance in PAD due to impaired circulation and metabolism. The diet-induced obese (DIO) mouse provides a robust model of humans at risk for PAD and diabetes. This study evaluated the expression of growth factors, cytokine signaling pathways, and regulators of adipocyte differentiation in DIO mice in the setting of DI.

Methods: DIO C57BL6 mice underwent unilateral femoral artery ligation followed by two weeks recovery. Mice were then randomized into two groups. The first group (n=8) was subjected to 1hr of daily treadmill exercise (12m/min, 10° incline) for 4 weeks to induce DI, and a parallel group (n=7) remained sedentary (SI) for 4 weeks. Hindlimb muscles were examined histologically for capillary density (CD31 immunohistochemistry) and muscle cross sectional area (CSA). Hindlimb skeletal muscles were analyzed for the levels of the pro-angiogenic cytokines G-CSF and VEGF using multiplex assay while the phosphorylation of the signaling transduction molecules, pSTAT3 and pERK1/2 and the brown adipocyte marker uncoupling protein-1 (UCP-1) levels were measured by western blotting. Real-time qPCR analysis was used to assess gene expression of the energy metabolism transcription factor PGC1-α. Statistical analysis was performed with student t-test.

Results: (see table) Exercise significantly enhanced capillary density (p=0.0086) and markedly lowered CSA (p=0.0012) in DI muscle compared to SI. These findings were paralleled by a significant increase in G-CSF (p= 0.0006) and pSTAT3 (p=0.0114) protein levels as well as enhanced PGC1-α (p= 0.014) gene expression. There was no difference in the pERK1/2 (p=0.23) or VEGF (p=0.2) protein levels between the two groups. Furthermore, DI muscle had significantly diminished UCP-1 protein levels compared to SI muscle.

Conclusions: These data suggest that DI enhances capillary density but lowers muscle CSA. Since VEGF and ERK1/2 levels were not different between DI and SI muscles, the enhanced capillary density may be primarily dependent on G-CSF and STAT3 signaling pathway after 4 weeks of DI. Exercise also appears to modulate adipogenic differentiation and muscle energy metabolism, evident by the decrease in UCP-1 protein and increase in PGC1-α gene expression. Ongoing regular exercise in patients with PAD is important to augment microvascular perfusion and decrease fatty degeneration, thereby enhancing muscle adaptation to ameliorate claudication and improve limb function.

47.02 atRA Polymeric ePTFE Grafts Inhibit Intimal Formation in a Rat Aortic Interposition Graft Model

Posted on January 18, 2016

E. K. Gregory4, A. R. Webb2,3, J. M. Vercammen4, M. E. Flynn4, W. Jiang4, R. van Lith2, G. A. Ameer2,4, M. R. Kibbe4 2Northwestern University,Biomedical Engineering/ McCormickSchool Of Engineering,Chicago, IL, USA 3University Of Florida,Department Of Materials And Science Engineering,Gainesville, FL, USA 4Feinberg School Of Medicine – Northwestern University,Surgery/ Vascular,Chicago, IL, USA

Introduction: The poor performance of expanded polytetrafluoroethylene (ePTFE) vascular grafts demonstrates the need for alternative prosthetic graft materials. All-trans retinoic acid (atRA) has many beneficial effects on the vasculature, including inhibition of proliferation. We previously demonstrated that localized delivery of atRA through a poly (1,8) octamethylene citrate (POC) periadventitial membrane significantly reduced neointimal hyperplasia after carotid balloon injury in rats. The objective of this study was to develop an atRA-POC coated ePTFE graft and evaluate the biocompatibility and efficacy of this graft in the vasculature. We hypothesize that atRA-POC ePTFE grafts will be biocompatible and inhibit intimal formation after aortic interposition grafting in rats as compared to standard ePTFE grafts.

Methods: To evaluate biocompatibility ex vivo, compliance was evaluated via compression testing, platelet aggregation was evaluated using plasma from Sprague Dawley rats (n=3), and complement activation was assessed using human serum (n=3). To establish efficacy and biocompatibility in vivo, an aortic interposition bypass was performed on 12-week-old Sprague Dawley rats. Treatment groups included ePTFE (n=6), POC ePTFE (n=7), atRA ePTFE (n=6) and atRA-POC ePTFE (n=6). At 28 days the grafts with 1 cm of native aorta and blood were collected. Morphometric analysis was performed on H&E stained cross sections using ImageJ software. Analysis was performed on collected blood samples to evaluate renal and liver function.

Results: All 3 grafts (POC, atRA, and atRA-POC) ePTFE exhibited similar compliance as compared to standard ePTFE grafts. There were no differences in platelet aggregation or complement activation between the treatment groups. Rats who received the atRA-POC ePTFE grafts were found to have 40% less intimal formation at the proximal graft anastomosis and 56% less intimal formation at the distal graft anastomosis compared to control ePTFE grafts (P<0.03). atRA ePTFE grafts resulted in a 53% decrease in intimal formation at the distal graft anastomosis compared to ePTFE control (P< 0.01); however, atRA ePTFE grafts did not reduce intimal formation at the proximal graft anastomosis. There were no clinically significant differences in the liver function tests and renal chemistries between treatment groups.

Conclusion: atRA-POC ePTFE grafts resulted in less intimal formation at both the proximal and distal graft anastomoses compared to ePTFE grafts. Assessment of biocompatibility revealed no alteration of graft compliance, platelet aggregation, complement activation, or hepatic or renal toxicity. This novel drug-eluting prosthetic vascular graft has great potential to impact prosthetic graft patency rates. Further study in a large animal model is warranted.

47.03 Targeted Therapeutics for the Prevention of Restenosis Following Vascular Interventions.

Posted on January 18, 2016

M. A. Wasserman1,2,3, J. S. Rink1, C. S. Thaxton1,4, M. R. Kibbe1,2,3 1Simpson Querrey Institute For BioNanotechnology,Chicago, IL, USA 2Northwestern University,Department Of General Surgery,Chicago, IL, USA 3Northwestern University,Department Of Vascular Surgery,Chicago, IL, USA 4Northwestern University,Department Of Urology,Chicago, IL, USA

Introduction:
Current surgical and endovascular therapies for severe atherosclerotic disease often fail due to the development of neointimal hyperplasia with resultant arterial restenosis and occlusion. The objective of this study is to evaluate the targeting specificity, cellular binding, and internalization patterns of a novel synthetically engineered targeted delivery vehicle that can subsequently be designed to deliver a therapeutic agent to prevent arterial restenosis. Our hypothesis is that the targeted gold nanoparticle will bind with specificity to the site of arterial injury and demonstrate cellular binding and internalization to the cells that comprise the vascular wall.

