62.03 Green Plasma Has a Superior Hemostatic Profile Compared with Standard Color Plasma

Posted on December 22, 2014

B. A. Cotton1, J. C. Cardenas1, E. Hartwell1, C. E. Wade1, J. B. Holcomb1, N. Matijevic1  1University Of Texas Health Science Center At Houston,Acute Care Surgery/Surgery,Houston, TX, USA

Introduction:  While the transfusion of plasma has increased over the last decade, the

availability of this product has seen dramatic changes that continue to threaten the current

supply. The conversion to male predominant plasma has further limited potential donors

of emergency release products used for intial resuscitaion in trauma. During this same

period, investigators have demonstrated a sexual dimorphism in response to sepsis and

injury, with less multiple organ failure and improved survival observed in premenopausal

females. Plasma from female donors who are pregnant or taking oral contraceptives often

has a green appearance. This green discoloration (due to increased ceruloplasmin levels)

frequently results in these units being discarded or removed from the donor pool for

commercial use, purely based on its appearnace. The purpose of this pilot study was to

evaluate the hemostatic potential and capacity of green plasma compared to standard

color plasma.

Methods:  We obtained plasma from 12 blood group-matched female donors from our

local blood center. Six of these units had a normal appearing hue (STANDARD) and six

were grossly green-appearing plasma (GREEN). These units were then evaluated by

thrombelastography (TEG), calibrated automated thrombogram (CAT) and factor level

measurements. Univariate analysis was then performed using Wilcoxon rank sum and

values are expressed in medians with 25th and 75th interquartile range.

Results: GREEN plasma had a more hypercoagulable TEG profile for all values (r-value:

1.6 vs. 3.1 min, p=0.004; k-time: 2.2 vs. 4.8 min, p=0.088, angle: 69 vs 42 degrees,

p=0.004; mA: 38 vs. 25 mm, p=0.054) when compared to STANDARD plasma.

Differences were also observed with coagulation factor levels comparison, with

GREEN plasma having higher levels than STANDARD (factor II: 107 vs. 96%, p=0.004;

factor VII: 124 vs. 106%, p=0.149; factor IX: 145 vs. 114%, p=0.077; factor X: 125 vs.

102%, p=0.006; factor XI: 121 vs. 101%, p=0.025). Using CAT, GREEN plasma had

higher lag time (4.1 vs. 3.6 min, p=0.037) and increased endogenous thrombin potential

(1669 vs. 1280 nM/min, p=0.114).

Conclusion: This pilot study demonstrates that plasma from female donors that has a green color has a

superior hemostatic profile than standard color plasma. Current AABB recommendations

for male-predominant plasma have further reduced the availability of emergency release

plasma for life-threatening bleeding. GREEN plasma should be further investigated for

its safety profile and hemostatic potential. Should it prove to be a safe and functionally

non-inferior (and potentially superior) product, GREEN plasma should be actively

re-introduced into the medical community for transfusion of critically injured and

bleeding patients.

62.04 Carnosol and Derivatives have Potential as Novel Organ Protective Agents

Posted on December 22, 2014

T. KAWAMURA1,2, T. MOMOZANE1,2, S. FUNAKI1, Y. SHINTANI1, M. INOUE1, M. MINAMI1, K. SUGIMURA2, O. IIDA2, H. FUCHINO2, N. KAWAHARA2, H. TAKEMORI2, M. OKUMURA1  1Osaka University Graduate School Of Medicine,General Thoracic Surgery,Suita, OSAKA, Japan 2National Institute Of Biomedical Innovation,Ibaraki, OSAKA, Japan

Introduction:  Oxidative stress is thought to be involved in various disease states related to organ transplantation such as ischemia/reperfusion injury and its abrogation in affected patients is critical. Carnosol is a plant-derived phenol that functions as an antioxidant and its mechanism of action involves activation of nuclear factor E2 p45-related factor 2 (Nrf2), which plays a key role in regulation of genes driven by antioxidant response element (ARE). Although it is considered to have potential for treatment of oxidative stress, disadvantages include instability in water and high cost, making it difficult to employ in clinical practice. In the present study, we investigated the lung protective effects of carnosol, extracted from Callicarpa longissima, and its derivatives in vivo using a warm lung ischemia model and in vitro with a lung cell line.

Methods:  C57BL/6J mice received 0.01% carnosol chow for 2 weeks, after which the left lungs were excised and cultured in DMEM at 37˚C. The concentration of lactate dehydrogenase (LDH) in medium was measured as a marker of lung damage. Following pre-treatment with carnosol, we assessed the expression of heme oxygenase (HO)-1 and H2O2 treatment tolerance in a lung cell line (NCI-H1975), and also evaluated activation of Nrf2 using a cell-based ARE-luciferase reporter assay. Furthermore, we investigated the stability of carnosol derivatives (methylated-, acetylated-, propionylated-carnosol) in a water solution.

Results: In the excised ischemic lungs, carnosol was shown to increase the amount of HO-1 protein and delayed the leakage of LDH (Fig. A). In NCI-H1975 cell cultures, pretreatment with carnosol induced HO-1, increased the amount of intracellular Nrf2, and protected against H2O2 treatment (Fig. B). Carnosol also increased ARE-luciferase activity in a dose-dependent manner. In addition, our results showed that carnosol induced HO-1 to a much greater degree than the same concentration of tBHQ, a representative Nrf2 inducer (Fig. C). Together, these findings suggest that carnosol induces HO-1 via multiple signaling pathways including Nrf2. Furthermore, carnosol derivatives were stable in a water solution and induced HO-1 up to 1 week after preparation.

Conclusion: Although additional investigation is required, carnosol and its derivatives can significantly reduce organ and cell damage by inducing HO-1, and may have potential as novel organ protective agents.

 

60.16 Valproic Acid Protects Endothelial Cells from Hypoxia-induced Injury

Posted on December 22, 2014

I. Halaweish1, C. Tafatia1, M. Mohamed1, B. Liu1, W. He1, Z. Chang1, Y. Li1, H. B. Alam1  1University Of Michigan,Surgery,Ann Arbor, MI, USA

Introduction: Treatment with histone deacetylase inhibitors, such as valproic acid (VPA), has been shown to be protective in models of hemorrhagic shock; however, the precise mechanism remains unclear.  We simulated the cellular effects of shock by subjecting human umbilical vein endothelial cells (HUVEC) to hypoxia-induced injury in-vitro, to better characterize the mechanistic actions of VPA.

Methods: HUVECs (ATCC HUVEC-EC-C CRL-1730) were grown to 90% confluency in F12K medium supplemented with 20% fetal bovine serum and 5 mg/mL endothelial cell growth factor.  After addition of either phosphate buffered saline (control) or 4 mmol/L VPA (treatment), cells were incubated in either normoxic or hypoxic conditions for 48 hours at 37°C.   Cellular viability was evaluated by redox dye assay (n=4).  Cellular morphology was assessed by light microscopy. Levels of acetylated histone H3, hypoxia inducible factor 1α (HIF-1 α), cleaved caspase 3, vascular adhesion molecule 1 (VCAM1), and vascular endothelial growth factor receptor 1 (VEGFR1) were measured by Western blotting.

