80.20 Alterations in Biomarkers of Coagulation and Fibrinolysis Following Burn Injury

Posted on January 26, 2017

M. Vigiola Cruz2,3, K. E. Brummel-Ziedins4, T. Orfeo4, L. Moffatt2, J. W. Shupp2,3  2MedStar Health Research Institute,Firefighters’ Burn And Surgical Research Laboratory,Washington, DC, USA 3MedStar Washington Hospital Center,The Burn Center,Washington, DC, USA 4University Of Vermont,Department Of Biochemistry,Colchester, VT, USA

Introduction: There is limited understanding of the alterations in the coagulation cascade caused specifically by burn injury. Considering the fine balance between clot formation and degradation that is necessary for normal coagulation, further insight into the dynamics of pertinent biomolecules following thermal injury is crucial. Our aim is to analyze specific factors involved in clot formation and fibrinolysis over time to improve the current understanding of altered coagulation pathways in burn patients.

Methods: Blood samples were serially collected from 29  burn patients to quantify various biomarkers implicated in coagulation and fibrinolysis. Patients were grouped by injury severity, <10% (n=19) or 10-30% TBSA (n=10). Sampling began within four hours of burn injury, occurring every 2-4 hours in the initial 12 hours and subsequently twice daily for 7 days or until hospital discharge. Concentrations of fibrinogen, D-dimer, Plasminogen (PLG), Tissue Factor Pathway Inhibitor (TFPI), and Thrombin Activatable Fibrinolysis Inhibitor (TAFI) were quantified using ELISA.

Results:Fibrinogen levels on all patients were within normal limits on admission (mean 314mg/dL), and increased until they reached plateau at approximately 96 hours, a 2.3-fold elevation from initial evaluation (p=0.0007). Compared to the 10-30% TBSA group, the <10% cohort trended to have higher levels of fibrinogen measured at all timepoints; the differences were not statistically significant. D-dimer levels increased from presentation (mean 0.4µg/dL), and in the first 24 hours elevated to nearly 3-fold (p=0.0003), with markedly higher levels in the more severe burns as early as hour 12. Conversely, plasminogen levels initially decreased to the lower end of normal range in the initial 36 hours. Modest elevations were seen in TFPI, with fluctuations within a narrow range compared to early values. Mean TAFI concentrations peaked at hour 12 in the 10-30% TBSA group and returned to initial values after 36 hours. All patients survived.

Conclusion:The patterns in factor dynamics (e.g. fibrinogen) demonstrated by our analyses are not consistent with current concepts of coagulopathy, showing aberrancies that necessitate additional exploration. This observation emphasizes the suboptimally understood variation between hyper and hypocoagulability that follows burn injury, pointing to the complexity and multifactorial nature of the coagulation and fibrinolysis processes.  We have previously established that alterations in these cascades may not be detected by lab assays routinely used in the clinical setting, and increases in various acute phase reactants have been associated with higher risk of mortality in trauma and infection. Therefore, further work should continue to integrate specific factor data with clinical observations and measurable outcomes to improve resuscitative interventions in burn patients.

 

80.19 Angiogenin Regulates COX-2 Expression by TNF-α and Bradykinin in the Human Colonic Myofibroblast

Posted on January 26, 2017

E. Chu1, T. Liu1, N. Vanli1, G. F. Hu1, J. Yoo1  1Tufts Medical Center,Colon And Rectal Surgery,Boston, MA, USA

Introduction: The myofibroblast is an important stromal cell of the gastrointestinal (GI) tract that is a target of tumor necrosis factor-alpha (TNF-α ), a potent pro-inflammatory cytokine that has been strongly implicated in the pathophysiology of colitis-associated cancer.  Crosstalk mechanisms are known to exist between TNF-α and other pro-inflammatory mediators, including multiple G protein-coupled receptor (GPCR)-mediated agonists, that amplify inflammatory signaling.  However, the mechanism has not been previously determined.  Angiogenin (ANG) is a 14-kDa member of the ribonuclease superfamily that was the first tumor-derived angiogenesis protein. Like TNF-α , ANG levels are elevated in patients with inflammatory bowel disease (IBD) and colorectal cancer.  However, the role of ANG on inflammatory mediator crosstalk in the myofibroblast is unknown.

Methods: The human colonic myofibroblast cell line 18Co was grown to confluence on 35×10 mm cell culture dishes and was used from passages 8-14. 18Co cells were exposed to TNF-α  (10 ng/ml) and bradykinin (100nM) for varying times. ANG was quantified from the supernatant of serum-starved 18Co cells by ELISA.  The monoclonal antibody 26-2F was used to block the activity of ANG. The expression of cyclo-oxygenase-2 (COX-2) was assessed by Western Blot. 

Results:We have previously reported that 18Co cells exposed to both TNF-α  and the pro-inflammatory GPCR bradykinin (BK) lead to the synergistic expression of COX-2, evident after 4 h (P<0.05).  To determine whether ANG was involved in this process, we first measured ANG levels in the cell culture supernatant of 18Co cells by ELISA.  18Co cells secrete high levels of ANG (265.5 ± 4.7 pg/ml in serum-free media over 24 h).  Exposure of 18Co cells to TNF-α  (10ng/ml) led to a rapid (4 h, 127.8 ± 9.7 pg/ml, P<0.05) and sustained (24 h, 124.6 ± 25.1 pg/ml, P<0.05) reduction in the concentration of ANG in the supernatant, corresponding to an uptake of ANG by these cells.  The anti-ANG monoclonal antibody 26-2F, which neutralizes the activity of ANG, inhibited the synergistic expression of COX-2 induced by TNF-α  and BK at 4 h (P<0.05). 

Conclusion:TNF-α  stimulates ANG uptake by the myofibroblast, and inhibition of ANG blocks synergistic COX-2 expression induced by TNF-α  and BK.  Crosstalk signaling between TNF-α  and BK appears to be mediated by ANG.  Angiogenin may play an important role in the regulation of COX-2 expression in the setting of inflammation, and may be a novel therapeutic target for the management of colitis-associated cancer. 

