4.07 The Effects of Exercise on Soleus Function in Severe Burn with Muscle Disuse Atrophy

Posted on December 22, 2014

M. R. Saeman1, K. DeSpain1, M. Liu1, B. Carlson1, L. A. Baer2, J. Song1, C. E. Wade2, S. E. Wolf1  1University Of Texas Southwestern Medical Center,Surgery,Dallas, TEXAS, USA 2University Of Texas Health Science Center At Houston,Surgery,Houston, TEXAS, USA

Introduction:
Muscle loss is a known sequela of severe burn and critical illness that increases the risk of complications such as sepsis and prolonged recovery time. A prior study in a rat model of hindlimb unloading after burn supports that bedrest contributes significantly to muscle atrophy. The aim of our study was to evaluate if exercise mitigates the loss of muscle in this animal model.

Methods:
Two groups of 24 Sprague-Dawley rats were randomly assigned to burn ambulatory (B/A), burn hindlimb unloading (B/H), sham ambulatory (S/A), or sham hindlimb unloading (S/H). One group was trained to perform twice daily weighted resistance climbing of 1 meter with 5 repetitions; the other group had no exercise. Rats received a full thickness scald burn of 40% total body surface area or sham and were allowed to ambulate or were placed in a tail traction system for hindlimb unloading. On day 14 in situ isometric forces were measured on the left soleus muscle. Statistical analysis was performed with Sigma Plot using Student’s t-test, Mann Whitney, or ANOVA with Holm-Sidak method where appropriate. 

Results:
The soleus wet weight was lower in the hindlimb (144 mg) and the exercise (136 mg) versus the ambulatory (190 mg, p<0.001), and no exercise (180 mg, p=0.01) groups. There was no difference in weights between burn and sham. Twitch was significantly lower in the hindlimb group: 31 vs 12 g (p<0.001). Compared to no exercise, the B/H exercise group had a significantly higher twitch force 14 vs. 8 g (p=0.04). Across all other factors there was no significant difference in the twitch between exercise and no exercise. There was a significantly lower tetanic force in the hindlimb group: 55 vs 148 g (p<0.001). B/A had a lower tetanic force in the exercise group versus no exercise: 118 vs 165g (p=0.02). In B/H no difference in tetanic force was seen with or without exercise. All hindlimb groups had significantly lower specific tetanic force than ambulatory: 12 vs. 22 N/cm2 (p<0.001). The specific tetanic force in B/H was significantly higher in exercise versus no exercise: 14 vs. 7 N/cm2 (p=0.008). Fatigue index was significantly lower in the ambulatory (55%) and exercise (52%) groups versus hindlimb (69%) and no exercise (73%) groups (p=0.03, p=0.002 respectively). Muscle function of all groups included in table.

Conclusion:
Hindlimb unloading is a significant factor in muscle atrophy with or without burn. Exercise increased soleus twitch and specific force in this model. However, there was a surprising decrease in muscle mass with exercise in all groups and a decrease in the fatigue index. These findings suggest that exercise contributes to a functional muscle change in a model of disuse and critical illness. 
 

4.08 Stress Conditions Modulate Adipose Tissue Inflammatory Response and Subsequent Organ Injury

Posted on December 22, 2014

D. A. Edelman1, D. M. Liberati1, L. N. Diebel1  1Wayne State University,Surgery/School Of Medicine,Detroit, MI, USA

Introduction: Obesity is an independent risk factor for ARDS and organ failure after severe trauma.  Adipose tissue (AT) composed of adipocytes, macrophages, and other immune cells is a source of pro-inflammatory mediators that are associated with a chronic low grade inflammatory state in the obese patient.  Obesity is also associated with activation of the sympathetic nervous system, which has been linked to shock induced gut and lung injury in the trauma setting.  We hypothesized that sympathomimetic stimulation of adipose tissue would augment inflammatory signaling from adipose tissue and contribute to lung injury and ARDS after trauma.