Results: High levels of acetylated histone H3 were detected in the VPA-treated cells.  As expected, the hypoxic cells demonstrated increased levels of HIF-1α compared to normoxic cells.  Redox dye assay showed that the number of viable cells increased 26% with VPA treatment in normoxic conditions and 22% in hypoxic conditions (n=4, p=0.002, and p=0.001 respectively).  Hypoxia caused structural alteration in the morphology of HUVECs, which was prevented by VPA treatment (Figure). There was a significant decrease in apoptosis in VPA-treated cells as evidenced by decrease in cleaved-caspase 3 expression in both normoxic and hypoxic conditions (58%, p=0.05 and 48% p=0.002, respectively).  Hypoxia increased the levels of VCAM1 by 50% compared to normoxic control (n=3, p=0.003); which was normalized by VPA treatment. Finally, VPA treatment decreased the expression of VEGFR1 by 18% compared to hypoxic control (n=3, p = 0.02).
 

Conclusion: This is the first study to demonstrate that VPA treatment increases the viability and stabilizes the structural morphology of endothelial cells in a hypoxic-injury model.  In addition, it decreases the expression of adhesion molecules (VCAM1 and VEGFR1) responsible for leukocyte recruitment and inflammatory response.

 

60.17 Viscoelastic Modeling of Human Saphenous Vein Grafts after Surgical Manipulation

Posted on December 22, 2014

K. M. Hocking1,2, E. Wise1, B. Evans2, C. Duvall2, C. M. Brophy1  1Vanderbilt University Medical Center,Nashville, TN, USA 2Vanderbilt University Medical Center,Biomedical Engineering,Nashville, TN, USA

Introduction:
An underappreciated feature of healthy saphenous vein (SV) is its ability to conform to viscoelastic parameters. This feature of biologic tissue may be lost in pathological processes such as calcification or particularly, in surgical preparation when used as graft. The consequences of deformation, while not fully understood, include promotion of intimal hyperplasia and perhaps development of a thrombogenic surface. In fact, external stents are becoming increasingly studied as a method to maintain normal flow conditions in autografted human SV. In this study, the viscoelasticity of unmanipulated SV is compared to that of graft that was fully prepared prior to implantation into CABG patients, manipulations that include supraphysiologic distension, marking with a toxic surgical skin marker and preservation in normal saline.

Methods:
Unmanipulated (UM) human saphenous vein (HSV) and HSV that had been surgically prepared (SP) were obtained from CABG patients, dissected free of fat and cut into rings. Rings were then suspended on a muscle bath in bicarbonate buffer at 37 degrees celsius and were challenged with 110 mM potassium chloride (KCl) to ensure viability of the tissue. The rings were also contracted with phenylephrine and relaxed with sodium nitroprusside, a traditional smooth muscle vasodilator. Data was recorded on LabChart and imported into Eureqa where it was fit to solutions based on the nonlinear least square error of the equation. The equation with a complexity under 10 that had the best fit was used to model the equation. A correlation coefficient of above 0.99 was achieved for each modeled set with a mean absolute error of under 0.03.

Results:
In response to KCl, the UM tissue responded with a force equation of (a+x)(b+mx) resembling a Hill elastic muscle model for 12 out of 12 samples. SP tissue responded to KCl contraction with a force equation of (a+mx) resembling an elastic model for 10 out of 12 samples and 2 out of 12 samples exhibited the Hill elastic model (χ2=17.1, P<0.0001, Figure 1 A,B) . In response to sodium nitroprusside the UM tissue had a relaxation profile of a/(b+mx) whilst the SP tissue responded with a relaxation profile of (a-mx) (χ2=7.273, P=0.007, Figure 1 C,D). The Hill elastic muscle model is represented by a contractile element and two non-linear spring elements where one is in series with the contractile element and the other in parallel.

Conclusion:
It is shown that healthy unmanipulated SV corresponds to a viscoelastic model both when contracting and relaxing. Surgical preparation causes a loss of confirmation to the Hill elastic muscle model in human SV. The use of less deformative techniques may mitigate intimal hyperplasia in autografts.
 

60.18 Preservation in Acidic Normal Saline Solution Decreases Saphenous Vein Viability

Posted on December 22, 2014

E. S. Wise1, K. M. Hocking1, P. Komalavilas1,2, J. Cheung-Flynn1, C. M. Brophy1,2  1Vanderbilt University Medical Center,Vascular Surgery,Nashville, TN, USA 2VA TN Valley Healthcare System,Vascular Surgery,Nashville, TN, USA

Introduction:
Human saphenous vein (HSV) remains the most common conduit for CABG. A recent retrospective analysis on the PREVENT IV cohort suggested that normal saline (NS), relative to buffered salt solutions and blood, may be associated with a higher incidence of graft failure.  We hypothesized that the acidic pH of NS leads to injury to the conduit. 

Methods:
Unmanipulated (UM) HSV samples and a vial of arterial blood were obtained from CABG patients. Two rings were placed into heparinized (10U/mL) solutions for two hours, including control (UM), Plasma-Lyte A (PL), NS, and autologous whole blood (AWB). Smooth muscle (SM) contractility and endothelial-dependent relaxation (EDR) were assessed via potassium chloride (KCl, 110 mM) and carbachol (0.5 µM) following phenylephrine (1-5 µM) pre-contraction, respectively. The colorimetric MTT assay for cellular viability was performed on two additional rings. Finally, rings of rat aorta (RA) were placed into NS, PL and PL adjusted to various pH levels for two hours and endothelial-dependent relaxation responses were determined. Data was analyzed via paired student t-tests; P<0.05 was considered significant.

Results:

HSV preserved in NS demonstrated decreased KCl-induced SM contractility relative to UM and PL (n=16-17, *P<0.03, Figure 1A). AWB SM contractility was decreased relative to PL (n=6, *P<0.03, Figure 1A). NS HSV also demonstrated decreased EDR relative to UM and PL (n=9-10, *P<0.03, Figure 1B). In the MTT assay, NS had decreased cellular viability relative to UM, PL and AWB (n=11-12, *P<0.03, Figure 1C). Using a rat aorta model, pH dependence on endothelial function was determined; pH 5.5-PL and NS were significantly lower than UM, and pH 5.5-PL was significantly lower than PL (n=5; #P<0.05, Figure 1D). 

Conclusion:
Preservation of HSV in NS leads to impaired functional responses and viability of the SM and endothelial layer. This may be due to the acidic pH of NS. The use of a pH-balanced, buffered solution for HSV is recommended for storage after harvest. 