 

80.18 Circulating Plasma MicroRNAs in Colorectal Neoplasia: A New Role in Assessing Response to Therapy

Posted on January 26, 2017

J. Carter1, U. Netz1, K. Feagins1, V. States1, M. R. Eichenberger1, S. Galandiuk1  1University Of Louisville,Department Of Surgery,Louisville, KY, USA

Introduction:

Recurrence following treatment for colorectal cancer is common. Current blood-based tests, such as serum carcinoembryonic antigen (CEA), are used both for post-operative surveillance and for monitoring response to therapy. CEA; however, lacks sufficient sensitivity and specificity to accurately detect recurrence of CRC or its precursor lesion, advanced adenoma (CAA). microRNAs (miRNAs) have been associated with both the diagnosis and regulation of different disease processes. They are short, non-coding RNAs that play an important role in gene expression. We believe miRNAs may have a potential role in monitoring therapy following removal of an adenoma or cancer. We have previously identified longitudinal changes in plasma miRNA in patients with CAA and CRC. Our aim is to confirm significantly dysregulated miRNAs identified from screening studies in plasma samples obtained from patients with CAA and CRC prior to and after endoscopic or surgical removal.

Methods:

Plasma was isolated from 24 patients, 12 with CAA (>0.6cm diameter) and 12 with stage II-III CRC prior to treatment and 4-6 weeks following endoscopic removal or surgical resection. Total RNA was extracted and RNA quality and quantity were assessed. Reverse transcription and quantitative real-time polymerase chain reaction was performed using specific primers and probes for the miRNAs of interest. A total of 11 miRNAs were included for assessment: 4 miRNAs identified from our screening cohort to be significantly dysregulated between pre-treatment and post-removal samples and 7 miRNAs that were significantly different between CAA and controls and CRC and controls in a prior study. Data was analyzed comparing pre-treatment samples to post-removal samples using paired t-tests after normalizing raw cycle threshold data to endogenous RNU6 and miR-16.

Results:

Of the 11 analyzed miRNAs, miR-29c (sensitivity 50% 95% CI:25-75, specificity 83% 95% CI:54-97%, AUC 0.67 95% CI:0.45-0.90) was found to be downregulated in pre-treatment plasma samples compared to post-removal samples in CRC (p<0.05 at α=0.05). miR-374a (sensitivity 50% 95% CI:25-75, specificity 83% 95% CI:54-97%, AUC 0.66 95% CI:0.43-0.89) was found to be downregulated in pre-treatment plasma samples compared to post-removal samples in CAA (p<0.05  at α=0.05).

Conclusion:

The expression levels of miR-29c and miR-374a, were different in pre-treatment as compared to post-removal plasma samples in patients with CRC and CAA, respectively. These findings may help provide for a relatively non-invasive method of monitoring therapy or assessing response to treatment. Future considerations should include standardized protocols for assessing miRNA pre- and post-neoadjuvant therapy in patients with rectal cancer in order to determine the effect of such treatment on tumor regression.

80.17 CD36 as a Potential Modifier Gene in Familial Adenomatous Polyposis

Posted on January 26, 2017

M. Holmes1, K. Bolton2,3, B. Talseth-Palmer2,3, P. Pockney1,4, R. Scott1,2,3  1Hunter New England Local Health District,Newcastle, NSW, Australia 2Hunter Medical Research Institute,Newcastle, NSW, Australia 3University Of Newcastle,Discipline Of Medical Genetics, School Of Biomedical Science And Pharmacy,Newcastle, NSW, Australia 4University Of Newcastle,School Of Medicine And Public Health,Newcastle, NSW, Australia

Introduction:  Familial Adenomatous Polyposis (FAP) is a hereditary syndrome that predisposes individuals to developing hundreds to thousands of adenomatous polyps predominantly in the colon. There is an almost 100% lifetime risk that these individuals will go on to develop colorectal cancer (CRC). The development of FAP is largely due to a germline mutation in the adenomatous polyposis coli (APC) gene. In recent years significant variability in phenotypic expression of FAP has been found in individuals carrying the same APC mutation. Ongoing research has supported the theory that this variability in phenotype may be the result of modifier genes. Identifying modifier genes in human populations has proven difficult and mouse models have been used to help identify a number of possible modifier genes. Recently a potential candidate gene for a mouse gene modifier was identified, CD36, with a demonstrated impact on tumorigenesis1. This study aimed to investigate if single nucleotide polymorphisms (SNPs) in the CD36 gene could act as modifier genes for colon cancer development in human FAP patients.

Methods:  DNA from 143 FAP patients with an APC germline mutation was obtained from the Hunter Area Pathology Service, Newcastle, NSW, and genotyped using 3 SNP assays. Polymerase chain reaction (PCR) was performed and read. Data recording extent of disease, presence of genetic mutations, age at diagnosis and intervention had been previously collated.

Results: Kaplan-Meier analysis was suggestive of a protective effect in patients carrying the homozygote variant genotype of the SNP rs1984112, with none of these patients developing CRC in the follow up period. 

Conclusion: 
These findings are suggestive of the existence of a disease modifiying gene which may have a significant impact on the clinical management of  these patients. A larger validation cohort is currently being sought to further investigate. This method could be used to investigate other potential modifier genes.

 

Reference:

1. Otterpohl KL, Gould KA: Genetic dissection of the Mom5 modifier locus and evaluation of Mom5 candidate genes. Mamm Genome. 2015 Jun;26(5-6):235-47.

80.16 Which Appendicoliths are More Likely to Cause Appendicitis?

Posted on January 26, 2017

M. Khan1, M. B. Chaudhry2, N. Shahzad1, M. Wajid1, W. A. Memon2, A. Alvi1  1Aga Khan University Medical College,Surgery,Karachi, Sindh, Pakistan 2Aga Khan University Medical College,Radiology,Karachi, Sindh, Pakistan

Introduction:
Appendicoliths are frequently considered as the cause of appendicitis. However, not all appendicoliths lead to appendicitis and are commonly detected incidentally on computed tomographic scans in uninflamed appendixes. The differences between appendicoliths associated with appendicitis and ones that are found incidentally have not yet been studied. The objective was to determine if greater number, greater diameter and a more proximal location of appendicoliths in appendix is associated with appendicitis

Methods:
A retrospective case-control study of patient with appendicoliths identified on Computed-Tomographic (CT) scan from 01/2008-12/2014 was completed. Patients were divided into two groups; those with appendicitis and appendicoliths (AA) (cases) and those with incidentally discovered appendicoliths (IA) without evidence of appendicitis (controls). The number, diameter and position of the appendicolith/s were ascertained and studied in relation to appendicitis at presentation.