Methods: Cell co cultures of mature adipocytes and macrophages (RAW264.7) were established and then incubated with either low or high concentrations of epinephrine (10-6 or 10-3  μM respectively). Cell culture supernatants (sup) were obtained at 12 hrs, and AT derived TNF α and IL-6 determined. In separate experiments, human microvascular endothelial cell (HMVEC) monolayers were incubated with adipocyte macrophage sup and HMVEC apoptosis (%apo), ICAM expression (MFI) and FITC-dextran permeability determined.

Results: No difference was seen in co culture data between the low dose epinephrine groups and the no dose epinephrine groups (p<0.001).

Conclusion: Both adipocytes and macrophages contribute to the "obesity related" proinflammatory state. Augmentation of this response after stress related sympathetic activation could contribute to lung injury and other remote organ failure in the injured obese patient. This response seems primarily due to stimulation of the adipocyte component of adipose tissue. The clinical impact likely depends on the magnitude of injury and the distribution/percent total body fat of the patient. Modification of the stress response following trauma may be a therapeutic target in this population. 

 

4.09 Inhibition of Histone Deacetylase 6 Protects the Intestinal Tight Junctions in Hemorrhagic Shock

Posted on December 22, 2014

Z. Chang1, W. He1, B. Liu1, I. Halaweish1, T. Bambakidis1, Y. Li1, H. B. Alam1  1University Of Michigan,Surgery,Ann Arbor, MI, USA

Introduction: We have previously shown that treatment with histone deacetylase (HDAC) 6 inhibitor Tubasatin A (Tub A) can improve outcome in a rat model of septic shock. Also, we have demonstrated that circulating levels of claudin 3, a tight junction (TJ) protein, increase after shock and return to normal after treatment with VPA (pan-HDAC inhibitor). This study investigated whether specific inhibition of HDAC 6 could protect intestinal cellular TJ and improve survival after HS.

Methods: Experiment I: Sprague Dawley rats underwent hemorrhagic shock (40% blood loss over 10 min) followed by treatment with Tub A (70 mg/kg), without any fluid resuscitation. The experimental groups were: (1) sham (no hemorrhage, no treatment), (2) control (hemorrhage, no treatment), and (3) treatment (hemorrhage with Tub A treatment). Animals were sacrificed 6 hrs later, and intestinal tissues were used to create whole cell lysate, which were analyzed for acetyl-tubulin, total tubulin, claudin 3 and zonula occludens 1 (ZO-1) proteins by Western blot. Experiment II: human intestinal epithelial cells (Caco-2) were divided into 3 groups: (1) sham (no hypoxia), (2) control (hypoxia, no treatment), (3) treatment (hypoxia, treatment with tub A). After 12 hours in a hypoxia chamber, cells were analyzed for viability, Lactate dehydrogenase (LDH) levels measuring cellular injury, and TJ protein (claudin 3 and ZO-1) levels.

Results: Hemorrhage decreased, whereas treatment with Tub A increased, expression of claudin 3 and ZO-1. Administration of Tub A also acetylated the tubulin protein. In vitro study showed that hypoxia decreased the viability of caco- 2 cells, which was prevented by Tub A treatment. Similar results were seen when cellular cytotoxicity was determined by the LDH activity. The expression of TJ protein claudin 3 and ZO-1 was significantly decreased by hypoxia, which was significantly prevented by  Tub A treatment. Immunofluorescent study further confirmed that Tub A attenuated the hypoxia-induced claudin 3 and ZO-1 signal loss in the caco-2 cells (Figure). 
 

Conclusion: Selective inhibition of HDAC6 with Tub A preserves the expression of intestinal TJ proteins in models of hemorrhagic shock in vivo and cellular hypoxia in vitro.