Methods:
A citrate-stabilized 13 nm gold nanoparticle (AuNP) was surface functionalized with a molecular fluorophore and a collagen-binding peptide (CBP) or a scrambled peptide (SCR). In vivo assessment of targeting specificity was accomplished utilizing the rat carotid artery balloon injury model in 10-week-old male Sprague-Dawley rats. After balloon angioplasty of the left carotid artery, systemic injection of the AuNP into the inferior vena cava was performed with either the CBP-AuNP (15-60 nM, n=14) or the SCR-AuNP (30-60 nM, n=7). Arteries were harvested 20 minutes post injection. Binding was detected using fluorescent microscopy. The right carotid artery served as the control (n=21). In vitro determination of cellular binding and internalization was performed on rat aortic endothelial cells (EC), adventitial fibroblasts (AF), and smooth muscle cells (SMC) exposed to 50 nM of the AuNP. Fluorescent uptake was quantified at 4 and 24 hours using the Cytation 3 Cell Imaging Multi-Mode Reader. Statistical analysis was performed using ANOVA on Ranks.

Results:
In vivo, the collagen-targeted CBP-AuNP bound to the site of arterial injury and brightly fluoresced, with the most prominent signal at 45 nM. No binding was noted to the contralateral, uninjured right carotid artery. Rats injected with the SCR-AuNP expressed no fluorescent signal in either the right or left carotid arteries. In vitro, the AuNP bound to all three cell types and fluorescence significantly increased over time (EC p<0.001, AF p<0.001, SMC p<0.001), suggesting internalization within the cell. Among the three cell types, the greatest fluorescence was noted with EC (2.0-fold increase vs. 1.6-fold AF and 1.3-fold SMC at 24 hours).

Conclusion:
Our collagen-targeted AuNP binds with specificity to the site of vascular injury and demonstrates binding to and internalization within the cells that comprise the vascular wall. These studies serve as the foundation for further evaluation and optimization of our delivery vehicle for the vasculature. Ultimately, our goal is to systemically administer a therapeutic agent in a targeted manner to prevent neointimal hyperplasia and arterial restenosis following vascular interventions.

47.04 Apicidin Inhibits Major Vascular Smooth Muscle Cell Pathogenic Phenotypes

Posted on January 18, 2016

C. J. Little1, A. Kent1, B. Wang1, M. A. Chaudhary1, K. Kent1, L. Guo1 1University Of Wisconsin School Of Medicine And Public Health,Department Of Surgery,Madison, WI, USA

Introduction: Intimal hyperplasia leads to failure of 20-50% of vascular reconstructions. The underlying cause is smooth muscle cell (SMC) pathophysiology characterized by proliferation, migration and dedifferentiation. Apicidin, a class I histone deacetylase (HDAC3) inhibitor, has been shown to have a potent anti-proliferative effect in a number of cancers. The mechanism of its effect appears to be through activation of the tumor suppressor factor P53. The purpose of this study is to evaluate the effectiveness of Apicidin in preventing recurrent stenosis after vascular reconstruction.

Methods: In vitro assays included proliferation (CellTiter-Glo), migration (scratch assay), and western blotting. In vivo analysis was conducted using a rat model of carotid balloon angioplasty.

Results: Rat SMCs were pretreated (2 hours) with Apicidin and then stimulated with 10% Fetal Bovine Serum (FBS) for 72 hours followed by measurement of proliferation. Based upon an initial dose response curve, we found the optimal concentration of Apicidin to be 500nM, which reduced proliferation by 72.9% ± 5.3% (p < 0.05). Pretreatment of SMCs with Apicidin also reduced SMC migration by 77.1% ± 2.0% (p < 0.01) at 24 hours in response to 10% FBS. We have previously shown that the combination of TGF-β/Smad3 is a potent stimulant of SMC dedifferentiation. To evaluate whether Apicidin can reverse this effect and promote differentiation, SMCs transfected with Smad3 were pretreated for 2 hours with Apicidin and then stimulated with TGF-β (10ng/mL) for 48 hours. Reduction of several proteins indicative of SMC differentiation by TGF-β/Smad3 was rescued by Apicidin by the following percentages: Calponin (22.3%), Myosin Heavy Chain (108.9%), and Smooth Muscle Actin (30.0%). Blots were re-probed for levels of acetylated histone, which were increased 14.3 ± 2.1 fold (p < 0.05), confirming the expected function of the HDAC inhibitor. Finally, adult rats underwent carotid balloon angioplasty with periadventitial application of Apicidin (500µg) in 23% pluronic gel. Cross-sectional carotid samples were harvested at 21 days, yielding preliminary results indicating that Apicidin reduces neointimal area by 55% while preserving luminal and vessel integrity.

Conclusion: The HDAC3 inhibitor, Apicidin, can reduce SMC proliferation and migration, promote SMC differentiation, and subsequently lead to a significant reduction in intimal hyperplasia. Inhibition of HDAC3 could be an effective therapeutic intervention for the prevention of recurrent stenosis leading to improved long-term outcomes in patients treated for vascular disease.

46.08 Bcl-2 induces paradoxical intrinsic and extrinsic enterocyte apoptosis in septic Bcl2xTAg mice

Posted on January 18, 2016

J. D. Lyons1, N. Klingensmith1, Z. Liang1, C. Coopersmith1 1Emory University,Surgery,Atlanta, GA, USA

Introduction: Changes in intestinal epithelial cell apoptosis and proliferation have been associated with mortality in animal models of sepsis, and preventing gut cell death by local overexpression of Bcl-2 has repeatedly been shown to increase survival. However, our unpublished observations indicate that when anti-apoptotic Bcl-2 is overexpressed in conjunction with the pro-proliferative transgene SV40 T-antigen, the epithelium is not protected from sepsis-induced apoptosis, but instead displays a paradoxical increase in cell death. We here attempt to shed light on this phenomenon by examining mechanisms of cell death in the gut of septic Bcl2xTAg animals.