60.19 Therapeutic Targets for Graft Survival: Hyperfibrinogenemia and Impaired Fibrinolysis in ESRD

Posted on December 22, 2014

M. P. Chapman2,4, E. E. Moore1,2, H. B. Moore1,2, D. Burneikis2, E. Gonzalez1,2, A. Slaughter1,2, A. P. Morton2, A. Banerjee2, C. C. Silliman2,3  1Denver Health Medical Center,Aurora, CO, USA 2University Of Colorado Denver,Aurora, CO, USA 3Children’s Hospital Colorado,Aurora, CO, USA 4Georgia Health Sciences University,Augusta, GA, USA

Introduction:

Patients in end-stage renal disease (ESRD) display various derangements of coagulation. Our previous work has demonstrated a a mixed pattern of hypo- and hypercoagulability in these patients, with a paradoxical prolongation of the enzymatic phase of clot formation followed by rapid clot growth and elevated final clot strength. We sought to clarify the detailed features of the hypercoagulable component of the coagulopathy of ESRD to develop targets for prophylactic therapy aimed at prevention of dialysis access graft thrombosis.

Methods:

Blood was collected from 16 consecutive ESRD patients at the time of dialysis access construction and compared to that of 53 healthy volunteers using multichannel thrombelastography (TEG). Rapid TEG and functional fibrinogen (platelet-inhibited) TEG were used to assess clot strength and the relative contributions of platelets and fibrinogen. tPA-challenged TEG was used to assess fibrinolysis susceptibility, using the clot lysis at 30 minutes (LY30) parameter of TEG, when the sample is challenged with exogenous tPA. Platelet function was assessed by aggregometry and TEG platelet mapping.

Results:

Overall clot strength, measured by Rapid TEG maximum amplitude (MA), was elevated at 71±6 mm in ESRD patients compared to 66±4 for healthy controls (p=0.0005, two-tailed Mann-Whitney test). Functional fibrinogen level (by platelet-inhibited TEG MA) was even more markedly elevated at 32 (IQR 29-37) mm in ESRD patients versus 20 (IQR 17-22) mm for controls (p<0.0001). ESRD patients also displayed increased resistance to fibrinolysis, with a tPA-challenged TEG LY30 of 29% (IQR 15-39%) compared to 56% (IQR 40-65%) for healthy controls (p=0.0004). Platelet function tests on ESRD patients were within normal limits.

Conclusion:

Hyperfibrinogenemia and impaired fibrinolysis are responsible for the hypercoagulability observed in ESRD and may contribute to graft/fistula thrombosis. As enzymatic clotting is already prolonged in ESRD and platelet function is generally normal, traditional agents such as heparin or aspirin are of limited prophylactic benefit in prevention of graft/fistula thrombosis. Therapeutic agents effecting fibrin clot strength and fibrinolysis (e.g factor XIIIa inhibitors, PAI-1 antagonists or low dose thrombolytics) may therefore be of greater utility for preservation of dialysis access.

 

60.20 Comparative Analysis of Polymers for siRNA Delivery in Vascular Smooth Muscle Cells

Posted on December 22, 2014

L. M. Bools1, D. J. Mountain1, R. K. Fisher1, S. S. Kirkpatrick1, J. D. Arnold1, S. L. Stevens1, M. H. Goldman1, M. B. Freeman1, O. H. Grandas1  1University Of Tennessee Graduate School Of Medicine,Department Of Surgery,Knoxville, TN, USA

Introduction: RNA interference (RNAi), using short interfering RNA (siRNA) as one of the strategies to degrade mRNA in the cytoplasm and transiently attenuate intracellular proteins, shows promise in the inhibition of vascular pathogenesis. A critical obstacle for its therapeutic application is a safe and effective delivery system, as unfavorable physiochemical properties limit intracellular siRNA delivery. Synthetic polymers are promising alternative molecular carriers due to their ability to deliver genetic material to cells in a predictable, non-toxic way. Here we aim to establish polymeric transfection as a feasible non-viral, less-toxic method for gene therapy in cells of vascular origin.

Methods: Human aortic smooth muscle cells (HASMC) were transfected in vitro with polyethylenimine and poly(B-amino ester) polymers conjugated to GAPDH or negative control (NC) siRNAs. Increasing siRNA:polymer ratios were tested for optimal transfection efficiency. DharmaFECT2 chemical transfection complexes were used for comparative analysis. Live/dead dual stain was used to measure cell viability and GAPDH gene silencing was measured by qPCR normalized to 18S.

Results:The highest rate of polyethylenimine-mediated silencing was achieved with a 9µl polymer:220pmol/ml siRNA conjugate (16±2% expression vs. NC; n=6; Fig1A). Comparable poly(B-amino ester)-mediated silencing could be achieved with a 1.95µl polymer:100pmol/ml siRNA conjugate (10±1% expression vs. NC; n=5; Fig1B). Transfection using polyethylenimines resulted in silencing equivalent to other methods, but with less efficiency and increased cell toxicity at 24h polymer exposure (Fig1C,D). Limiting polyethylenimine exposure to 4hr resulted in similar silencing efficacy (25±9% – 33±8% expression vs. NC, n=3) with decreased toxicity (Fig 1E,F).

Conclusion:Polymeric bioconjugates transfected HASMCs in a manner similar to chemical complexes, with comparable cell toxicity and silencing efficiency. Polyethylenimine bioconjugates demonstrated silencing equivalent to poly(B-amino ester) bioconjugates, though less efficient in terms of required polymer concentrations. Given the cost-to-benefit difference between the assayed polymers, and polyethylenimine’s ability to transfect HASMCs within a short duration of exposure with an improved toxicity profile, this study shows that polyethylenimine bioconjugates are a viable transfection agent for vascular tissue. Future studies will expand on this method of gene therapy for in vivo transfection in animal models of vascular disease. Our long term goal is to deliver molecular inhibitors in vivo, targeting the regulation of genes thought to play a significant role in intimal hyperplasia and restenosis development. 

 

61.01 UDCA Promotes Enterocyte Migration via Upregulation of COX-2 and Activation of the EP2 receptor

Posted on December 22, 2014

J. Golden1, A. Dossa1, P. Kavarian1, K. Goldstein1, H. Ford1, C. Gayer1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA

Introduction:  The secondary bile acid, ursodeoxycholic acid (UDCA), plays a role in intestinal epithelial cell signaling. Previous work in our lab has shown that UDCA promotes enterocyte migration possibly through an epidermal growth factor receptor (EGFR)-dependent pathway. However, the downstream signaling events remain unknown. Cyclooxygenase-2 (COX-2) and its product, prostaglandin E2 (PGE2), are key mediators of enterocyte cell signaling and act on four G-protein coupled receptors (EP1-4). PGE2 has been shown to promote enterocyte migration via EP2 receptor activation and transactivation of EGFR. Therefore, we hypothesized that UDCA promotes enterocyte migration by inducing COX-2, which leads to PGE2 production, and downstream activation of mediators such as PI3K, Akt, and ERK via EP2 receptor signaling.

Methods:  Rat intestinal epithelial cells (IEC-6) were treated with UDCA (0.1uM to 400uM) for 12 hours. Expression of COX-2 protein was examined using Western blot. IEC-6 cell migration was measured using a modified wound-healing assay by creating circular holes in cell monolayers and observing them over 6 hours. Cells were treated with or without 200uM UDCA, 10uM EP2 antagonist PF-04418949, 10uM PI3K inhibitor LY294002, 1uM AKT inhibitor IV, or 20uM ERK (MEK) inhibitor PD98059. Statistical analysis was performed using Student’s t-test.