4.11 Opioids Promote Anastomotic Leak Following Colo-rectal Surgery in Rats: The Role of the Microbiota

Posted on December 22, 2014

B. A. Shakhsheer1, J. R. Defazio1, J. N. Luo1, R. Klabbers2, I. D. Fleming1, N. Belogortseva1, A. Zaborin1, O. Zaborina1, J. C. Alverdy1  1Pritzker School Of Medicine, University Of Chicago,Department Of Surgery,Chicago, IL, USA 2Radboud University Nijmegen Medical Centre,Department Of Surgery,Nijmegen, , Netherlands

Introduction:  The most dreaded complication after resection of the gastrointestinal tract is anastomotic leak. The effects of opioids on outcomes after gastrointestinal surgery continue to be discovered. Use of patient-controlled analgesia pumps has been associated with increased deep surgical site infection rates by unknown mechanisms. Two findings from our lab may shed light on the mechanisms by which opioids increase anastomotic leak rates: 1. opioids (morphine) directly enhance the virulence of intestinal pathogens and 2. intestinal bacteria play a key causative role in the pathogenesis of leak. Therefore the aim of this study was to examine the effect of opioids on colonic anastomotic leak in a rat model.

Methods:  Rats undergoing one-centimeter colectomy at the peritoneal reflection and primary anastomosis were treated with slow release subcutaneous morphine pellets or placebo pellets implanted in the nape of the neck. Rats were sacrificed on post-operative day six and autopsied for gross signs of anastomotic leak. Microbial composition and phenotype (i.e. collagenase production) was investigated via culture of tissues and intraperitoneal lavage. Local anastomotic tissue was subjected to high magnification microscopy and phenotype analysis.

Results: Rats treated with high-dose (15-mg sustained release) subcutaneous morphine pellets developed a 56% leak rate compared with a 3% leak rate in the placebo treated group (n=73, p=0.0045) High powered images of the anastomotic site demonstrated mucosal ulceration in the morphine group with visible disruptions in the anastomotic integrity whereas no such findings were observed in the placebo treated group. Culture of local tissue and intraperitoneal lavage fluid identified the presence of gram-negative bacteria producing high levels of collagenase which may a play role in anastomotic disruption and non-healing. 

Conclusion: Morphine significantly increases anastomotic leak rates in rats. The role of microbial composition (i.e. gram negative pathogens) and phenotype (i.e. collagenase productions) remains to be investigated and their predictive value confirmed. Taken together these findings provide a rationale to limit opioid use following colorectal surgery and/or to block their peripheral effects with selective opioid antagonists as a countermeasure to prevent deep organ space infection and anastomotic leak.

 

4.12 Agent-based model of Non-Toxigenic Clostridium difficile as Prophylaxis for C. difficile Infection

Posted on December 22, 2014

D. Streicher2, G. An1  1University Of Chicago,Surgery,Chicago, IL, USA 2University Of Michigan,College Of Literature, Science And Arts,Ann Arbor, MI, USA

Introduction:  Non-Toxigenic Clostridium difficile (NTCD) is a generic term for several naturally occurring strains of C. difficile that lack the Pathogenicity Locus (PaLoc) Region, which contains the DNA sequences for Toxins A and B, which are responsible for the pathologic changes in Clostridium difficile Infection (CDI). Otherwise, NTCD shares behavioral properties with other strains of C. difficile, and depletion of commensal microbes by systemic antibiotics affect the suppression of NTCD spores and can trigger their shift from spore to germinated state. Pre-administration of NTCD has been suggested as a prophylaxis for CDI in high-risk population, with the initiation of early stage clinical trials for its study. However, safety concerns have been raised due to recent reports that NTCD is capable of undergoing horizontal gene transfers of the PaLoc region from toxigenic C. difficile. We investigate the feasibility and safety of NTCD prophylaxis with an Agent-based model (ABM) of the gut microbial ecology.

Methods:  A previously produced agent-based model of CDI (CDIABM) was expanded by adding NTCD with both its Germinated (NTCD-G) and Sporulated (NTCD-S) forms. Horizontal Gene Transfer of the PaLoc region was implemented as a stochastic contact effect with toxigenic C. difficile in its germinated form, resulting in the acquisition of toxin producing ability in the target NTCDs. Simulation experiments were performed to examine the prophylactic suppressive and treatment efficacy of NTCD on the development of CDI following systemic antibiotic administration. PaLoc gene transfer events were examined with respect to their impact on persistence and recurrence of CDI.