Results:
In total, 321 patients were found to have appendicoliths on abdominal CT scans. Of these, 103 (32%) patients were in the AA group while 218 (68%) patients were in the IA group. The proportion of patients with appendicitis significantly increased with multiple appendicoliths: 28 (23.5%) with 1 appendicolith, 31 (35.2%) with 2 appendicoliths, 13 (43.3%) with 3 appendicoliths and 19 (70.4%) with >3 appendicoliths (p<0.001). Mean diameter of appendicoliths in the AA group was significantly greater than the appendicoliths of the IA group (6.7+-2.8 vs 3.9+-1.2mm respectively, p<0.001). When analyzing the location of appendicoliths, the proportion of patients with appendicitis was greater for more proximal location of the largest appendicolith; 34 (50.7%) at the base, 28 (32.2%) at the proximal third, 28 (31.5%) at the middle third, and 5 (17.2%) at the distal third (p<0.001). However, 29 (75%) patients with sludge that filled the entire appendix were in the IA group. After multivariable logistic regression analysis it was found that 3 or more appendicoliths, diameter of largest appendicolith of greater than 4 mm and location of largest appendicolith at the base were significantly associated with appendicitis.

Conclusion:

Multiple appendicoliths, greater diameter and more proximal position of appendicoliths are more likely to cause appendicitis. These results support the proposed concept of the obstructive phenomenon underlying the occurrence of appendicitis. This knowledge will aid in the diagnosis of equivocal cases of appendicitis.

80.14 Hand to Hand Coupling: A Novel Mechanism of Unintentional Energy Transfer

Posted on January 26, 2017

D. M. Overbey1,2, S. A. Hilton1, N. T. Townsend1, B. C. Chapman1, C. D. Raeburn1,2, T. N. Robinson1,2, E. L. Jones1,2  1University Of Colorado,Surgery,Aurora, CO, USA 2Denver Veterans Affairs Hospital,Surgery,Denver, CO, USA

Introduction: Energy-based devices are used in nearly every laparoscopic operation. Radiofrequency energy can transfer to nearby instruments via antenna and capacitive coupling without direct contact. Previous studies have described inadvertant energy transfer through bundled cords and nonelectrically active wires.  The purpose of this study is to describe a new mechanism of stray energy transfer from the monopolar instrument through the operating surgeon to the laparoscopic telescope, and propose practical measures to decrease the risk of injury.

Methods: Radiofrequency energy was delivered to a laparoscopic L-hook (monopolar “bovie”), an advanced bipolar device, and an ultrasonic device in a laparoscopic simulator. The tip of a 10mm telescope was placed adjacent but not touching bovine liver in a standard four-port laparoscopic cholecystectomy setup. Thermal injury was measured as increased tissue temperature from baseline nearest the tip of the telescope which was never in contact with the energy-based device after a five second activation.

Results: The monopolar L-hook increased tissue temperature adjacent to the camera/telescope tip by 47±8°C from baseline (p<0.001). By having an assistant surgeon hold the camera/telescope (rather than one surgeon holding both the active electrode and the camera/telescope), temperature change was reduced to 26±7°C (p<0.001). Alternative energy devices significantly reduced temperature change in comparison to the monopolar instrument (47±8°C) for both the advanced bipolar (1.2 ±0.5 °C; p<0.001) and ultrasonic (0.6 ±0.3 °C; p<0.001) devices.

Conclusion: Stray energy transfers from the monopolar “bovie” instrument through the operating surgeon to standard electrically inactive laparoscopic instruments.  Hand-to-Hand coupling describes a new form of capacitive coupling where the surgeon’s body acts as an electrical conductor to transmit energy (Figure 1).  Strategies to reduce stray energy transfer include avoiding the same surgeon holding the active electrode and laparoscopic camera, or using alternative energy devices.

 

80.12 Periosteal Cells are Skeletal Progenitor Cells

Posted on January 26, 2017

T. V. Boyko1,2, Z. Wang1, M. T. Longaker1, G. P. Yang1,3  1Stanford University,Surgery,Palo Alto, CA, USA 2State University Of New York At Buffalo,Surgery,Buffalo, NY, USA 3VA Palo Alto Healthcare Systems,Surgery,Palo Alto, CA, USA

Introduction:  We have identified the mouse skeletal progenitor cells consisting of 8 distinct subpopulations capable of self-renewal and giving rise to all three components of the skeleton: bone, cartilage and stroma. The periosteum consists of a thin layer of cells that have been shown to expand and contribute to bone fracture healing. Separately, we recently reported the strong expression of Del1 in cartilage and its potential role in osteoarthritis. Del1 was also strongly expressed in skeletal progenitors, and histology had shown strong expression in the periosteum. We hypothesized that the expression of Del1 in the periosteum served as a marker for the presence of skeletal progenitors and that periosteal cells would be found to consist primarily of skeletal progenitor cells.

Methods:  Del1-LacZ mice were anesthetized, the left femur exposed, and the periosteum was injured by scraping with the side of a pair of scissors. The right femur was untouched and used as a control. On post-operative day 7, mice were harvested and processed for histology. To obtain cells for Fluorescent Activated Cell Sorting (FACS) analysis uninjured femurs were stripped of periosteum, which was digested to obtain a cell suspension. Cells were then stained for surface markers of skeletal progenitor cells and FACS was performed to analyze for presence of skeletal progenitor cells. 

Results: Strong expression of Del1 could be seen in uninjured periosteum. Using LacZ staining, we demonstrated that these cells underwent expansion with differentiation into hypertrophic cartilage and bone at the injury site. To identify skeletal progenitors within the periosteum, we harvested cells and subjected them to FACS analysis. FACS showed that the largest subpopulation of skeletal progenitors in the periosteum were Bone Cartilage Stroma Progenitor cell population (BCSPs), the skeletal progenitors found to contribute most to fracture healing.

Conclusion: We have previously shown that mouse skeletal progenitor cells play an integral role in bone fracture healing. It has long been known that the periosteum contributes cells to bone repair. We show here that the periosteum contains a large number of skeletal progenitor cells and that the largest population were BCSPs, the cells most involved in fracture repair. Furthermore, we have identified Del1 expression as a strong marker of skeletal progenitor cells. Separately, we have shown that Del1 deletion leads to fracture healing with a decreased bony callus. We conclude that Del1 represents a novel regulator of skeletal progenitor cell biology. 