Results: UDCA significantly increased COX-2 expression in a dose-dependent manner at doses greater than or equal to 10uM.  UDCA at 200uM increased COX-2 expression 2.7 ± 0.6 fold from control (p<0.05). UDCA at 200uM also increased wound closure 10% ±0.04 from control (p<0.05). Pre-treatment with an EP2 antagonist abrogated UDCA-induced IEC-6 cell migration. Similarly, pre-treatment with inhibitors of PI3K, AKT, or ERK respectively, eliminated UDCA-induced cell migration. 

Conclusion: These data suggest a novel role for COX-2 in UDCA-induced stimulation of intestinal epithelial cell migration. The mechanism likely involves induction of COX-2 followed by PGE2 production and downstream signaling via the EP2 receptor, PI3K-Akt-ERK pathway. This may represent a novel pathway to enhance epithelial restitution after injury.

 

61.02 Intestinal Alkaline Phosphatase Alters Specific Fat Metabolism and Insulin Secretion Pathways

Posted on December 22, 2014

M. Gharedaghi1, M. Najibi1, S. Morrison1, K. Economopoulos1, T. Phupitakphol1, S. K. Hyoju1, A. Osmani1, S. R. Hamarneh1, R. A. Hodin1  1Massachusetts General Hospital,Department Of Surgery,Boston, MA, USA

Introduction: Intestinal alkaline phosphatase (IAP) is a brush border enzyme that plays a key role in the development of obesity and the metabolic syndrome (MetS).  IAP-knockout (IAP-KO) mice are more susceptible to high fat diet (HFD)-induced MetS and oral supplementation with IAP has been shown to prevent the MetS in WT mice. The precise mechanism by which IAP works to prevent the MetS is not known. We therefore sought to compare the expression of metabolism-related genes between wild type (WT) and IAP-KO mice fed regular chow diet (CD) vs. HFD.

Methods: Six to eight week old IAP-KO and WT mice were fed with either CD or HFD for 23 weeks. The weight of the mice was registered weekly. Serum, liver and pancreatic tissue was harvested after sacrifice. Serum insulin levels were measured with enzyme linked immunosorbent assay. Expression of key metabolic genes in the liver and pancreatic tissues was determined by quantitative real time PCR. Data were expressed as mean ± standard error of mean (S.E.M.) and analysis of variance (ANOVA) and Tukey’s post hoc test was used to analyze the differences. P values less than 0.05 were considered as statistically significant. 

Results: The percentage of weight gain was significantly higher in the HFD-fed IAP-KO mice in comparison to the CD- or HFD-fed WT mice (P = 0.041 and P= 0.047, respectively; Figure 1A). Although serum insulin levels trended upward in the HFD-fed IAP-KO mice, the analysis failed to reach statistical significance (ANOVA: P = 0.135; Figure 1B). The hepatic expression of lipoprotein lipase (LPL), peroxisome proliferator-activated receptor-γ (PPAR-γ) and acyl co-A dehydrogenase (ACAD) was significantly decreased in HFD-fed KO mice (Figures 1C, 1D and 1E). Interestingly, the decrease in hepatic expression of LPL was limited to IAP-KO mice and was not seen in HFD-fed WT mice (Figure 1C). Pancreatic expression of the adenosine triphosphate-sensitive inward rectifying potassium channel (kcnj11) was increased in HFD-fed IAP-KO mice suggesting alterations in the insulin secretion pathways (Figure 1F).             

Conclusion:  IAP has a significant effect on the expression of the genes which are involved in fat degradation and insulin secretion. Based on the LPL decrease in the IAP-KO but not the HFD-fed mice, it appears that IAP may prevent the metabolic syndrome through a mechanism that is distinct from dietary fat intake.

61.03 EGFR Replenishment Does Not Correct Liver Regeneration in Leptin Receptor Deficient Mice

Posted on December 22, 2014

N. Valsangkar1, X. Jin3, Z. Zhang3, T. A. Zimmers2,3, L. G. Koniaris2,3  2Indiana University School Of Medicine,Surgery,Indianapolis, IN, USA 3Thomas Jefferson University,Surgery,Philadelphia, PA, USA

Introduction: Fatty liver of any degree is associated with propensity to liver failure in mice and patients. Normal, young lean mice tolerate 70% hepatectomy well, with 100% survival and complete recovery of liver mass by 7 days. In contrast, 70% hepatectomy in diet-induced obese mice is characterized by increased liver injury and hepatocyte necroapoptosis, delayed mitoses and slower recovery of liver mass. The genetically obese, leptin receptor mutant lepr-db/db mouse displays high mortality after 70% hepatectomy, due to increased injury and defective proliferation. Others have shown this is associated with reduced expression of EGFR. Here we sought to correct liver regeneration in lepr-db/db mice by enhancing EGFR expression .

Methods: Genetically obese Lepr-db/db and age-, strain- and sex-matched wild-type mice were subjected to sham surgery or 70% hepatectomy. Introduction of EGFR or control plasmids was conferred by hydrodynamic injection of naked plasmid DNA. Survival and regeneration were assayed by serum liver enzymes, histology, Western blotting analysis and recovery of liver mass.

Results:  Kaplan-Meier survival analysis of survival after 70% hepatectomy revealed significantly greater mortality in lepr-db/db mice (50% survival)  versus wild-type controls (100% survival, p>0.01). Among survivors, lepr-db/db mice demonstrated increased serum AST, ALT, and bilirubin (indicating cell death and liver dysfunction) versus wild-type mice. Histologic examination of lepr-db/db livers revaled areas of necrosis and hemorrhage, vacuolar change, and mitotic figures. Recovery of liver mass was delayed. After hepatectomy, lepr-db/db mice failed to increase EGFR expression as observed in the wild-type mice and furthermore showed little phosphorylated Y1068-EGFR.  At 6h post-hepatectomy, mouse liver extracts in db/db mice showed increased total hepatic protein carbonylation, indicating increased oxidative stress, and at 48h, reduced expression of pAkt, CyclinD1, and PCNA, demonstrating impaired hypertrophy and proliferation. Delivery of EGFR via transgenesis through hydrodynamic injection in db/db mice did not improve survival or regeneration.

Conclusion: Injury and impaired proliferation are very early events in the abnormal liver regeneration observed in db/db mice. This mechanism likely contributes to mitochondrial damage, impaired liver function, increased hepatocyte death and impaired proliferation in db/db mice. Regeneration in genetically obese mice cannot be corrected by EGFR replenishment alone.