Methods: Fabpi-Bcl2 mice were crossed to fabpl-TAg mice to produce bi-transgenic Bcl2xTAg mice with small intestinal enterocyte-specific overexpression of Bcl-2 and SV40 T-antigen. Mice aged 8-10 weeks old were subjected to cecal ligation and puncture (CLP) and sacrificed at 24 hours. Jejunal sections were collected at time of sacrifice for western blot analysis. Septic Bcl2xTAg mice were compared to septic TAg mice to determine how the additional expression of Bcl-2 might lead to the increased cell death seen in Bcl2xTAg enterocytes.

Results: Septic Bcl2xTAg mice expressed the Bcl-2 protein at a concentration 4.1-fold greater than septic TAg controls (p=0.001, n=9-10). Expression of the apoptosis effector caspase-3 was significantly increased in these animals (3.1-fold increase, p=0.017, n=5-6). Bcl2xTAg mice also displayed increased expression of caspase-9 (24.5-fold increase, p=0.005, n=7-9) and caspase-8 (1.8-fold increase, p=0.013, n=9-10), suggesting both intrinsic and extrinsic signaling upstream of caspase-3 activation. These changes were associated with trends in increased concentrations of the mitochondrial apoptosis mediators Bax (8.9-fold increase, p=0.051, n=9-11) and cytochrome C (2.0-fold increase, p=0.052, n-5-6), but not apoptosis inducing factor (1.06-fold increase, p=0.65, n=5-6).

Conclusion: The overexpression of Bcl-2 in the gut of Bcl2xTAg mice paradoxically results in increased sensitivity to sepsis-induced enterocyte apoptosis by both intrinsic and extrinsic mechanisms. The greatly increased caspase-9 expression suggests the intrinsic pathway may predominate. Overexpression of an anti-apoptotic factor, when coupled with overexpression of a pro-proliferative factor, may trigger a counter compensatory response that increases the likelihood of cell death rather than survival.

46.09 Cold-inducible RNA-binding Protein Promotes a Th1 T Cell Response via TLR4 in Sepsis

Posted on January 18, 2016

A. C. Bolognese1,2, A. Sharma3, W. Yang1,2,3, J. Nicastro1, G. F. Coppa1, P. Wang1,2,3 1Hofstra North Shore-LIJ School Of Medicine,Department Of Surgery,Manhasset, NY, USA 2Elmezzi Graduate School Of Molecular Medicine,Manhasset, NY, USA 3The Feinstein Institute For Medical Research,Center For Translational Research,Manhasset, NY, USA

Introduction: Cold-inducible RNA-binding protein (CIRP) was recently discovered as a proinflammatory mediator in sepsis that stimulates cytokine release from macrophages. In light of the immune dysregulation seen in sepsis, the effect of CIRP on the innate and adaptive immune response of T cells is an area of interest that has not yet been characterized. Here, we hypothesized that extracellular CIRP activates splenic T cells via a toll-like receptor 4 (TLR4)-dependent mechanism and promotes a proinflammatory Th1 response.

Methods: C57BL/6 wild type (WT) and TLR4 knockout (TLR4KO) mice received intravenous (IV) injection of recombinant murine CIRP (rmCIRP, 5 mg/kg BW) or PBS (vehicle). At 20 h after injection, splenocytes were isolated, labeled, and analyzed by flow cytometry. The early T cell activation marker CD25 was used to identify activated T cells. CD4 T cells isolated from the spleens of WT and TLR4KO mice were incubated on anti-CD3/anti-CD28-coated plates with or without rmCIRP (1 µg/ml) for 20 h, followed by flow cytometry or PCR array analysis.

Results: After rmCIRP injection in WT mice, the CD25+ CD4 T cell population was increased 4.1-fold compared to PBS; however, there was no significant change in the number of CD25+ CD4 T cells in TLR4KO mice (Table). Furthermore, incubation of WT CD4 T cells with rmCIRP and CD3/CD28 co-stimulation increased the population of CD25+ CD4 T cells by 11% compared to PBS control. In contrast, CD4 T cells isolated from TLR4KO mice showed a decreased percentage of CD25+ CD4 T cells with rmCIRP incubation (Table). Additionally, an 84-gene Th1 & Th2 response PCR array (QIAGEN) showed significant upregulation of seven genes in CD4 T cells treated with rmCIRP compared to PBS control (n=3, P < 0.05). Of these, four were Th1-related genes; interferon-γ, interleukin 12 receptor beta 2, T-bet and colony stimulating factor 2 were increased 7.1-, 5.3-, 3.7- and 2.3-fold, respectively. Two CD4 T cell markers, interleukin 6 and suppressor of cytokine signaling 3, were increased 3.9- and 2.9-fold, respectively. Lastly, the Th2 cytokine interleukin 13 was increased 3.7-fold. No genes were downregulated.

Conclusion: Our findings demonstrate that CIRP activates T cells via a TLR4-dependent mechanism. We have further shown that CIRP promotes a proinflammatory Th1 response profile in activated CD4 T cells. These findings demonstrate that CIRP plays an important role in T cell dysregulation in sepsis.

46.10 Protective Effect of Different HDAC Inhibitors on Hypoxia/Reoxygenation Induced Cardiomyocyte Injury

Posted on January 18, 2016

W. He1, Z. Chang1, B. Liu1, X. Cheng1, T. Bambakidis1, I. Halaweish1, Y. Li1, P. Patrick2, V. Nikolian2, H. B. Alam1 1University Of Michigan,General Surgery,Ann Arbor, MI, USA 2University Of Michigan Health System,Ann Arbor, MI, USA

Introduction: We have previously shown that histone deacetylase inhibitors (HDACIs) preserve organ function and improve survival in a rat model of hemorrhagic shock. It remains unknown whether the different types of HDACIs differ in their cytoprotective potential. Here we used an in vitro model of hypoxia/ reoxygenation (H/R) to assess whether different HDACIs, such as SAHA (pan), MS-275 (class I), MC-1568 (class IIa), Tubastatin-A (Tub-A, class IIb), and EX-527 (class III), could protect the cardiomyocytes from injury. The most effective agent was also subjected to a mechanistic study.