Results: NTCDs were successfully implemented into the existing CDIABM, and demonstrated qualitatively validated sporulation and germination dynamics. Simulations of different prophylactic NTCD regimens produced a dose dependent suppressive effect on the development of subsequent CDI; however, there was an inflection point identified in the dosing of NTCD and a late phase worsening of CDI. This infection point was associated with the number of horizontal gene transfer events of PaLoc, and demonstrated an exponential effect in low nutrient conditions. Post CDI administration of NTCD did not qualitatively change the trajectory of the CDI.

Conclusion: The modified CDIABM plausibly reproduced the behavior of NTCD in the gut. Prophylactic NTCD appeared to slow the rate and severity of CDI, though it does not prevent damage entirely. The risk of virulizing NTCD through horizontal gene transfer of PaLoc is low, though present with a NTCD dose dependent effect. NTCD was not demonstrated to be effective as a treatment of active CDI. As NTCD prophylaxis is currently being considered in clinical trials, computational investigations of this type can help to augment research and trial design by identifying potential negative effects, characterizing safety concerns, and establishing potential efficacy ranges.

 

4.13 The Anti-Cholinergic Pathway Protects Against Intestinal Barrier Dysfunction and DAMPs Release

Posted on December 22, 2014

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

Introduction: Intestinal barrier injury occurs following major trauma and leads to an  intestinal inflammatory response and subsequent remote organ dysfunction. This response may be modulated by either vagal nerve stimulation or pharmacologic stimulation of the alpha7-cholinergic receptor (nAChR) anti-inflammatory pathway. The downstream mediators in this pathway are relatively unknown. Gut injury is also associated with the release of endogenous damage associated molecular patterns (DAMPs) which may modulate shock induced organ dysfunction. The impact of stimulation of the intestinal cholinergic anti-inflammatory pathway on DAMPs release and resultant tissue injury was studied in vitro.

Methods: Intestinal epithelial cell (IEC-6) monolayers were subjected to hypoxia-reoxygenation (H/R) challenge. Cell subsets were treated after hypoxic challenge with nicotine or AR-R17779, a specific nAChR agonist. Nuclear factor kappa light-chain-enhancer of activated B cell (NFkB) activation was determined by ELISA and IEC monolayer integrity was indexed by permeability to an FITC-dextran probe (4,000 mw; FD-4). DAMPs production was indexed by high mobility group box 1 (HMGB-1) (western blot) and mitochondrial DNA (coxIII using RT-PCR) release. Human pulmonary microvascular endothelial cells (HMVEC) were then co cultured with IEC culture supernatants and monolayer permeability and ICAM-1 expression determined.

Results: mean ± S.D., N = 4 for each group

Conclusion: Pharmacologic stimulation of the nAChR pathway protected against H/R induced intestinal barrier derangement, NFkB activation and DAMPs release. Decreased IEC mediated DAMPs release was associated with protection against lung microvascular injury and ICAM-1 expression in this in vitro study. Modulation of this pathway may be helpful in the clinical setting.

 

3.06 Immunotherapeutic Virus GLV-1h153 Fascilitates 131I Radiotherapy and Imaging in Cholangiocarcinoma

Posted on December 22, 2014

C. Johnsen1, J. W. Ady1, K. Mojica1, A. Pugalenthi1, D. Love1, V. Longo6, P. Zanzonico6, N. G. Chen5, R. J. Aguilar5, Y. A. Yu5, A. A. Szalay5, Y. Fong2  1Memorial Sloan-Kettering Cancer Center,Surgery,New York, NY, USA 2City Of Hope National Medical Center,Surgery,Duarte, CA, USA 3University Of California – San Diego,4Department Of Radiation Medicine And Applied Sciences, Rebecca & John Moores Comprehensive Cancer Center,San Diego, CA, USA 4University Of Würzburg,5Department Of Biochemistry, Rudolph Virchow Center For Experimental Biomedicine, And Institute For Molecular Infection Biology,Würzburg, BAVARIA, Germany 5Genelux,Research And Development,San Diego, CA, USA 6Memorial Sloan-Kettering Cancer Center,Small Animal Imaging Core,New York, NY, USA