 

80.11 Modulation of Intestinal Epithelial and Microbiotal Homeostasis with Exercise in a Colitis Model

Posted on January 26, 2017

A. R. Munoz1, S. R. Hamarneh1, H. Albadawi2, M. Watkins2, F. Adiliaghdam1, J. M. Ramirez1, F. Kuehn1, R. Hodin1  1Massachusetts General Hospital,Department Of Surgery,Boston, MA, USA 2Massachusetts General Hospital,Division Of Vascular And Endovascular Surgery,Boston, MA, USA

Introduction: Ulcerative colitis (UC) is characterized by a chronic increase of intestinal pro-inflammatory cytokines. The brush-border enzyme intestinal alkaline phosphatase (IAP) functions as an anti-inflammatory factor, and its levels are decreased in UC patients. Furthermore, IAP supplementation has been shown to be protective against colitis in mice and humans.  Recently, attention has been drawn to the potential benefits of exercise on UC. We thus sought to study the effect of exercise on colitis development, while also investigating the potential role of IAP in the pathway.

Methods: Seven week old C57BL/6 mice were either exercised (EX) or not exercised (NX) for 4 weeks (n=6). Exercised mice were placed on a treadmill for 1 hour daily with 12 m/min speed and 10º incline. Stool was collected daily and used for pNPP phosphatase activity assays and bacterial colony quantification plating. Following the 4-week period, mice were treated with either 2% dextran sodium sulfate (DSS) or normal drinking water for 7 days (n=3). After the 7-day treatment, mice were all placed on normal drinking water for 2 days. Mice were then weighed to determine percent weight loss during DSS treatment and were gavaged with FITC for intestinal permeability determination. Five hours after gavage, mice were sacrificed and serum FITC levels were determined.

Results: After the 4-week exercise period, the EX group weighed less than the NX group (23.38±0.40 vs 26.17±1.05 g, p=.033), demonstrating exercise efficacy. Additionally, the EX group showed significantly increased IAP activity (299.19±30.76 vs 167.82±30.40 pmole pNPP hydrolyzed/min/ug protein, p=.038) and increased levels of aerobic bacterial colonies (440,133±93,597 vs 108,800±40,754 CFU/g stool, p=.0088). In response to the DSS exposure, EX mice lost less percent body weight compared to NX mice (7.3±2.1 vs 22.6±3.7 percent body weight loss, p=.024), indicating less severe colitis in the EX group. Furthermore, the EX mice maintained a healthier gut mucosal barrier, i.e., less FITC fluorescence in serum following DSS-treatment (569.9±199.3 vs 1376.3±26.2 fluorescence, p=.015).

Conclusion: Exercise appears to be protective against the development of colitis in mice, perhaps mediated in part by an upregulation of endogenous IAP.

80.10 Regulation of Cell Proliferation by the MicroRNA-200 Family in Colorectal Cancer Cell Lines

Posted on January 26, 2017

J. Burton1, J. Carter1, K. Ramos2, B. G. Oxford3, M. R. Eichenberger1, S. Galandiuk1  1University Of Louisville,Department Of Surgery,Louisville, KY, USA 2University Of Arizona,College Of Medicine,Tuscon, AZ, USA 3University Of Louisville,School Of Medicine,Louisville, KY, USA

Introduction:
Chromosomal instability (CIN) is the major molecular pathway associated with development of sporadic colorectal cancer (CRC). With increasing CIN, mutations in the KRAS gene contribute to the progression from normal mucosa to invasive CRC. K-Ras protein is a product of the KRAS gene and acts within the mitogen-activated protein kinase (MAPK) pathway, a major regulator of cell proliferation.The miR-200 family has been linked to several cancers, including CRC, and is known to target a negative regulator of K-Ras, RASSF2. We have previously identified increased expression of all members of the miR-200 family and decreased expression of RASSF2 in CRC cell lines as compared to a normal colon epithelial cell line. Therefore, we hypothesize that the miR-200 family regulates cell proliferation through targeting of this negative regulator and subsequent K-Ras activity in the MAPK pathway.

Methods:
A K-Ras wild type CRC cell line (HT29) and normal colon epithelial cell line (CCD841) were acquired (ATCC®, Manassas, VA). Cells were grown in appropriate culture medium until confluent. Once grown, cells were harvested and plated into separate wells on a 12-well plate at a concentration of 100,000 cells/mL culture media and then allowed a 24-hour period to adhere. At 24 hours, cells were serum starved for 2 hours and transfected with miR-200 family mimics or antagomirs, and their respective negative controls, using the Lipofectamine®RNAiMAX Transfection Reagent protocol (ThermoFisher Scientific, Waltham, MA). Transfection was stopped after 24 hours and cell number was measured each day for 5 days using an automated cell counter (TC20™ Bio-Rad, Hercules, CA).

Results:
Confirmation of successful transfection was performed. CCD841 cells had significantly increased proliferation when transfected with miRNA mimics of all members of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) both individually and combined, as compared to the negative control at day 5 (p<0.05). Conversely, we observed a significant decrease in cell proliferation at days 4 and 5 when HT29 cells were transfected with miR-200 antagomirs for the miR-200 family members both individually and combined, compared to the negative control (p<0.05). A representative growth curve is shown in Figure 1.

Conclusions:
These findings suggest that the gain and loss of function of the miR-200 family affects cell proliferation. Overexpression of the miR-200 family increases cell proliferation, an important process in tumor growth, whereas silencing of the miR-200 family decreases cell proliferation, reducing neoplastic progression. miR-200 should be investigated further as a potential therapeutic target in the treatment of colorectal cancer.

80.09 Effect of Biomechanical Stretch on Regulation of Extracellular Matrix Hyaluronan by Fibroblasts

Posted on January 26, 2017

M. Fahrenholtz1, X. Wang1, H. Li1, Y. Dhamija1, P. Duann1, M. Rae1, K. Grande-Allen2, S. Keswani1, S. Balaji1  1Baylor College Of Medicine,Surgery,Houston, TX, USA 2Rice University,Bioengineering,Houston, TX, USA

Introduction:  Mid gestation fetus heals cutaneous wounds without scar and its anti-fibrotic phenotype is defined by negligible resting tension and distinct extracellular matrix (ECM) with elevated levels of hyaluronan (HA) produced by fetal fibroblasts. In contrast, adult skin is characterized by relatively low levels of HA, and much higher resting tension. Biomechanical tension induces a pro-fibrotic phenotype in fibroblasts which is characterized by increased inflammatory cytokine and ECM production, which leads to the formation of scar tissue in vivo. The role of HA production by fibroblasts under biomechanical stretch has not been fully examined. We hypothesize that higher mechanical tension will alter HA metabolism of fibroblasts via differential regulation of HA synthases (HAS1-3) and hyaluronidases (Hyal1-2).