 

61.04 The role of luminal contents and IAP in modulating TLR-mediated pathways in a murine colitis model

Posted on December 22, 2014

S. A. Morrison1, S. Hamarneh1, T. Tantillo1, T. Phupitakphol1, M. Gharedaghi1, K. Economopoulos1, S. Hyoju1, S. S. Gul1, M. N. Kohnehshahri1, R. A. Hodin1  1Massachusetts General Hospital,Surgery,Boston, MA, USA

Introduction: Despite the increasing prevalence and morbidity of inflammatory bowel disease (IBD), the mechanistic pathogenesis remains elusive. Established clinical practice in the management of Crohn’s disease rest on the premise that exposure to the fecal stream is intimately correlated to disease expression. This tenet is supported by numerous studies in the literature; however, the mechanism underlying the toxicity of the luminal stream has never been thoroughly studied. This study was based upon the hypothesis that constituents within the fecal stream serve to aberrantly activate TLR-mediated pathways and that the brush border enzyme, intestinal alkaline phosphatase (IAP), known for its anti-inflammatory properties, may play an antagonistic role in attenuating this dysregulated immune response.

Methods: 8 week old female C57BL/6 wild type mice were used in this study. Study groups consisted of a control and experimental group. Colitis was induced in the experimental group by the addition of 3% Dextran Sodium Sulfate (DSS) to the drinking water for 7 days, at which time all animals were sacrificed, and luminal fluid samples were collected. These samples were then applied to HEK-293 cell lines, engineered to express a specific Toll-Like Receptor (TLR); 2, 4, 5, or 9. Prior to application, samples were incubated +/- IAP. Resulting IL-8 production was then assayed.

Results: Specific TLR cell lines were first tested in vitro to demonstrate an appropriate response to known target ligands, as well as antigens that served as negative controls.  These cell lines demonstrated significant IL-8 production as anticipated in response to known receptor ligands, and this response was counteracted by incubation with IAP. In studying the activation of TLR pathways in response to luminal fluid, TLR-4 and TLR-9 pathways appeared relatively quiescent when incubated with luminal contents from the control group; however incubation with luminal samples from the colitis group demonstrated a hyperactivation of these pathways (TLR 4: 640.5pg/ml v. 1675.9pg/ml, p=0.009, TLR9: 138.4pg/ml v. 1063.5pg/ml, p=0.005; Control v. DSS), suggesting a role in the pathogenesis of disease expression. This response was significantly attenuated by incubation with IAP (TL4: 1675.9pg/ml v. 1083.5pg/ml, p=0.03, TLR 9:1063.5pg/ml v. 438.4pg/ml, p=0.01; DSS v. +IAP). The TLR-2 pathway was noted to be activated by luminal contents from both groups, implying some basal activation of this pathway (TLR2: 1670.8pg/ml v. 2216.6 pg/ml, p=0.68; Control v. DSS). The TLR-5 pathway was relatively inactive in response to luminal contents from both control and colitis groups (TLR5: 29.1pg/ml v. 20.9pg/ml, p=0.17; Control v. DSS).

Conclusion: Following the initiation of DSS colitis, the luminal contents of mice contribute to the generation of inflammation, at least in part through the aberrant over-stimulation of TLR-4 and TLR-9 pathways. This response was attenuated by incubation with IAP.

 

61.05 Cyclooxygenase-2 is Induced by Prostaglandin E2 via EP2/EP4 Receptor Activation in Enterocytes

Posted on December 22, 2014

J. Golden1, P. Kavarian1, L. Illingworth1, J. Lim1, J. Wang1, A. Grishin1, H. Ford1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA

Introduction:  Cyclooxygenase-2 (COX-2) and its product, prostaglandin E2 (PGE2), have been identified as critical factors in inflammatory gut barrier failure. PGE2 is known to act on four receptors (EP1-EP4) all of which are present in the intestinal epithelium. Previous work in our lab has shown that high levels of COX-2 and PGE2 cause intestinal barrier breakdown in experimental peritonitis. Therefore, we hypothesized that COX-2 is induced by its end product, PGE2, via EP receptor activation resulting in runaway inflammation. 

Methods:  Intestinal epithelial cells (IEC-6) were treated for 12 hours with PGE2, PGE2 following EP receptor antagonist pre-treatment, and with PGE2 following pre-treatment with inhibitors of various downstream mediators of the EP1-4 pathways (PKC, PI3K, MEK). Expression of COX-2 protein was examined using Western blot. Results were obtained using quantitative immunofluorescent protein detection and statistical analysis was performed using Student’s t-test.

Results: COX-2 protein levels were significantly increased 2.6 (±0.5) fold from control after treatment with 100uM PGE2. PGE2-induced COX-2 expression was attenuated when cells were pre-treated with EP2 antagonist PF-04418948 and EP4 antagonist GW627368, but not with EP1 or EP3 antagonists. Pre-treatment with a PKC inhibitor, a downstream mediator of EP1, did not attenuate PGE2-induced COX-2 expression. Inhibiting mediators downstream of EP2 and EP4, such as PI3K and MEK, eliminated PGE2-induced COX-2 induction. 

Conclusion: COX-2 is induced by its end product PGE2 in IEC-6 cells. This induction is attenuated if cells are pre-treated with EP2 and EP4 antagonists or with inhibitors of their downstream mediators, MEK and PI3K. This attenuation is not seen with EP1 or EP3 antagonists or inhibition of PKC. Our data suggest that PGE2 causes induction of COX-2, likely via an EP2/EP4-dependent pathway, contributing to the runaway inflammatory response seen in inflammatory intestinal disorders.
 

61.06 Plasma HouseKeeping Gene Free For All: A Need for Standardization

Posted on December 22, 2014

J. D. Rice1, H. Roberts1, M. R. Eichenberger1, J. Pan2, S. Rai2, S. Galandiuk1  1University Of Louisville,Hiram C. Polk, Jr., MD Department Of Surgery,Louisville, KY, USA 2University Of Louisville,2Department Of Bioinformatics And Biostatistics,Louisville, KY, USA

Introduction:

Plasma miRNAs have been shown to be promising biomarkers for cancer. A large problem is the lack of standardization in current data acquisition and reporting. Part of this problem lies in the numerous different housekeeping genes (HKG) used in real time PCR data acquisition. With multiple accepted HKG, data comparison and validation between different investigators becomes difficult. Due to issues of data reproducibility and standardization, it is crucial that a plasma HKG 1) be expressed in all samples, 2) have medium to high levels of expression and 3) have consistent levels of expression. 

Methods:

We screened for 380 miRNAs using microfluidic array technology (Applied Biosystems), in a discovery cohort of 20 colorectal cancer (CRC) patients, 10 patients each with colorectal adnoma (CRAd), breast cancer (BC), lung cancer (LC), pancreatic cancer (PC) and controls. We investigated Let-7a, Let-7d, Let-7g, miR-16, RNU6, RNU48, miR-191, miR-223, miR-484 and miR-520d-5p as potential HKG’s based both upon our previous data and reports in the literature.  The mean ∆CT and standard deviation were calculated for each HKG. Only microRNAs with >90% expression were included in this statistical analysis. 

Results:

RNU6, miR-520d-5p, miR-16, miR-191, miR-223, miR-484 were expressed in all samples. Let-7a, Let-7d, Let-7g and RNU48 were not expressed in 26%, 7%, 10% and 8% of samples respectively. U6 and miR-520d-5p had the most consistent ∆CT and lowest standard deviation.