Introduction:  Cholangiocarcinoma (CC), a deadly carcinoma of the bile ducts, is clinically silent in the majority of patients until curative surgery is no longer an option. With most patients succumbing to the disease within 6 months of diagnosis, novel treatment options are needed.  Oncolytic viruses are promising cancer therapy agents because they selectively infect, replicate within, and kill cancer cells.  In this study, we assess the ability of the human sodium iodide symporter (hNIS) expressing recombinant oncolytic vaccinia virus GLV-1h153 to kill and image CC when combined with 131I radiotherapy.

Methods:  Three human CC cell lines were assayed for infectivity, cytotoxicity and viral replication in vitro.  hNIS mediated 131I radiouptake was assayed in vitro. Flank CC xenografts were treated with intratumoral GLV-1h153 alone and/or with 131I to assess tumor burden reduction. SPECT/CT was performed to visualize 131I uptake in infected tumor in vivo.

Results: All cell lines demonstrated infectivity and oncolysis in a time and concentration dependent manner. Significant viral replication was supported in all cell lines. Flank xenografts treated with GLV-1h153 combined with 131I demonstrated significant tumor reduction as compared to controls.  131I mediated SPECT/CT demonstrated significant uptake in infected tumors. 

Conclusion: GLV-1h153 efficiently kills human CC in vitro. When combined with 131I, GLV-1h153 allows for SPECT/CT imaging of CC tumors and significantly reduces tumor burden in vivo. These results indicate that GLV-1h153 is a promising novel imaging and therapeutic agent for patients with CC.

 

3.07 A Flavonoid and Midkine. Novel Targeted Therapeutic Approach for Hepatocellular Carcinoma

Posted on December 22, 2014

K. M. Sokolowski1, S. Kunnimalaiyaan1, M. Balamurugan1, S. T. Koprowski1, T. C. Gamblin1, M. Kunnimalaiyaan1  1Medical College Of Wisconsin,Surgical Oncology/Surgery/Medical College Of Wisconsin,Milwaukee, WI, USA

Introduction: Despite improvement in therapeutic strategies, median survival in advanced hepatocellular carcinoma (HCC) remains less than one year. Therefore, molecularly targeted compounds with less toxic profiles are needed. Xanthohumol (XN), a prenylated chalcone flavonoid has been shown to have anti-proliferative effects in various cancers types in vitro. XN treatment in healthy mice and humans yielded favorable pharmacokinetics and bioavailability. Although a report providing therapeutic potential of XN in prostate cancer in transgenic mice, the effects of XN on HCC proliferation is unknown. A potential molecular target, midkine (MK- heparin-binding growth factor) is negatively correlated with HCC prognosis and readily detected in serum. Therefore, investigating the effects of XN on HCC cellular proliferation and the evaluation of midkine as a possible therapeutic biomarker is integral.

Methods: The effects of XN on a panel of HCC cell lines were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colometric and colonogenic assay. Cell lysates were analyzed via Western blotting for pro-apoptotic (c-PARP and cleaved caspase-3) and anti-apoptotic proteins (Bcl2, Survivin, and Mcl-1). Mechanism of XN on HCC cellular proliferation was also examined. Midkine expression was evaluated following XN treatment of conditioned HCC media.

Results: Up to 2 µM concentration of XN resulted in cell viabilities exceeding 90% in HCC cell lines. However, 5 µM and above of XN significantly reduced cell viability in a dose-dependent manner. Colonogenic ability also decreased with increasing concentrations of XN (Figure). Additionally, growth suppression due to apoptosis was evidenced by increased expression of pro-apoptotic and reduced anti-apoptotic proteins. Importantly, MK secretion into media was significantly reduced following XN treatment. Furthermore, XN inhibited Notch1 signaling.