Methods:  Primary murine adult dermal fibroblasts (AFb) were cultured on collagen-coated silicone membranes +/-10% static strain. AFb expression of HA synthesis(HAS1-2), remodeling(HYAL1-2), and receptor(CD44) genes, as well as phenotype(ASMA) were assessed by qPCR. Total HA production was measured by ELISA. Data is presented as mean+/-SD, n=3/group, p values by ANOVA with post-hoc Tukey HSD.

Results: Mechanical tension induced differential HA gene regulation in AFb, with significantly increased HAS1 gene expression (1.67+/-1.03 vs 5.13+/-0.70, p < 0.01) and decreased HAS2 expression (0.92+/-0.10 vs 0.063+/-0.009, p < 0.01), but no change in HAS3 expression after 24h application of stretch, as compared to unstretched condition. Both Hyal1 and Hyal2 were down-regulated under tension (1.72+/-0.84 vs 0.17+/-0.10, p < 0.05, and 1.64+/-0.70 vs 0.13+/-0.05, p < 0.01, respectively). Total HA quantification at 24 h showed no significant influence of stretch on AFb production of HA, despite differential regulation of HA synthesis and HA remodeling genes, indicating a need to assess additional time points and hyaluronidase activity. ASMA gene expression was not influenced by tension at 24h. Tension downregulated CD44 gene expression (1.18+/-0.32 vs 0.33+/-0.08, p < 0.05), which may influence AFb ability to interact with HA produced under mechanical stretch.

Conclusion: Our data suggest that biomechanical forces have a significant role in influencing the dermal fibroblasts’ cell-matrix interaction and their regulation of the ECM-specifically HA. Understanding the time course of these effects on fibroblast responses to tension, especially under the influence of exogenous wound factors and chemokine imbalance, may yield novel therapeutic interventions to promote anti-fibrotic healing. Understanding the contribution of mechanical environment via morphological and phenotypic alterations may yield novel therapeutic targets in recapitulating fetal regenerative healing in postnatal tissues.

80.08 A novel Alkaline Phosphatase in Pancreatic β-cells

Posted on January 26, 2017

J. M. Ramirez Decrescenzo1, A. R. Munoz1, A. S. Liss1, F. M. Kuehn1, F. Adiliaghdam1, S. R. Hamarneh1, R. A. Hodin1  1Massachusetts General Hospital,Surgery,Boston, MA, USA

Introduction: Type 2 diabetes is characterized by insulin resistance, inadequate insulin secretion and declined pancreatic β-cell mass. Intestinal alkaline phosphatase (IAP) is an endogenous anti-inflammatory factor which is thought to be exclusively expressed in the gut. Mice lacking IAP are more prone to the development of metabolic syndrome and hyperglycemia. Given the embryological association between the intestinal epithelia and the pancreas, we explored the expression and functional role of IAP in pancreatic beta cells.

Methods:  Pancreatic and colon tissue from 17 unique human donors were used to measure the expression level of IAP mRNA by qPCR. IAP expression levels were additionally assessed in pancreatic and duodenal tissue from the same donors (n=3). Cell specific localization of IAP in the pancreas was performed by immunohistochemistry using an antibody against human IAP (n=3). Furthermore, 12-week-old C57BL/6 wild-type and IAP-knockout mice (n=6) were used to study the levels of IAP in pancreatic tissue, its activity and its effect on inflammation.

Results: In human tissues, IAP mRNA levels in the pancreas were abundant compared to the intestinal tissues (Pancreas vs. Duodenum, 2.21 ± 1.45 vs. 1.02 ± 0.21 Relative Expression = 0.5). Remarkably, immuno-histochemistry staining showed specific localization of IAP to pancreatic β-cells, whereas it was absent in acinar cells. Deletion of the IAP gene in mice resulted in the expected absence of IAP mRNA expression as well as a nearly four-fold lower activity of alkaline phosphate enzyme in pancreatic tissue when compared to WT mice (IAP-KO vs. WT, 12.57 ± 6.56 vs. 46.21 ± 2.3 PNPP p= <0.01). Additionally, mice deficient for IAP had dramatically higher inflammatory cytokine mRNA levels in pancreatic tissue: TNF-α, IL1-β (IAP-KO vs. WT, 4.00 ± 0.53 vs 1.18 ± 0.49 Relative Expression = 0.017) and IL-6 (IAP-KO vs. WT, 10.11 ± 1.83 vs. 1.20 ± 0.43 Relative Expression = <0.01) compared to WT-littermates.

Conclusion: IAP is specifically expressed in pancreatic β-cells and likely exerts an anti-inflammatory activity within that tissue. Given the importance of the anti-inflammatory pathway in regard to beta cell dysfunction, the IAP pathway may represent a novel therapeutic target for the prevention or treatment of diabetes in humans.

 

80.07 Sphingosine Demonstrates Effective Killing of E. coli in Models of Urinary Tract Infection

Posted on January 26, 2017

R. M. Boudreau1, G. E. Martin1, C. Couch1, A. E. Mahdy1, M. J. Edwards1, E. Gulbins1,2, A. P. Seitz1, P. L. Jernigan1  1University Of Cincinnati,Department Of Surgery,Cincinnati, OH, USA 2Universitat Duisburg-Essen,Division Of Molecular Biology,Essen, NORTH RHINE-WESTPHALIA, Germany

Introduction:  Catheter-associated bacteriuria is the most frequently diagnosed nosocomial infection globally. The conventional treatment of these infections with systemic antibiotics creates an opportunity for the selection of antimicrobial resistance, adds to burgeoning medical costs, and increases the risk for antibiotic-related side effects. Furthermore, this approach remains suboptimal, with approximately one-quarter of acutely infected patients developing recurrent infection. Sphingosine, a membrane sphingolipid with broad-spectrum anti-microbial properties, has been described as an important part of the innate immunity of the respiratory epithelium to bacterial invasion. Our group has recently discovered high amounts of sphingosine in the transitional epithelium of mouse bladders. We hypothesize that sphingosine is important in the bladder’s innate immunity and that exogenous sphingosine may serve as an effective anti-microbial in murine models of E. coli UTI.