Conclusion:

Let-7a, Let-7d, Let-7g and RNU48 were not expressed in all samples and were therefore not considered reliable HKG’s. Investigating the remaining HKG with 100% expression in all samples, RNU6 and miR-520d-5p are excellent HKG candidates for studies of plasma miRNA due to consistent ∆CT in all samples and a very narrow standard deviation. 

 

61.07 SIGIRR/TIR8 Predicts Biochemical Recurrence After Prostatectomy in Low-grade Prostate Carcinomas

Posted on December 22, 2014

T. M. Bauman1, A. J. Becka1, P. D. Sehgal1, W. Huang2, W. A. Ricke1  1University Of Wisconsin School Of Medicine And Public Health,Department Of Urology,Madison, WI, USA 2University Of Wisconsin School Of Medicine And Public Health,Department Of Pathology And Laboratory Medicine,Madison, WI, USA

Introduction: Single Ig IL-1-related receptor (SIGIRR) is a negative regulator of toll-like receptor (TLR) 4 and IL-1 mediated activation of NF-κB. A tumor suppressive role of SIGIRR has previously been established in certain carcinomas, but the role and expression of SIGIRR in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer (PCa) has yet to be investigated. The purpose of this study was to characterize SIGIRR protein expression in human prostate tissues.

 

Methods: Nuclear and cytoplasmic SIGIRR expression were quantified in glandular prostate tissue using immunohistochemistry and multispectral imaging. Expression was compared between tumor-adjacent normal prostate (n=48 patients), BPH (n=24), high-grade prostatic intraepithelial neoplasia (HGPIN; n=25), PCa (n=73), and metastases (n=22), and SIGIRR expression was evaluated in relation to clinico-pathological features of PCa (Gleason score, pathological stage, tumor volume, surgical margin status, serum PSA). Kaplan-Meier analysis and Cox proportional hazards regression was used to investigate the association of SIGIRR expression and PSA biochemical recurrence. Patient outcomes were reanalyzed in subgroupings of low Gleason score (≤6 or 3+4) and high Gleason score (4+3 and ≥8).

 

Results: Compared to normal prostate, cytoplasmic SIGIRR expression was similar in BPH (p=0.37), HGPIN (p=0.20), PCa (p=0.40), and metastases (p=0.31). No significant changes in nuclear SIGIRR expression were found in BPH (p=0.07), HGPIN (p=0.37), or PCa (p=0.06), but a significant decrease in expression was found in metastasis samples (p=0.04). Decreased nuclear SIGIRR expression was observed in patients with high Gleason score (4+3 and ≥8; p=0.03), but no significant changes were found in cytoplasmic SIGIRR expression (p=0.08). Both cytoplasmic and nuclear SIGIRR expression were not related to pathologic stage, tumor volume, surgical margin status, or serum PSA (p>0.05).

Nuclear SIGIRR expression (p=0.96) and cytoplasmic SIGIRR expression (p=0.89) as continuous variables were not associated with biochemical recurrence in univariable analysis when all patients were analyzed. In sub-analysis of low Gleason score patients, high cytoplasmic SIGIRR expression was associated with increased biochemical recurrence in both univariable (p=0.01) and multivariable (HR 2.31 [95% CI 1.05-5.06] p=0.04) analysis, independent of pathologic stage, tumor volume, and margin status.

 

Conclusions: SIGIRR predicts biochemical recurrence in patients with low Gleason score prostate cancer, but is not associated with recurrence in high Gleason score patients. These findings highlight a potential mechanistic role of NF-κB signaling specifically in low grade PCa.

 

 

61.08 Curcumin-Mediated Regulation of Notch1/HES1/Survivin: Molecular Targeting in Cholangiocarcinoma

Posted on December 22, 2014

S. T. Koprowski1, K. M. Sokolowski1, S. Kunnimalaiyaan1, T. C. Gamblin1, M. Kunnimalaiyaan1  1Medical College Of Wisconsin,Surgical Oncology/Department Of Surgery/Medical College Of Wisconsin,Milwaukee, WI, USA

Introduction: Cholangiocarcinoma (CCA) is highly malignant and characterized by poor prognosis with chemotherapeutic resistance. Therefore, continued development of novel, effective approaches are needed. Notch1 is highly expressed in CCA, but the utility of Notch1 inhibition is not defined. Based on recent findings, we hypothesized that curcumin, a polyphenolic phytochemical, suppresses CCA growth in vitro via inhibition of Notch1 signaling.  

Methods: Established CCA cell lines CCLP-1 and SG-231 were treated with varying concentrations of curcumin (0-20µM). Viability was assessed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colonogenic assays. Cell lysates were analyzed via Western blotting for Notch1/HES1/Survivin pathway expression, apoptosis, and cell cycle progression. Following curcumin treatment, quantitative real-time polymerase chain reaction (qPCR) was utilized to determine mRNA expression of Notch signaling components.

Results: Curcumin-treated CCA cells exhibited reduced viability compared to control treatment. Statistically significant reductions in cell viability were observed with curcumin treatment at concentrations of 7.5, 10, and 15µM by approximately 10%, 48%, and 56% for CCLP-1 and 13%, 25%, and 50% for SG-231 respectively. Upon Western analysis, concentrations of 10µM and above showed reductions in Notch1, HES1, and Survivin. Apoptosis was evidenced by an increase in expression of cleaved PARP (Poly [ADP] ribose polymerase). Cyclin D1 (cell cycle progression) expression levels were also reduced with treatment. Data collected from qPCR similarly indicated significant reductions in Notch1, HES-1, and Survivin mRNA expression in CCA treatment groups.

Conclusion: Curcumin effectively induces CCA (CCLP-1 and SG-231) growth suppression and apoptosis at relatively low treatment concentrations when compared to previous research. A concomitant reduction of Notch1, HES1, and Survivin expression in CCA cell lines provides novel evidence for a potential anti-tumorigenic mechanism-of-action. To our knowledge, this is the first report showing reduction in HES-1 expression via both mRNA and protein analysis following treatment with curcumin. Such findings merit further investigation of curcumin-mediated inhibition of Notch signaling in CCA either alone or in combination with chemotherapeutic agents.