Methods: Experiment I: H9c2 cardiomyocytes from ATCC were subjected to an in vitro model of H/R (12/8 hours), and treated with or without SAHA, MS-275, MC-1568, Tub-A, or EX-527. Cell viability was measured by MTT assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Lactate dehydrogenase (LDH), an indicator of cellular toxicity, in cell culture media was detected by a LDH Cytotoxicity Assay Kit. The levels of acetylated histone H3 and acetylated α-tubulin were measured by Western blots. Experiment II: Tub-A was chosen for further investigation to analyze the underlying protective mechanisms. Cultured H9c2 cells were randomly divided into Sham control, H/R, and H/R+Tub-A groups. Expression of phospho-Akt (p-Akt)/Akt, phospho-mTOR (p-mTOR)/mTOR, phospho-p70s6k (p-p70s6k), phospho-rpS6 (p-rpS6)/rpS6 proteins were determined by Western blots. In addition, the changes in expression of microtubule-associated protein 1 light chain 3 (LC3)-II, a marker of autophagy, were assessed.

Results: High levels of acetylated histone H3 were detected in all of the HDACI treated-groups, while acetylated α-tubulin were only seen in Tub-A or SAHA treated-groups. Moreover, MC-1568, Tub-A, or EX-527, but not SAHA, or MS-527, markedly improved cell viability and decreased LDH release (Fig 1, p<0.05). Pro-survival effect of Tub-A was further confirmed by TUNEL assay. In addition, Tub-A treatment stimulated expression of p-Akt, p-mTOR and p-p70S6 kinases, as well as p-rpS6, in H9c2 cells. Expression of LC3-II was also significantly decreased in the Tub-A group.

Conclusion: HDACIs improve cell viability in a model of cardiomyocyte hypoxia/reoxygenation, with class IIa, IIb, and III inhibitors showing superior efficacy. This effect, at least in part, is due to the activation of AKT- mTOR- rpS6 signaling pathway.

47.01 Utilization of I-domain of LFA-1 to Mediate Binding of Nanocarrier-coated EPC to Inflamed EC

Posted on January 18, 2016

Z. Liu1, L. Zhang1, Y. Li1, S. Joel1, S. Deo1, S. Daunert1, O. C. Velazquez1 1University Of Miami,Miami, FL, USA

Introduction: Intercellular adhesion molecule-1 (ICAM-1) is induced on inflamed endothelial cells (iEC) under several pathological conditions, such as atherosclerosis and inflammation. Integrin LFA-1 recognizes ICAM-1 as its ligand, and the I-domain of LFA-1 (idLFA-1) is responsible for bind to ICAM-1. idLFA-1/ICAM-1 pair is thus an attractive target for delivery of therapeutic cells to tissues where iEC are presented. We are aimed to develop a safe and targeted cell delivery method for therapeutic administration of stem/progenitor cells, which carry repair function, to atherosclerotic lesion to stave off the development of atherosclerosis. We have recently developed a novel cell delivery platform by coating the surface of the cells to be delivered with nanocarriers composed of nanoparticle–adhesion molecule complex. These nanocarriers guide the coated cells to their destination via recognition and association with their counterpart adhesion molecules, which are highly or selectively expressed on the diseased tissue, and execute their therapeutic roles. Here, we tested effectiveness of idLFA-1 complexed nanocarriers in mediating interaction of nanocarrier-coated endothelial progenitor cells (EPC) to iEC in vitro.

Methods: Recombinant human idLFA-1 protein was denatured, refolded, and purified by gel filtration. Binding activity of idLFA-1 to ICAM-1 was validated by testing association of FITC-conjugated idLFA-1 with human aorta endothelial cells (HAEC) pre-transduced with Notch1IC/lentivirus to induce high ICAM-1 (ICAM-1hi) expression. Nanocarriers were created by conjugation of idLFA-1 with dendrimers. Human EPC were labeled by transduction with DsRed/Lentivirus. DsRed+EPC were then coated with idLFA-1-dendrimers and BSA-dendrimers (control), respectively. The effectiveness of idLFA-1 complexed nanocarriers in mediating interaction of DsRed+EPC to ICAM-1hiHAEC (Notch1IC/lentivirus transduced) vs ICAM-1loHAEC (LacZ/lentivirus transduced) was tested using in vitro cell-cell binding assay. Interaction of DsRed+EPC-HAEC was quantified by measuring fluorescence intensity.

Results: Purified human idLFA-1 protein was characterized based on its size. HAEC transduced with Notch1IC/lentivirus are ‘inflamed’ since expression of ICAM-1 along with a panel of inflammatory citokines, including IL-1α, IL-6, IL-8 and RANTES, are elevated significantly. FITC-conjugated idLFA-1 preferentially bound to ‘inflamed’ HAEC compared to control HAEC (p<0.01). idLFA-1-complexed nanacarriers mediated increased interaction of DsRed+EPC to ICAM-1hi HAEC compared to ICAM-1lo HAEC (>1 fold, p<0.01).

Conclusion: We demonstrated effectiveness of idLFA-1 complexed nanocarriers in mediating interaction of nanocarrier-coated EPC to iEC in vitro. This work paves way to test feasibility of idLFA-1 complexed nanocarriers for targeted delivery of therapeutic cells to atherosclerotic lesion or other inflammation tissues in in vivo model.

46.05 Sepsis Enhances TH2 Cytokine Phenotype, but TH1 Function Remains Robust Post-Sepsis

Posted on January 18, 2016

B. N. Jacobs1, M. J. Delano1 1University Of Michigan,General Surgery,Ann Arbor, MI, USA

Introduction:
Sepsis is the leading cause of death in the critically ill.. Poor outcomes are attributed to immune dysregulation and susceptibility to nosocomial infection. Sepsis produces a TH1 to TH2 immune shift associated with poor outcomes. The specific impact of theTH1 to TH2 shift on immune function is unknown. We investigate the timing and impact of the TH1 to TH2 response on immune function.