Methods:  E. coli was incubated with normal saline control or varying concentrations of sphingosine in vitro for 2 hours at 37C and 125 rpm agitation.  Bacterial growth was quantified by the plate-dilution method. To evaluate sphingosine’s effectiveness in vivo, wild type mice were sterilely catheterized and inoculated with E. coli for one hour before undergoing bladder irrigation with normal saline control or varying concentrations of sphingosine. Bladders were then harvested and homogenized; and bacterial load was quantified by the plate-dilution method.

Results: Sphingosine demonstrated impressive killing of E. coli compared to saline control in our in vitro study (94% reduction, p<0.001). Similarly, in our murine model of UTI, the bladder irrigations showed maintenance of this antimicrobial effect (Table 1, 95% reduction vs. saline control, p=0.007). 

Conclusion: It was demonstrated previously that sphingosine both plays a crucial role in innate mucosal immunity and possesses antimicrobial activity against E. coli in solution. Here, we present the first study to demonstrate that exogenous sphingosine causes effective bacterial killing in a murine model of UTI. Our data support the need for further investigation toward a possible role for sphingosine-based bladder irrigation in the management of UTIs.

80.06 Characterization of the Abdominal Adhesion Fibroblast

Posted on January 26, 2017

C. D. Marshall1, M. S. Hu2, R. C. Ransom1, L. A. Barnes1, A. A. Moore1,3, T. D. Leavitt1, H. P. Lorenz1, M. T. Longaker1  1Stanford University School Of Medicine,Department Of Surgery,Stanford, CA, USA 2University Of Hawai’i John A. Burns School Of Medicine,Department Of Surgery,Honolulu, HI, USA 3Brigham And Women’s Hospital,Department Of Surgery,Boston, MA, USA

Introduction:
Abdominal adhesions resulting from surgery are the most common cause of small bowel obstruction. Their presence complicates subsequent operations and contributes to infertility. Adhesion formation depends on fibroblast collagen production. The precise cell populations and molecular signals that induce adhesion formation are not known. As a result, no effective pharmaceutical anti-adhesion therapies exist. An improved understanding of the cellular and molecular basis of adhesions would allow for the development of improved treatments.

Methods:
Laparotomy was performed on wild type mice and the cecum and abdominal sidewall were abraded using sandpaper. The resulting adhesion tissue was examined with immunohistochemistry and was digested with collagenase, allowing adhesion fibroblasts to be subjected to immunocytochemical analysis. In an abdominal wall transplant model, the abdominal muscular wall of a pan-RFP mouse was sutured onto the inner surface of the abdominal wall of a pan-GFP mouse before adhesions were created. Fluorescent imaging of adhesion tissue forming within the red-green interface was used to determine the origin of adhesion cells. Finally, adhesions were created in transgenic inducible multi-color Rainbow mice, allowing for the assessment of adhesion cell clonality.

Results:
Adhesion fibroblasts expressed known fibroblast markers: vimentin, fibronectin, FSP, PDGFRα , and collagen. Additionally, many fibroblasts expressed the myofibroblast and smooth muscle marker α SMA, the mechanical transduction mediator FAK, and CD26, a surface marker implicated in fibrosis. After two weeks, smooth muscle cells migrated out of the intestinal wall into the adhesion space. Abdominal wall transplantation revealed that >80% of cells in the adhesion tissue were green and hence derived from the bowel surface, rather than red and derived from the abdominal wall (Figure 1). Assessment of cell proliferation in the adhesion using rainbow mice showed that individual intestine surface cells multiply after injury and expand clonally into the adhesion space.

Conclusion:
Adhesion fibroblasts express several cell markers associated with fibrosis that may provide molecular targets for future anti-adhesion therapies. Smooth muscle cells of the intestinal wall migrate into the adhesion and may contribute to inflammation and the formation of permanent adhesions. There is likely significant overlap between cells traditionally defined as fibroblasts and smooth muscle cells in the adhesion. Cells of the intestinal surface are substantially more active in adhesion formation than cells of the abdominal wall surface. Individual progenitor cells of the intestinal surface produce progeny that proliferate clonally and populate the adhesion. 
 

80.05 A Novel Blood Coagulation Assay: Optical Detection of Clot Kinetics Between Matched Surface Areas

Posted on January 26, 2017

M. J. George1, C. Cox1, K. Aroom1, T. Sharma1, M. Skibber1, B. Gill1  1The Univeristy Of Texas Health Science Center At Houston,Department Of Surgery,Houstn, TX, USA

Introduction: Various devices exist capable of detecting platelet activity and an effect of antiplatelet therapy. However, these devices only offer qualitative data without basis in direct measurement of platelet activity. There is no existing clinical assay capable of quantitatively detecting platelet contractile forces. The purpose of this study is to create a novel assay to detect anti-platelet drug effects on clotting by measuring contractile forces of platelets in clotting whole blood.

 

Methods: After appropriate IRB approval whole blood samples were collected from healthy human subjects before and after taking 325 mg of oral aspirin. Calcium chloride was added to citrated samples to initiate clotting. Samples were placed in a temperature controlled glass test chamber with acrylic inserts of matched surface areas at the top and bottom creating a cylindrical blood sample of known height and radius. A camera recorded deflection of a bent wire attached to the top acrylic insert. Using beam equations, force generated by the contracting clot was recorded with time. Kinetic metrics such as clot activation, rate of contraction and clot volume change are recorded. Student t-tests compared metrics taken from the force curves.

 

Results: Qualitative analysis of force curves identified an activation phase prior to a clot reaching a steady state rate of contraction. Student t-tests comparing rates of steady state clot contraction demonstrated aspirinated blood contracted slower, thus generated force at a slower rate than control blood (20.24 versus 23.92 micro-Newtons per second, p = 0.032). Time to reach steady state contraction also was longer for aspirinated blood compared to control blood (588 versus 435 seconds, p = 0.043).

 

Conclusion: This novel blood coagulation assay detects force generated by platelets in a contracting clot with time and demonstrates the kinetics of blood clotting. Aspirinated blood develops force at a slower rate and takes more time to reach a steady state of contraction than control blood.

 

80.03 Hydrogen peroxide promotes polarization of macrophages to the M1 phenotype

Posted on January 26, 2017

J. Xu1, J. Hu1, C. Zgheib1, M. Hodges1, K. W. Liechty1  1Laboratory For Fetal And Regenerative Biology, Children’s Hospital Colorado And The University Of Colorado Anschutz Medical Campus,Department Of Surgery,Aurora, CO, USA

Introduction: Macrophages play an essential role during wound healing and have the ability to dynamically transition between M1 and M2 phenotypes in response to signals from the surrounding microenvironment. Reactive oxygen species (ROS), including hydrogen peroxide, are one of many stimuli that have the potential to polarize macrophages. However, the role of hydrogen peroxide in macrophage polarization remains poorly defined.   We hypothesize that treatment of macrophages with hydrogen peroxide will polarize the macrophage into M1 phenotype with induction of M1 gene expression and reduction of M2 gene expression.