 

 

61.09 S1P forms a feed-forward loop with the Ang2 and VEGF-C pathways in lymphangiogenesis

Posted on December 22, 2014

L. J. Fernandez1, W. Huang1, K. P. Terracina1, A. Yamada5, T. Aoyagi3, S. Spiegel2, M. Nagahashi4, K. Takabe1,2  1Virginia Commonwealth University,Surgical Oncology,Richmond, VA, USA 2Virginia Commonwealth University,Biochemistry And Molecular Biology,Richmond, VA, USA 3Chiba University,Surgery,Chiba, , Japan 4Nigata University,Digestive And General Surgery,Nigata, , Japan 5Yokohama City University,Surgery,Yokohama, , Japan

Introduction:
It is well established from prior studies that both Angiopoietin2 (Ang2) and vascular endothelial growth factor-C (VEGF-C) in the human lymphatic endothelial cell (HLEC) play important roles in the induction of lymphangiogenesis in inflammation and cancer. Ang2 acts by activating the Tie-2 receptor and VEGF-C through VEGF receptor 3 (VEGFR-3). Sphingosine-1-phosphate (S1P), a lipid mediator, has emerged as an important player in cancer progression that promotes cell proliferation, survival, migration, angiogenesis and lymphangiogenesis. S1P is generated inside cells by sphingosine kinase 1 (SphK1), which is exported and exerts its functions by binding to its specific G protein coupled receptors. In HLECs, S1P binds to S1PR1 and causes lymphangiogenesis. To date, evidence of cross-talk between these pathways has not been reported

Methods:
HLECs were obtained from LONZA, and used after less than 4 splitting-passages. Protein levels of Ang2 and VEGF-C in the supernatant of cell culture were measured by ELISA, mRNA expression by qPCR, and phosphorylation was detected by Western Blot assay. SphK1 activity assay was used to compare the activity after treatment with Ang2 or VEGF-C

Results:
We found that both Ang2 and VEGF-C mRNA expression increased by S1P treatment, with a corresponding significant increase in VEGF-C and Ang2 in the supernatant of cell culture after S1P treatment. This implies that S1P promotes Ang2 and VEGF-C production and secretion from HLECs. Furthermore, with Western-blot we found that Tie2 was phosphorylated after S1P treatment as was VEGFR-3, which implies that Ang2 and VEGF-C bind to their receptors after S1P-induced secretion. We found that Ang-2 treatment to HLEC cells not only resulted in phosphorylated Tie-2 but also in phosphorylated SphK1. This result was similar with VEGF-C treatment which resulting not only VEGFR-3 phosphorylation but also in phosphorylated SphK1. This result is in agreement with that of an S1P activity assay that showed a 1.5 fold increase in SphK1 activity when HLEC were treated by Ang2 or VEGF-C when compared to control. These findings imply that both Ang2 and VEGF-C activate SphK1, which we know produces S1P, via activation of their receptors. Together with our previously published findings, we found that there is an amplification loop type cross-talk between S1P signaling and Ang2 and VEGF-C signaling in LECs

Conclusion:
Our results suggest that there is a feed-forward amplification loop in HLEC where S1P increases the levels and activities of both Ang2 and VEGF-C, then in turn, Ang2 and VGEF-C activate SphK1 that produces S1P. S1P as well as Ang2 and VEGF-C have been independently implicated to be involved in lymphangiogenesis. The feed-forward relationship between these previously separate pathways, will likely potentiate lymphangiogenesis when these pathways are activated. This may have important ramifications in the pathogenesis and treatment of inflammatory and neoplastic conditions

61.10 Differences in Treatment-Induced Perfusion Following Flap Surgery

Posted on December 22, 2014

T. Ramesh1, N. Patel1, G. Aaron1, J. Warram1, E. Rosenthal1  1University Of Alabama At Birmingham,Department Of Surgery,Birmingham, AL, USA

Introduction:  Minimizing surgical morbidity after local flap reconstruction is important in the management of cutaneous defects. In the past, many treatment modalities have been used to mitigate the disease burden and preserve the function of vital head and neck structures. Controversy exists in the literature regarding the effects of two such modalities—radiation and chemotherapy—on flap perfusion. Neoadjuvant treatments have the potential to cause damage to the microvasculature of the surgical bed through fibrosis, endothelial cell damage, and reduced cell proliferation, all of which increase the likelihood of postoperative flap failure. The aim of the present study is to examine the effects of neoadjuvant radiation and chemotherapy on blood perfusion following dorsal flap surgery in an athymic female mouse model. 

Methods:  Animals were divided into three treatment groups: No pre-flap treatment (negative control, n=4), 36-Gy electron-beam radiation administered to dorsal skin (radiation group, n=4), and 2 mg/kg intraperitoneal cisplatin (chemotherapy group, n=4). Treatments were completed on mice 15 days prior to undergoing random-pattern dorsal flap surgery with a length-to-width ratio of 4:1 (4x1cm2). Flap perfusion was assessed via laser-assisted indocyanine green dye angiography and by standard clinical assessment.

Results: LUNA perfusion imaging performed on post-operative day 1 showed 56% distal end flap perfusion relative to healthy skin in chemotherapy group mice, compared to 69% and 71% distal end perfusion in control and radiation groups, respectively. LUNA perfusion imaging performed on post-operative day 4 showed 75% and 72% perfusion relative to healthy skin in control and radiation group flaps, respectively. By post-operative day 5, all chemotherapy group flaps experienced full flap loss. In contrast, 3 of 4 control group flaps and all radiation group flaps survived to the conclusion of the experiment. Clinical assessment of flap perfusion by two physicians on post-operative day 2 revealed that elevated skin flaps of the chemotherapy group were more poorly perfused throughout than flaps in control and radiation groups.

Conclusion: Both LUNA intraoperative imaging and clinical judgment indicated that distal ends of chemotherapy group flaps were most poorly perfused. In addition, complete flap loss occurred faster in chemotherapy group mice than in radiation group and control group mice, suggesting that chemotherapy treatment has the most detrimental effect on flap viability.

 

61.11 Tumor Necrosis Factor Signaling Is Critical For Chronic Hypoxia-Conditioned Tumorigenicity

Posted on December 22, 2014

C. V. Romain1, J. Qiao1, D. H. Chung1  1Vanderbilt University Medical Center,Pediatric Surgery,Nashville, TN, USA

Introduction:  Solid tumors, having outgrown their blood supply, are known to exhibit central area of tumor necrosis and cell death. In the tumor microenvironment, a process of selection occurs where signaling pathways are activated which promote tumor growth and resistance to anti-tumor strategies despite relative hypoxic environment. Hypoxia is known to upregulate cell signaling through tumor necrosis factor receptor (TNFR)-1 and induces aggressive phenotype in certain cancer cells; however, its role in neuroblastoma is largely unknown. Here, we developed an in vitro system to select out a subclonal population of human neuroblastoma cells conditioned by exposure to chronic hypoxia. The purpose of this study was to characterize the exact cell signaling pathways contributing to the survival and growth of virulent hypoxia-conditioned human neuroblastoma cells.

Methods:  Human neuroblastoma BE(2)-C cells were plated under normoxic conditions to 70% confluence in 100 mm dish. They were then transferred to hypoxic chamber (1% O2) until > 90% cells had undergone cell death (detached) for a period of 7-10 days. The remaining viable cells, labeled as HS1, were plated and incubated under normoxia and allowed to re-grow to 70% confluence. This cycle was repeated and the recovered subclonal cell population was designated as HS2. Gene expression for death receptor pathway was performed on BE(2)-C parental vs. HS2 cells. Transcription activity of TNFR superfamily member1A (TNFR1) and TNF-α  were assessed using RT-PCR for BE(2)-C and HS2 cells under normoxia and hypoxia, as well as for BE(2)-C and HS1 cells after TNFR1 silencing. Cell viability was measured in BE(2)-C and HS1 cells with or without treatment with vincristine (200 nM) or cisplatin (2 μ M).