Methods:
C57Bl/6 mice underwent cecal ligation and puncture (LD10) or sham procedure. Plasma cytokine measurements were taken over 10 days. On days 3 and 7 post sepsis, baseline antibody production was measured. Mice were immunized with NP-KLH. T-cell dependent B-cell class-switching and antigen-specific immunoglobulin production were measured. On day 3 or 7 TH1 and TH2 functions were tested by inoculating mice with a lethal dose of L. monocytogens or P. aeruginosa. Spleen and liver were evaluated for bacterial colonization. At days 3 and 7 post sepsis mice were euthanized, and spleen and bone marrow monocyte number and function assessed.

Results:
TH1 (IFN, GM-CSF, TNF, IL-2, IL-12p40, IL-3, IL12p70) and TH2 (IL-4, GM-CSF, IL-5, IL-10, IL-6, IL-13) cytokines were elevated in a biphasic pattern. T-cell dependent B-cell class-switching and antigen-specific production showed equivalent increases in both TH1 and TH2 antibody subtypes. Septic mice eradicated and survived lethal Listeriosis at both 3 and 7 days post sepsis while sham and control mice did not. Mice treated with lethal Pseudomonas on day 3 post sepsis died, whereas mice treated on day 7 preferentially survived. After sepsis mice were able to eradicate Listeria from the spleen and bone marrow, but not Pseudomonas form the lung. Monocyte phagocytic function was greatly increased in CLP vs sham mice.

Conclusion:
The data indicate that sepsis produces both functional TH1 and TH2 immune responses. There is a transient deficit in the TH2 immunity early after sepsis that may contribute to nosocomial infection development.

46.06 Urine Intestinal Fatty Acid Binding Protein (I-FABP) Predicts Acute Mesenteric Ischemia in Patients

Posted on January 18, 2016

S. Y. Salim1, P. Young1, Y. Li1, T. A. Churchill1, R. G. Khadaroo1,2 1University Of Alberta,Div. Gen Surgery, Dept. Surgery,Edmonton, AB, Canada 2University Of Alberta,Div. Critical Care Medicine/ Dept Surgery,Edmonton, AB, Canada

Introduction:
Acute mesenteric ischemia (AMI) has a high morbidity and mortality and often presents as a diagnostic challenge. Currently there are no blood, urine or radiological tests that provide a definitive diagnosis of AMI. The aim of this study was to evaluate if intestinal fatty acid binding protein (I-FABP), especially detected in urine, can predict AMI in patients.

Methods:
Eighteen patients referred to the Acute Care Surgery service at University of Alberta Hospital with suspected AMI taken to the operating room for definitive diagnosis, were recruited. Pathological findings from surgical specimens confirmed gold standard diagnosis for intestinal ischemia. The patients found to be non-ischemic became the internal controls. ELISAs for I-FABP and IL-6 were done on blood and urine samples collected at the time of surgery.

Results:
Ten patients were diagnosed with AMI while 8 patients were non-ischemic. There was no difference in age or gender between ischemic and non-ischemic patients (65+21 vs. 53+17 years old, respectively; 4 females with no ischemia and 5 females in the ischemic group). Additionally, there was no difference in serum lactate and creatinine between the 2 groups. Serum IL-6 levels in patients with AMI were significantly higher than non-ischemic controls (0.6+0.2 ng/mL vs. 0.08+0.04 ng/mL, respectively, p<0.006). There was an increase in serum I-FABP in AMI patients, though it was not statistically significant compared to internal controls (11+4 ng/mL vs. 1.9+0,6 ng/mL, respectively, p=0.06). Alternatively, urine I-FABP was significantly higher in patients diagnosed with AMI than in controls (8.7+1.4 ng/mL vs. 2.5+0.7 ng/mL, respectively, p<0.002). The receiver operating characteristic (ROC) illustrated that urine I-FABP does discriminate between patients with AMI and controls (area under ROC=0.9, p<0.003).

Conclusion:
The traditional clinical marker, lactate, was not able to differentiate AMI from non-ischemic bowel. However, we found that urine I-FABP can be used as a non-invasive biomarker with high specificity and sensitivity for accurately diagnosing AMI in patients. A non-invasive accurate tool for AMI would facilitate a rapid treatment, while preventing unnecessary surgical interventions in high-risk patient populations.

46.07 Diffuse Brain Injury Induced Circuit Reorganization Coincides With Thrombospondin-1 Expression

Posted on January 18, 2016

S. Ogle1,2,3, H. G. May2,4, P. Adelson1,2, J. Lifshitz2,3, T. Currier Thomas2,3, S. B. Johnson1 1Banner University Medical Center- Phoenix,Surgery,Phoenix, AZ, USA 2University Of Arizona,College Of Medicine, Child Health,Phoenix, AZ, USA 3Barrow Neurological Institute At Phoenix Children’s Hospital,Phoenix, AZ, USA 4University Of Bath,Bath, UK, United Kingdom

Introduction: Traumatic brain injury (TBI) disrupts neuronal processes and connections (circuits), which initiate reparative events to rebuild damaged circuits (synaptogenesis). Aberrant synaptogenesis, however, can lead to circuit reorganization which is thought to manifest as persistent neurological dysfunction. In rodents, circuit reorganization in the thalamocortical circuit leads to the development of late-onset sensory sensitivity to whisker stimulation; similar to light/sound hypersensitivity in TBI survivors. Developmental synaptogenesis is, in part, stimulated by astrocyte-secreted thrombospondin-1 and 2 (TSP) via interaction with the α2δ-1 subunit on neuronal voltage-gated calcium channels (α2δ-1). TSP levels are high during development, but diminish with age. After focal trauma in rodent models, TSP levels increase and experimental TSP antagonism decreases epileptiform activity. We, therefore, hypothesize that TBI-induced TSP expression will coincide with circuit reorganization in the thalamocortical circuit.

Methods: Adult male Sprague-Dawley rats underwent sham or moderate midline fluid percussion brain injury. At multiple time points over 2-months post-injury, expression of TSPs and synaptic markers were quantified from thalamocortical circuit biopsies using qPCR and automated capillary westerns, respectively. Tissue sections were stained for glial acidic fibrillary protein and processed using silver and Golgi staining. GFAP and silver pixel density were quantified over time in cortical and thalamic regions. Golgi stained neurons were 3D reconstructed and analyzed for neuronal process length, number and surface area with Neurolucida software.