Methods: To test our hypothesis, Raw cells from the murine macrophage cell line RAW264.7 were treated with 0, 10 uM and 100 uM hydrogen peroxide for 1 hour.  Additional macrophages were treated with100 uM hydrogen peroxide for 5, 20, or 60 minutes.  M1 and M2 gene expression was analyzed using real-time PCR. 

Results: Macrophages treated with hydrogen peroxide showed significantly increased gene expression of the M1 macrophage markers (iNOS, and IL1-beta), while demonstrating decreased gene expression of M2 macrophage markers (CD206) in a dose and time dependent manner.

Conclusion: These findings provide evidence that hydrogen peroxide can polarize macrophages to the M1 phenotype. Furthermore, our results demonstrate that increased reactive oxygen species can perpetuate chronic inflammation through persistent M1 macrophage polarization and decreased M2 macrophage polarization. The use of anti-ROS therapies may help to create a microenvironment that decreases M1 polarization and promote M2 polarization and resolution of the inflammatory response.

 

80.04 Whole Blood Mitigates Acute Coagulopathy of Trauma by Avoiding the Coagulopathy of Resuscitation

Posted on January 26, 2017

A. R. Macko1, L. J. Schaub1, J. J. Glaser1, H. B. Moore2,3, E. E. Moore2,3, F. R. Sheppard1  1Naval Medical Research Unit-San Antonio,Expeditionary Trauma And Medicine,Ft. Sam Houston, TX, USA 2Denver Health Medical Center,Aurora, CO, USA 3University Of Colorado Denver,Aurora, CO, USA

Introduction: The contribution of fluid resuscitation to the development of coagulopathy remains controversial. We hypothesize that resuscitation accentuates the Acute Coagulopathy of Trauma (ACOT).

Methods: NHPs (n=8) underwent poly-traumatic (soft tissue injury and femur fracture) severe hemorrhagic shock (MAP=20 mmHg). Resuscitation was accomplished via a combination of whole blood (WB) and normal saline (NS), with resuscitation volume determined by shed blood volume (SBV). Animals were randomized to receive one of two, 3-stage sequences of WB and NS: 1) NS-WB-NS or 2) WB-NS-NS.  Blood samples were collected at baseline (BSLN), end-of-shock (EOS), end of first (R1), second (R2) and third (EOR) resuscitation phases, and 360 minutes after the initiation of shock (T360) for assessments of clinically-relevant indicators of coagulopathy including:  viscoelastic clotting properties in response to extrinsic pathway activation (EXTEM®) via rotational thromboelastometry (ROTEM®): clotting time (CT), -angle, and maximal clot firmness (MCF).  Data presented as mean ± SEM; statistical analysis via paired sample t-test and 2-way ANOVA; significance at p<0.05.

Results: Baseline lab values, blood pressure, percent blood loss, duration of shock, and survival were equivalent between groups.  EXTEM CT, α-angle and MCF were minimally altered at EOS compared to BSLN; however in the NS-first group CT significantly increased, and -angle and MCF decreased significantly (p<0.05) at R1 (Table 1). CT, α-angle and MCF returned to baseline following WB.

Conclusion: In this model of traumatic hemorrhage, significant alterations in coagulation function consistent with clinically described ACOT did not manifest until saline resuscitation commenced.  WB-first resuscitation was protective in maintaining coagulation function and effectively reversed the apparent acute coagulopathy induced by crystalloid resuscitation as determined by EXTEM.  This data supports not only blood first but potentially blood only resuscitation with regards to prevention of full blown ACOT and supports the concept that an acute coagulopathy of resuscitation (ACOR) is the largest driver of ACOT.

 

80.02 Direct Peritoneal Resuscitation Decreases Lung ICAM and MPO After Resuscitated Hemorrhagic Shock

Posted on January 26, 2017

M. A. Eid1, P. J. Matheson1,2, V. S. Graham1, C. D. Downard1,2, R. N. Garrison1,2, J. W. Smith1,2  1University Of Louisville,Department Of Surgery,Louisville, KY, USA 2Robley Rex Veterans Affairs Medical Center,Research,Louisville, KY, USA

Introduction:  Potential complications of hemorrhagic shock (HS) include gut and liver hypoperfusion, splanchnic hypoxia, gut cytokine storm, acute lung injury (ALI), and/or acute respiratory distress syndrome (ARDS).  While ALI/ARDS pathophysiology is multifactorial, lung polymorphonuclear neutrophil (PMN) infiltration occurs early in the cascade via increased intracellular adhesion molecule-1 (ICAM-1).  We hypothesized that DPR treatment improves gut and liver perfusion to prevent these sequelae to decreased ALI/ARDS, and might mitigate ALI/ARDS following HS/CR.

Methods:  Anesthetized male Sprague-Dawley rats (225-250g) were randomized to groups (n=8/group): 1) Sham, 2) HS/CR, 3) HS/CR+DPR (0), or 4) HS/CR+DPR (120).  HS was 40% of baseline MAP for 60 minutes.  CR was shed blood plus two volumes of normal saline over 30 minutes.  DPR was intraperitoneal injection of 30mL pre-warmed 2.5% dextrose peritoneal dialysis solution.  Serum and tissue were collected at 4 hours post-CR.  Lung ICAM-1 ELISA and MPO activity assay were performed.  Lung H&E and IHC for ICAM-1, VCAM-1, and MPO were blindly graded.  

Results: HS/CR increased ICAM-1 levels and MPO activity compared to Sham, while DPR diminished these effects (see Table).   In HS/CR, ICAM-1, VCAM-1, and MPO IHC staining increased compared to Sham, which was decreased with DPR.  MPO IHC revealed increased PMN extravasation and increased absolute number per high-powered field in HS/CR groups compared to Sham, which both decreased below HS/CR levels with DPR. 

Conclusion: Lung ICAM-1, VCAM-1, and MPO expression following hemorrhagic shock are modulated by peritoneal resuscitation using hypertonic peritoneal dialysis solution.  These data suggest that resuscitation applied to the peritoneal space has a remote effect on lung pathophysiology associated with hemorrhagic shock.  This study supports the finding of resuscitation with DPR in prior human trauma patient studies.