Conclusion: Xanthohumol effectively inhibits HCC growth in cell culture and reduces MK secretion. This may provide XN as a novel therapeutic agent and midkine as a potential biomarker. We report for the first time that XN targets Notch1 signaling in HCC cells. The favorable cytotoxic profile based on both healthy mice and human studies along with these findings warrant further preclinical in vivo analysis. Evaluation of Notch1 reduction and its effect on HCC cells following XN treatment is innovative. In addition, confirmation of MK as a therapeutic marker for HCC to XN treatment will be novel. To our knowledge, this is the first characterization of MK and Notch1 inhibition in HCC in response to XN treatment.

 

3.08 Bidirectional TSTA of SHIP-DTA Enhances the Effect of Gene Therapy for Insulinoma

Posted on December 22, 2014

S. Liu1, J. Wu1, G. Zhou1, J. Yu1, R. Sanchez1, F. Brunicardi1  1University Of California – Los Angeles,Department Of Surgery, David Geffen School Of Medicine,Los Angeles, CA, USA

Introduction: Two limitations of gene therapy are tissue specificity and tissue expression of the transgene. We hypothesize that synthetic promoters have greatly enhanced activities over endogenous promoters to drive gene expression and can be used to enhance the effect of gene therapy without loss of target specificity. We have demonstrated that the synthetic human insulin promoter (SHIP) has far greater activity than HIP or rat insulin promoter (RIP) in driving gene expression with subsequent enhanced effect of gene therapy in neuroendocrine tumors which overexpress pancreatic and duodenal homeobox1 (PDX1) transcription factor. Bidirectional two-step transcriptional amplification (TSTA) has been shown to enhance gene expression. In this study we sought to determine that TSTA enhances the expression and therapeutic effect of SHIP-diphtheria toxin A (SHIPTSTA-DTA) in PDX1 expressing cells, such as insulinoma.

Methods: SHIP-eGFP vs SHIPTSTA-eGFP, SHIP- firefly luciferase (FLuc) vs SHIPTSTA-FLuc and SHIP-DTA vs SHIPTSTA-DTA vectors were constructed using subcloning techniques. Activity and specificity of vectors were determined by bioluminescence and eGFP assays in mouse insulinoma (βTC6) cells, human primary pancreatic (HPP) cells, pancreatic ductal epithelial (HPDE) cells and PDX1-HPDE cells, since PDX1 is the primary activator of SHIP, with or without PDX-1 shRNA co-transfection. Cytotoxicity was determined by MTS assay and glucose-stimulated insulin secretion (GSIS) was determined by ELISA. Statistical analysis was performed via paired T test; p<0.05 = significant.

Results: SHIPTSTA-FLuc activity in βTC6 cells was 8 fold higher than that of SHIP-FLuc (p<0.05)(Fig. A). SHIP-FLuc was equal to CMV-driven expression, however was specific for cells expressing PDX1. PDX-1 shRNA co-transfection resulted in decreased SHIPTSTA-FLuc activity.  Similarly, increased cell numbers of eGFP and expression intensity of eGFP were observed in SHIPTSTA-eGFP transfected βTC6 cells vs controls. No FLuc activity was detected in both SHIPTSTA-FLuc and SHIP-FLuc transfected HPP and HPDE cells. SHIPTSTA-DTA resulted in enhanced cytotoxicity and enhanced suppression of proliferation and GSIS from βTC6 cells compared to SHIP-DTA (p<0.05)(Fig. B).

Conclusions:  SHIPTSTA markedly enhanced SHIP activity without loss of tissue specificity; SHIPTSTA-DTA in PDX1+ insulinoma cells enhanced cytotoxicity and suppressed proliferation and insulin secretion. The data support the hypothesis that synthetic promoters of the target gene have greatly enhanced expression efficiency over endogenous promoters and can be used to enhance the effect of gene therapy without loss of target specificity. 
 