Results: In the thalamus, TSP-1 gene and protein expression significantly increases 7 days post-injury (DPI). Gene and protein expression of α2δ-1, pre- and post-synaptic markers also significantly change over time. In the cortex, TSP-1 gene expression increases at 7 DPI which coincides with significant changes of expression of α2δ-1, pre- and post-synaptic markers. Protein analysis of the cortex is still on going. Histologically, there is increasing neuronal pathology and astrocytosis at 7 DPI. Neuronal process length, number, and complexity also differ significantly from shams in both relays over time.

Conclusion: Changes in synaptogenic and synaptic marker expression, pathology and neuronal morphology coincide with the development of late-onset of symptoms. This supports post-TBI reparative events leading to aberrant synaptogenesis and circuit reorganization resulting in neurological dysfunction. Persistent astrocytosis after TBI provides a source of the increased expression of TSP, which likely mediates synaptogenesis and ongoing events. Understanding the unique time course and mechanism of chronic neurological dysfunction after TBI is critical to development of treatment strategies.

46.03 Redox Changes Following Tri-Laser Synergy with Mesenchymal Stem Cells (MSC) in Renal Fibrosis

Posted on January 18, 2016

J. Jaramillo1, R. V. Patil1, J. Yu1, A. Bartholomew1 1University Of Illinois At Chicago,Chicago, IL, USA

Introduction:
The critical organ shortage dictates a need for innovative strategies in addressing organ replacement therapies. Strategies to enhance the regenerative potential of dysfunctional kidneys, may delay or reverse organ fibrosis. While mesenchymal stem cell therapy can improve acute kidney injury, it has little effect on fibrosis. Reversal of detrimental redox states in fibrotic tissues may be necessary to fully harness the regenerative potential of these cells and to inhibit myofibroblasts which receive differentiative cues based on cell redox state. We have previously observed low level light laser therapy of 635, 532, and 405nm wavelengths to synergize with MSC in enhancing angiogenesis and reducing apopotosis. We hypothesized the pro-regenerative effects observed would correlate with an improved redox state and tested representatives of various redox pathways.

Methods:

Unilateral ureteral obstruction was used to induce fibrosis; C57BL6 fibrotic kidneys were treated in vivo with either vehicle control, or thrice weekly Low Level Light tri- Laser Therapy (635nm, 532nm, and 405 nm) with or without weekly intravenous MSC treatment for 4 weeks. For analyses, kidneys were explanted, histologically examined for quantity of fibrosis and apoptosis and expression of the following pathways implicated in redox signaling were quantitated using Q-PCR: superoxide generating oxidases, ie, NOX 1 and NOX 4, Cystathionine-β-synthase(CBS) and cystathionine-γ-lyase(CSE), which produce reactive sulfur species critical to normal mitochondrial function, NRF 1 and NRF 2 which encode anti-oxidative enzyme genes through the anti-oxidant response element (ARE), peroxiredoxins, (PRDX) which catalyze the reduction of peroxides, and SIRTUINS and HIF, which are implicated in cell signaling and survival but dependent on NAD+ as key players in the fibrosis sequence.

Results:
Compared to UUO alone, gene expressions of superoxide generators NOX1 and NOX4, respectively, were decreased by 0.72 and 0.8 times in the MSC alone group,and 0.2 and 0.5 times in the tri-laser plus MSC treatment group. Mitigation was observed by elevated PRDX5 expression, 2.5 and 3.5 times in MSC and laser+MSC treatment group, respectively, whereas PRDX1 and 2 remained unaffected. CBS and CSE expression were increased 1.5 and 2 times in MSC and laser+MSC treatment group, respectively. Sirtuins 1 and 5 were 1.5 times increased in MSC alone group. NRF1 and NRF2 decreased 0.5 times with Laser + MSC treatment. MSC or laser+MSC did not alter expression of HIF.

Conclusions:
Beneficial effects of MSC and tri-laser therapy appear to reduce superoxide generating oxidases, NOX1 & 4 and favor maintenance of cellular mitochondrial function via the hydrogen sulfide pathway as evidenced by increased CBS and CSE expression. These data will guide future strategies in optimizing the ideal redox conditions to facilitate in situ kidney regeneration.

46.04 The Effects of Clonidine on Prolonged Bone Marrow Dysfunction after Severe Trauma and Chronic Stress

Posted on January 18, 2016

I. G. Alamo1, K. B. Kannan1, T. J. Loftus1, H. Ramos1, P. A. Efron1, A. Mohr1 1University Of Florida,Trauma,Gainesville, FL, USA

Introduction:
Following severe trauma, critically injured patients develop a persistent injury-associated anemia associated with a prolonged hypercatecholamine state. Propranolol has been previously been shown to improve bone marrow (BM) progenitor cell growth, reduce hematopoietic progenitor cell (HPC) mobilization, and improve anemia. Clonidine, an alpha 2-adrenergic agonist, is sympatholytic and inhibits norepinephrine release. This study sought to investigate the effect of daily clonidine administration on BM dysfunction in a combined lung contusion (LC), hemorrhagic shock (HS), and chronic stress (CS) rodent model.

Methods:
Male Sprague-Dawley rats (n=4-6/group) were subjected to CS, LCHS, and LCHS/CS ± daily clonidine (75ug/kg). Animals underwent two hours of daily restraint stress until the day of sacrifice (day 7). BM cellularity and growth of BM CFU-GEMM, BFU-E and CFU-E colonies were assessed. Peripheral blood was analyzed for hemoglobin (Hgb) and flow cytometry for mobilization of HPCs (CD71+CD117+). Data was presented as mean±SD; *p<0.05 vs. untreated counterpart by t-test.