 

80.01 Damage-Associated Molecular Patterns Are Mitigated by DPR in Resuscitated Hemorrhagic Shock

Posted on January 26, 2017

M. A. Wilson1,2, P. J. Matheson1,2, J. L. Weaver1, C. D. Downard1,2, R. N. Garrison1,2, J. W. Smith1,2  1University Of Louisville,Department Of Surgery,Louisville, KY, USA 2Robley Rex Veterans Affairs Medical Center,Research,Louisville, KY, USA

Introduction:  Hemorrhagic shock (HS), a significant cause of mortality in trauma patients, has traditionally been resuscitated with intravenous blood and fluid infusion (CR).  While central hemodynamic variables can be restored with CR, vital organ blood flow can often drop causing intestinal hypoperfusion, hypoxia, gut inflammation, and remote organ dysfunction.  The addition of Direct Peritoneal Resuscitation (DPR) can prevent intestinal and hepatic hypoperfusion and inflammation.   We hypothesized that DPR would improve lung function in resuscitated HS (HS/CR) by altering levels of serum and lung inflammatory mediators (DAMPs).

Methods:  Anesthetized Sprague-Dawley rats were randomly assigned to groups (n=8/group):  1) Sham (matching timeline but no HS, CR, or DPR) 2) HS/CR (HS=40% MAP for 60min, CR=shed blood + volumes NS); and 3) HS/CR+DPR.  All groups were followed for 4hr post-RES.  ELISA was used to measure serum and/or lung lipopolysaccharide (LPS), cytokines, hyaluronic acid (HA), high mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), MYD88, TRIF.  Statistics were by analysis of variance and Tukey-Kramer test a priori P value of 0.05.

Results: HS/CR increased serum levels of LPS, HA, pro-inflammatory cytokines (IL-1a, IL-1b, IL-6, and interferon-g), and HMGB1, and lung levels of TLR4 and MYD88 were increased but not TRIF compared to Shams.  HS/CR+DPR decreased LPS, HA, cytokines, HMGB1, TLR4 and MYD88 levels but did not alter TRIF levels compared to HS/CR alone.  

Conclusion: Gut-derived mediators of systemic inflammation can be modulated by peritoneal application of hypertonic DPR to prevent activation of lung inflammatory processes.  DPR after hemorrhagic shock improved visceral blood flow, reduced tissue injury, reduced DAMP formation and serum levels of multiple inflammatory cytokines and chemokines.  Direct peritoneal resuscitation has the potential to significantly improve morbidity and mortality by downregulating the systemic inflammatory response following hemorrhagic shock

 

79.20 The Temporal Response of Endothelial Dysfunction Following Shock Conditions: An In vitro Model

Posted on January 26, 2017

J. V. Martin1, D. M. Liberati1, L. N. Diebel1  1Wayne State University,Marian And Michael Ilitch Department Of Surgery,Detroit, MI, USA

Introduction: The role of the endothelium in the regulation of hemostasis and inflammation has been recognized for some time. More recently the effect of hemorrhagic shock (HS) on endothelial dysfunction (endotheliopathy of trauma, EOT) has been described. Key factors include hypoxia and/or reactive oxygen species production and sympathoadrenal activation. However, the parameters of endothelial dysfunction and the time course of EOT are not well described. Human umbilical vein endothelial cells (HUVEC) were used to characterize EOT in vitro.

Methods: Confluent HUVEC were subjected to either control conditions (21% O2) or exposure to hypoxia (5% O2) followed by oxidant challenge (H2O2) (H/O insult), epinephrine (10-3 μM) or both H/O and epinephrine exposure. Syndecan-1 shedding was analyzed to determine glycocalyx disruption, PAI-1 and tPA were used to index coagulation profile (thrombosis and fibrinolysis, respectively). Angiopoietin-2/angiopoietin-1 ratio (APO-2/APO-1) and soluble thrombomodulin (TM) were used to index microvascular integrity and endothelial injury, respectively. 

Results: See table.

Conclusion: Experimental conditions that mimic the vascular endothelial mileau following HS depict an "early" fibrinolytic profile (increased tPA) and a "later" prothrombotic profile (decreased tPA and increased PAI-1/tPA ratio). Microvascular integrity was impaired (increased APO-2/APO-1) early and remained so as did evidence of endothelial injury (TM). This study identifies temporal phenotypes of EOT and confirms an "early window" for the therapeutic use of antifibrinolytic tranexamic acid in the clinical setting.

 

79.19 Long non-coding RNA Lethe regulates NOX2 expression through inhibiting ERK pathway in macrophages

Posted on January 26, 2017

J. Xu1, C. Zgheib1, J. Hu1, M. Hodges1, K. W. Liechty1  1Laboratory For Fetal And Regenerative Biology, Children’s Hospital Colorado And The University Of Colorado Anschutz Medical Campus,Department Of Surgery,Aurora, CO, USA

Introduction: Recent studies reveal that long non-coding RNAs (lncRNAs) play important regulatory roles in many biological processes. We have previously shown that lncRNA Lethe is down-regulated in diabetic wounds and is involved in the regulation of Reactive oxygen species (ROS) production through modulation of NOX2 gene expression. We hypothesize that Lethe regulates NOX2 expression through the ERK pathway.

Methods: To test our hypothesis, we incubated the murine macrophage cell line RAW264.7 with media containing 5 mM glucose (low glucose), or 25 mM glucose (high glucose) for 24 hours. Overexpression of Lethe was achieved by plasmid transfection. Western blot analysis was used to measure ERK protein level and Real-time PCR used to quantify relative gene expression.

Results: NOX2 was significantly upregulated in high glucose conditions and was associated with significantly decreased Lethe gene expression. Overexpression of Lethe significantly reduced NOX2 gene expression but did not affect the levels of SOD2, SOD3, or Catalase. Western blot analysis showed significantly increased levels of phosphorylated ERK1/2 in high glucose conditions, and that the overexpression of Lethe significantly reduced the levels of phosphorylated ERK1/2.

Conclusion: These findings demonstrate that the lncRNA Lethe regulates NOX2 gene expression and ROS production in macrophages via the ERK pathway.  Furthermore, these results suggest a potential role of lncRNA Lethe in the pathogenesis of the diabetic wound healing impairment and may represent a potential novel therapeutic target to correct the impaired diabetic wound healing response.

 

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