3.09 Mitigation of Hypertrophic Scar Contraction in vivo via a Biostable Polyurethane Scaffold

Posted on December 22, 2014

M. M. Ibrahim1, E. R. Lorden2, K. J. Miller1, L. Bashirov1, E. Hammett2, C. Quiles1, A. Rastegarpour1, A. Selim3, K. W. Leong2, H. Levinson1,3  1Duke University Medical Center,Department Of Surgery, Division Of Plastic Surgery,Durham, NC, USA 2Duke University Medical Center,Department Of Biomedical Engineering,Durham, NC, USA 3Duke University Medical Center,Department Of Pathology,Durham, NC, USA

Introduction:

Over 2.4 million Americans and tens of millions of patients worldwide suffer from hypertrophic scar (HSc) contraction following thermal injuries. HSc contraction is a debilitating condition that results in disfigurement and decreased range of motion in affected joints. The current standard of care involves skin grafting with or without the placement of a collagen based, biodegradable, bioengineered skin equivalent (BSE). Commercial BSEs assist in tissue regeneration, but do not focus on mitigating the debilitating effects of HSc and they are marginally effective. To overcome this significant unmet medical need, we have created and tested an elastomeric, polyurethane (PU) based BSE that will last throughout the remodeling phase of repair.  In unwounded skin, collagen is arranged randomly, myofibroblasts are absent, and dermal stiffness is low. Conversely, in scar contractures, collagen is arranged in linear arrays, myofibroblast density is high, and dermal stiffness increases. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that will promote random cellular orientation, decrease myofibroblast formation, and mitigate HSc contraction. 

Methods:

Electrospun PU scaffolds were fabricated, covalently coated with bovine type-1 collagen, characterized for their tensile mechanical properties, and tested in vivo. Scaffolds were surgically inserted beneath skin grafts in a validated immune competent murine burn and HSc model we have previously developed.  Wounds were excised at day 30 post-surgery. Collagen was assessed with Masson. Macrophages were detected with F4/80 immune. Vascularity was assessed with CD31 immune. Myofibroblasts were assessed with alpha-smooth muscle actin (ASMA) immune. Elastic moduli were analyzed using a microstrain analyzer. All were compared to Integra™.

Results:

In vivo, at D30, collagen coated PU scaffolds integrated into the host tissue and restricted wound contraction to 70±4% of the original wound size. Meanwhile, control mice treated with skin graft alone contracted to 45±2%, while wounds treated with Integra™ beneath skin graft contracted down to 28±1.5% of the original size. Histological analysis demonstrated that PU scaffolds promote fibroblast invasion, angiogenesis, and macrophage recruitment compared to skin graft alone and Integra™. ASMA stains showed decreased numbers of myofibroblasts in PU scaffolds compared to control skin grafts and Integra™. Elastic modulus of excised PU treated wounds was significantly lower than skin-grafted control scars.  

Conclusion:

These data suggest that collagen-coated elastomeric electrospun PU scaffolds provide mechanical support to prevent wound bed contraction during healing, and decrease myofibroblast activation associated with HSc . Our long-term goal is to develop a rationally designed, translational medical therapy to mitigate HSc,

3.11 Apigenin Reduces PTHrP-Mediated Inflammation, PSC Proliferation, and ECM Synthesis in Pancreatitis

Posted on December 22, 2014

A. A. Mrazek1, V. Bhatia2, M. Falzon2, M. R. Hellmich1, C. Chao1  1University Of Texas Medical Branch,Surgery,Galveston, TX, USA 2University Of Texas Medical Branch,Pharmacology,Galveston, TX, USA

Introduction: Chronic pancreatitis (CP) is characterized by repeated bouts of acute pancreatitis, resulting in inflammation, glandular necrosis, and irreversible stromal fibrosis. Apigenin (Api) is a natural compound which we have shown to preserve acini and significantly inhibit interstitial fibrosis in a preclinical mouse model of CP. We found that Api down-regulates parathyroid hormone related protein (PTHrP), a pro-inflammatory mediator responsible for amplifying acinar and pancreatic stellate cell (PSC) response to injury. In vitro, Api reduced the viability and induced apoptosis PSCs, the cells responsible for secretion of inflammatory cytokines and the over-deposition of extracellular matrix (ECM). The aim of this study is to determine if Api acts at a transcriptional level, limiting the synthesis of inflammatory mediators (IL-6 and IL-8), markers of PSC proliferation (TGF-β and PCNA), and major components of the ECM, collagen type I and fibronectin (COL1A1 and FN).