Results:
Seven days after LCHS alone, BM cellularity and CFU-GEMM, BFU-E, and CFU-E growth remains 10-15% suppressed as compare to naïve (Table). With the addition of clonidine, BM cellularity improved by 19% and BFU-E and CFU-E colony growth was restored to naïve levels. In the more severe and clinically relevant model of LCHS/CS, after seven days there is a 27% suppression of BM cellularity and persistent anemia. Daily administration of clonidine following LCHS/CS resulted in a 26% improvement in BM cellularity and a significant recovery in BM CFU-GEMM, BFU-E, and CFU-E colony growth by 16, 37, and 32% (Table). Furthermore, the use of clonidine following LCHS/CS led to an 85% decrease in HPC mobilization to the peripheral blood and significantly improved Hgb (13.9±0.5* vs. 10.6±0.8 g/dL).

Conclusion:
Following lung contusion, hemorrhagic shock, and chronic stress, the use of clonidine increased BM cellularity, prevented prolonged suppression of BM HPC growth, reduced mobilization of HPC, and improved anemia. Clonidine has been shown to be protective by reducing the sympathetic effects on BM function following trauma and chronic stress. Therefore, reduction of the prolonged hypercatecholamine state following trauma is therapeutic goal that is beneficial for BM function.

45.10 Human Adipose Stromal Cells Increase Survival and Mesenteric Perfusion After Intestinal Ischemia

Posted on January 18, 2016

D. L. Doster1, A. R. Jensen1, E. B. Hunsberger1, M. C. Yoder1, T. A. Markel1 1Indiana University School Of Medicine,Surgery,Indianapolis, IN, USA

Introduction: Intestinal ischemia can quickly escalate to bowel necrosis and perforation. Transplantation of stem cells presents a novel treatment modality for this problem. Recent work suggests that bone marrow-derived stromal cells can protect the intestine from ischemia. However, the low proliferative potential and difficulties in isolating these cells make them difficult to implement on a large scale. Human adipose-derived stromal cells (ASC) have shown greater proliferative potential and are more readily isolated. We hypothesized that: 1) ASCs would increase seven day survival following intestinal ischemia, and 2) improved survival with ASC therapy would be associated with improved post-ischemic mesenteric perfusion.

Methods: Cells were cultured on polystyrene flasks at 37C in 5% CO2 in air. Human ASCs were grown in EGM2 with FBS and human nTERT keratinocytes (differentiated cell control) were cultured in Epilife media with keratinocyte growth factor. Two million cells in 250ul of PBS were used for experimentation. Adult male C57Bl6J mice (8-12 weeks, 20-30g) were anesthetized and a midline laparotomy performed. The intestines were eviscerated, the small bowel mesenteric root was identified, and baseline intestinal perfusion was determined using Laser Doppler Imaging. Intestinal I/R was established by temporarily occluding the superior mesenteric artery for 60 minutes with a non-crushing clamp. Following ischemia, the clamp was removed and the intestines reperfused. Immediately prior to abdominal closure, vehicle, ASCs, or keratinocytes were injected into the peritoneum. Animals were allowed to reperfuse for 12 or 24 hours (perfusion studies), or 7 days (survival studies). After these designated times, animals were reanesthetized and a final perfusion scan was obtained. Perfusion data is expressed as a percentage of baseline. Groups were compared with student’s t test and p<0.05 was significant.

Results: Animals administered ASCs following intestinal ischemia had significantly greater 7 day survival compared to vehicle or keratinocytes (ASC: 80% survival, keratinocytes: 20%, vehicle 40%, p<0.05). Additionally, ASCs facilitated better post-ischemic recovery of mesenteric perfusion compared to vehicle or keratinocytes at 24 hours (ASC: 66.0+/-11.8%; PBS: 25.6 +/- 6.1%; keratinocytes: 25.0 +/-11.4%, p<0.05) but not at 12 hours post-ischemia.

Conclusion: Adipose stromal cells attenuate post-ischemic intestinal injury and promote survival by enhancing mesenteric vascular perfusion. Further studies are needed to identify the mechanism that these cells utilize to promote improved perfusion. Understanding these mechanisms may allow for the design of stem cells that promote maximum effect during therapeutic use.

46.01 Cells from In Vivo Models of Heterotopic Ossification Exhibit Increased Osteogenic Properties

Posted on January 18, 2016

J. Drake1, S. Agarwal1, K. Shigemori1, S. Loder1, C. Hwang1, S. Li1, Y. Mishina1, S. Wang1, B. Levi1 1University Of Michigan,Ann Arbor, MI, USA

Introduction: Large burns and high-energy trauma can lead to heterotopic ossification (HO), a process by which pathologic, ectopic bone forms within soft tissue. Management of HO is limited by efficacy of available treatments, difficulty identifying at-risk patients, and high recurrence rates following surgical excision. The cellular and molecular basis of HO is unknown. Here we investigate whether the cells themselves or their environment drive HO formation. We demonstrate that human and mouse cells isolated from sites of HO retain increased osteogenic capacity when cultured outside of an inflammatory environment.

Methods: Human cells were cultured from HO and surrounding normal bone. Mouse cells were obtained from two models including trauma-induced and genetic HO. Cells from the trauma induced model were isolated from the tendon transection site of mice which had undergone a dorsal burn with tendon transection (burn/tenotomy) at 1, 2, and 3 weeks after injury, a model that reliably produces HO. In the genetic HO model (Nfatc1-cre/caACVR1fl/wt) normal and HO-derived osteoblasts were isolated from 1, 2, and 3 week old mice. Osteogenic differentiation was assessed for by alkaline phosphatase production, alizarin red stain for mineral deposition, RNA expression, and protein expression. Cell proliferation was also assessed.

Results: Human HO cells showed increased osteogenic signaling compared to human osteoblasts from non-HO bone (Fig. 1). Cells isolated from the burn/tenotomy mice 2 and 3 weeks after injury demonstrated significantly increased cell proliferation, alkaline phosphatase, alizarin red stain, and pSmad 1/5 expression when compared with controls. Similarly, HO-derived cells from our genetic HO model in 2 or 3 week old mice exhibited increased cell proliferation, alkaline phosphatase, alizarin red stain, and pSmad 1/5 expression when compared with non-HO osteoblasts from the same mice (Fig. 2). Finally, targeting these cells with inhibitors of smad5 phosphorylation (LDN-193189) decreased osteogenic capacity by alkaline phosphatase and alizarin red quantification (p<0.05), consistent with its effect on HO formation in our trauma model.