Results: Focused gene array showed >100-fold increase in the expression of TNFR1 in HS2 cells as compared to control. RT-PCR confirmed nearly 8-fold increase in mRNA levels of TNFR1 and TNF-α  in HS1 and HS2 cells as compared to BE(2)-C parental cells. After silencing of TNFR, there were increases in TNF-α  in both BE(2)-C/siTNFR1 as well as HS1/siTNFR1 when compared to control cells. An increased HS1 cell proliferation was found when compared to BE(2)-C cells under either normoxic or hypoxic conditions at 24 h with or without the chemotherapeutic agents. This trend continued at 48 h time point under normoxia but was lost under hypoxic conditions.

Conclusion: Upregulation of TNFR1 was observed in HS1/2, subclonal populations of human neuroblastoma cells created via chronic hypoxia cycling. Increased expression of TNFR/TNF-α  mRNA in both HS1 and HS2 suggests that activation of this signaling pathway may contribute to more robust cell survival in hypoxia-conditioned subclonal neuroblastoma cells. HS1 cells exhibited refractoriness to chemotherapy agents, as well as enhanced cell proliferation, mimicking chemoresistance patterns often seen in high-risk neuroblastoma. 

 

60.07 The Small Molecule BMP Inhibitor DMH1 Inhibits proliferation of calcifying Smooth Muscle Cells

Posted on December 22, 2014

T. Lin1, X. Wang1, R. Guzman1  1Beth Israel Deaconess Medical Center,Surgery,Boston, MA, USA

Arterial calcification is associated with increased cardiovascular morbidity and mortality. It is also associated with increased rates of restenosis after angioplasty. During arterial calcification, medial smooth muscle cells (SMCs) undergo phenotypic transformation into a more osteogenic cell type and it is thought that Bone Morphogenetic Proteins (BMPs) act as regulators of this process. We sought to evaluate the effects of the synthetic, non-peptide, BMP inhibitor DMH1 on migration and proliferation of calcifying SMCs.

Methods:   Human aortic SMCs were induced to calcify by stimulation with 3mM inorganic phosphate (Pi) in DMEM containing 10%FBS or control medium without Pi. Medium was changed every other day with or without increasing doses of DMH1. Calcium content was measured using the o-Cresolphthalein Complexone method normalized to protein content after 7 days. Total RNA from SMCs was isolated at 4 and 7 days using the RNeasy mini kit (Qiagen, USA) and qRT-PCR was performed with respective gene-specific primers. Changes in alpha-SM-actin, alkaline phosphatase (ALPL) and BMP signaling proteins p-Smad1/5/8 were assessed by western blot. Proliferation was assayed using the BrdU cell proliferation assay kit (EMDMillipore, USA). Migration was assayed by using transwell migration assay (Costar, USA).

Results:  Cells grown in Pi-containing medium had increased amounts of calcification compared with controls. DMH1 decreased calcium accumulation in Pi-stimulated SMCs in a dose dependent manner. Addition of DMH1 decreased the expression of smooth muscle cell markers (alpha-SM actin and SM22-alpha) while increasing the expression of the bone markers ALPL, RANK ligand and Runx2. In calcifying SMCs, DMH1 inhibited proliferation more significantly than in non-calcifying SMCs suggesting that calcifying SMCs are more sensitive to BMP receptor activation. In a transwell assay, addition of DMH1 increased migration in calcifying but not uncalcified cells. 

Conclusions:  We find that the synthetic BMP inhibitor DMH1 inhibits Pi-induced calcification and reduces proliferation of calcifying SMCs more strongly than untransformed cells. This suggests that cells from calcifying arteries are different, and that they may respond to BMP stimulation more like osteocytes than smooth muscle cells. DMH1 or similar BMP inhibiting compounds may have clinical utility in decreasing SMC proliferation after endovascular interventions in patients with calcified arteries due to diabetes and renal disease.

60.08 Sox18 is Upregulated in Neointima following Rat Carotid Angioplasty and is Stimulated by TGFβ/Smad3

Posted on December 22, 2014

S. Franco1, X. Shi1, L. Guo1, D. Direnzo1, C. Kent1  1University Of Wisconsin,Surgery,Madison, WI, USA

Introduction: Restenosis is the re-narrowing of the vessel lumen post vascular reconstructive procedures such as balloon angioplasty and bypass and occurs in ~50% of patients undergoing these procedures. Intimal hyperplasia (IH) is a complex process involving smooth muscle cell (SMC) migration and proliferation, and is the primary contributor to restenosis. SOX18 is a transcription factor important in developmental processes. It was previously shown that down regulation of SOX18 prevents proliferation and reduces migration in in cultured SMCs that were stimulated by serum. However, the role of SOX18 in the development of IH is not known. What factors regulate SOX18 in this process and the mechanism by which SOX18 regulates pro-IH SMC behaviors are poorly defined. We have previously shown that elevated TGFβ and its signaling protein Smad3 in injured arteries stimulate SMC proliferation and migration and IH. These results led us to hypothesize that TGFβ/Smad3 regulate SOX18 expression, resulting in enhanced SMC proliferation and migration, and IH following injury.

Methods: To determine the expression of Sox18 and Smad3 in vivo, carotid artery balloon injury was performed in male Sprague-Dawley rats to induce IH/restenosis, and immunohistochemistry was then carried out using anti-SOX18 and anti-Smad3 antibodies on sections from injured carotid arteries or uninjured controls collected at 3, 7, or 14 days post injury. In order to mimic up-regulation of Smad3 and TGFβ in vivo, primary rat aortic SMCs were infected with adenovirus expressing Smad3 (or GFP control) and then stimulated with TGFβ (5ng/ml) or solvent for 24 hours. Total RNA and protein were extracted and used for microarray analysis, real-time PCR, and western blotting.

Results: Using immunohistochemistry we observed that Sox18 positive cells (versus total cells)  were substantially increased in the neointima layer of balloon injured rat carotid arteries at 7 and 14 days post-angioplasty compared to those in the media layer and uninjured control. Smad3 expression was up-regulated in a similar pattern. Our Affymetrix gene expression array data revealed TGFβ/Smad3-stimulated differential expression of multiple genes including Sox18 which was up-regulated 3.19 fold (p=0.02). Real time PCR confirmed Sox18 up-regulation (28 fold, p<0.01) in response to TGFβ/Smad3 stimulation compared to control. Furthermore, Western blot results demonstrated Sox18 protein was also up-regulated following TGFβ/Smad3 stimulation.

Conclusion: In conclusion, we observed up-regulation of Sox18 in neointima following arterial injury where Smad3 is also increased. We confirmed that Sox18 up-regulation was stimulated by TGFβ/Smad3 in SMCs in vitro. Further studies are warranted to investigate a possible mechanism of Sox18-mediated IH stimulated by TGFβ/Smad3 up-regulation.

 

« Previous Page
Next Page »