Conclusion: In vitro analysis demonstrates significant differences in cellular behavior with regard to proliferation and osteogenic differentiation in HO models when cells are cultured separate from their in vivo environment. This data suggest that changes in cell behavior drive the process of HO as cell characteristics are preserved after they are removed from their environment. Furthermore, the striking differences in these HO-derived cells from normal cells suggests that they may be used for in vitro assays to study potential therapies targeting HO development.

46.02 Combined Effects of Insulin and Exercise on Muscle Function in Severe Burn

Posted on January 18, 2016

M. R. Saeman1, K. DeSpain1, L. A. Baer2, J. Song1, C. E. Wade2, S. E. Wolf1 1University Of Texas Southwestern Medical Center At Dallas,Surgery,Dallas, TEXAS, USA 2University Of Texas Health Science Center At Houston,Surgery,Houston, TEXAS, USA

Introduction: Muscle loss is a consequence of severe burn and critical illness increasing sepsis risk and recovery time. Previously, a rat model of hindlimb unloading after burn found bedrest contributes significantly to atrophy. In this model, we found that exercise increased the function and changed the fiber composition of slow-twitch muscle. We hypothesize that insulin with exercise will further diminish the loss of muscle function in burn with bedrest.

Methods: Twenty-four male Sprague-Dawley rats received a full thickness 40% total body surface area (TBSA) burn and were randomly assigned (n=6) to vehicle without exercise (V/No), insulin (pro zinc 40U daily) without exercise (I/No), vehicle with exercise (V/Ex), or insulin with exercise (I/Ex). All animals were placed in a tail traction system for hindlimb unloading to mimic bed rest immediately following burn. The exercise group was trained to perform twice daily weighted resistance climbing of 1 meter with 5 repetitions. On day 14 in situ isometric forces of the left soleus and plantaris muscles were measured. Fatigue measurement was performed in only the soleus. Statistical analysis was performed with Sigma Plot using Student’s t-test or ANOVA where appropriate.

Results: There was no significant change in animal body mass between treatments. The physiological cross sectional area (PCSA) of the plantaris increased with combined insulin and exercise. The tetanic (Po) and twitch (Pt) muscle functions were significantly elevated in the plantaris of I/Ex. However, there was no change in the tetanic force when normalized to PCSA (Po/CSA). The soleus had significant elevation of Po, Pt, Po/CSA, fatigue maximum, and fatigue minimum in I/Ex. Please refer to the table for specific values and significance.

Conclusion: Insulin and resistance exercise have a positive combined effect on the hindlimb muscle function in this model of critical illness. The plantaris muscle demonstrated increased physiological cross sectional area with increased force suggesting a net increase in muscle fibers. Previously, we found no change in plantaris with exercise only. The soleus demonstrated a change in the specific force of the muscle and fatigue functions indicating a change in the composition of muscle fiber types.

45.07 Targeting the ECM: An Innovative Strategy to Improve Pulmonary Hypertension in CDH.

Posted on January 18, 2016

S. Balaji1, M. Shah3, M. Phillips3, X. Wang1, C. Moles1, P. L. Bollyky2, S. E. Mclean3, S. G. Keswani1 1Baylor College Of Medicine & Texas Children’s Hospital,Pediatric Surgery,Houston, TX, USA 2Stanford Univeristy School Of Medicine,Division Of Infectious Diseases,Palo Alto, CA, USA 3University Of North Carolina,Division Of Pediatric Surgery,Chapel Hill, NC, USA

Introduction:
The management of pulmonary arterial hypertension (PAH) is challenging in patients with congenital diaphragmatic hernia (CDH) and is a significant source of morbidity and mortality in these patients. In CDH, the pulmonary arteries have thick walls due to smooth muscle cell hyperplasia, increased collagen deposition, and marked inflammation, leading to fibrotic remodeling of the perivasculature. Adult models of PAH and fibrosis demonstrate a dysregulation of hyaluronan (HA) metabolism in the pulmonary vasculature, which may contribute to increased inflammatory response. The role of HA has not been examined in neonatal PAH and CDH.

Methods:
The main challenge in studying CDH is the lack of a viable congenital murine model that develops PAH. We are characterizing the phenotype of the Slit3-/- mouse which shows a CDH at birth with a resultant increase in right ventricular pressures (surrogate for pulmonary arterial pressures) and evidence of vascular wall remodeling in the pulmonary arteries that mimics the human phenotype. To determine the dysregulation of HA metabolism in CDH, lungs from Slit3-/- or WT-control mice at 8-12 weeks (n=3-4/group) were harvested. RNA was isolated from frozen lung samples, and hyaluronan synthases (HAS1-3) and hyaluronidases (Hyal1-2) were measured (RT PCR). HA expression was determined in paraffin-embedded sections (HA-binding protein, staining). Data represented as mean+/-SD; p<0.5 denotes significance; t-test.

Results:
Compared to WT mice, the Slit3-/- mice progressively develop right ventricular remodeling, thickening of the pulmonary arteries, increased collagen, and TGFβ1 expression. Slit3-/- murine lungs expressed a significant (5-fold) increase in HAS1 gene expression (11.11+/-3.34 vs. 2.3+/-1.93, p<0.05) compared to WT-controls. There was no significant difference in the other 2 hyaluronan synthases, HAS2 (0.84+/-0.41 vs. 0.84+/-0.34) or HAS3 (0.69+/-0.24 vs. 1.15+/-0.8). Interestingly, the gene expression of hyaluronidases was also significantly higher in Slit3-/- murine lungs compared to WT-controls, including Hyal1 (3.24+/-0.68 vs. 1.02+/-0.11, p<0.01) and Hyal2 (2.58+/-0.57 vs. 0.84+/-0.15, p<0.01). HA staining demonstrated significantly more and dysregulated HA in the perivasculature in the Slit3-/- murine lungs.

Conclusion:
Using a Slit3-/- murine model, we demonstrated altered HA metabolism in CDH lungs. We also showed an increase in HA expression in the perivasculature in the lungs of Slit3-/- mice. These data provide evidence for a possible role of HA metabolism in the development of PAH in CDH, and HA may represent a novel therapeutic target. Targeting the ECM of the pulmonary vasculature would represent a paradigm shift in treatment of PAH in CDH patients.

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