Methods: Following an IRB-approved protocol, human PSC were isolated from discarded OR tissue by a standard outgrowth method. The PSC were pretreated with Api 50μM for 1h or vehicle (DMSO) and then stimulated with PTHrP (Bachem, Torrance, CA) 1ng/ml for 12h. Total RNA was isolated (Ambion, Austin, TX); RT-PCR was performed using SYBR Green Supermix (Applied Biosystems, Carlsbad, CA). The relative expression of transcripts was normalized to 18S expression. One-way ANOVA and post-hoc Tukey’s test were assessed with SPSS (IBM, Armonk, NY), and significance was set at p<0.05.

Results: Pretreatment with Api significantly reduced basal mRNA expression of IL-6 and IL-8, TGF-β, PCNA, and fibrillar protein COL1A1 (p<0.001) [Table 1], but not FN. When PSCs were stimulated PTHrP, Api significantly decreased the transcriptional response of all transcripts evaluated (p<0.001).

Conclusions: Api significantly lowers the threshold of basal transcriptional activity in PSC of IL-6 and IL-8 (pro-inflammatory cytokines), TGF-β and PCNA (measures of cellular proliferation), and COL1A1 (the most abundant ECM protein). Additionally, Api reduced PTHrP-stimulated increases in all factors evaluated. Pretreatment with Api did not decrease the basal mRNA levels of FN at 12h, an ECM protein which has been shown to be overabundant in CP. Changes in FN mRNA may be revealed at longer time intervals. These RT-PCR results offer mechanistic insight into the action of Api and confirm the preclinical findings in our murine model of CP, where Api reduced pancreatic fibrosis while preserving normal acinar unit architecture. This further supports apigenin’s development as a therapeutic in pancreatitis.

3.12 IAP Decreases Intestinal Permeability Prior to Developing NEC through Stabilization of Claudin-1

Posted on December 22, 2014

S. Dillman1,2, N. Heinzerling1,2, K. Fredrich1,2, J. Fawley1,2, D. M. Gourlay1,2  1Medical College Of Wisconsin,Pediatric Surgery,Milwaukee, WI, USA 2Children’s Hospital Of Wisconsin,Children’s Research Institute,Milwaukee, WI, USA

Introduction: Recent studies indicate an increase in intestinal permeability precedes necrotizing enterocolitis (NEC).  Prior research has shown supplemental enteral Intestinal Alkaline Phosphatase (IAP), an endogenous protein expressed by the intestines, decreases intestinal permeability and the severity of NEC in a rodent model. The tight junction proteins, claudins, are essential in creating a functional epithelial intestinal barrier. Increased expression and altered distribuiton of Claudin-1 (Cl-1) during intestinal inflammation was associated with increased intestinal permeability leading to a breach in mucosal barrier. We hypothesize that IAP decreases intestinal permeability prior to the development NEC through modification of CL-1. 

Methods: One day preterm Sprague Dawley rat pups were delivered via cesarean section and fed formula. Select pups received 4 units/kg of IAP in their formula. Pups were sacrificed on day of life 2-4 and the terminal ileum was harvested. Intestinal permeability was measured by filling loops of intestine with a fluorescein isothiocyanate-dextran (FITC-dextran) solution and measuring flux into the incubating media. Three day old dam-fed wild-type, heterozygotes, and IAP KO rat tissue was also harvested. To examine the effect of IAP on intestinal epithelial cell, in vitro experimentation involved FHs 74, a fetal epithelial cell culture. Cells were given either formula or formula plus 4 units of IAP. ZO-1, CL-1, 2, and 3 were measured by RT-PCR and Western Blot on all cells and ileal intestinal homogenates. Statistical analysis was performed using a paired t-test and a p<0.05 considered significant. 

Results: Intestine loops exposed to lipopolysaccharide and FITC-dextran had a greater statistically significant increase in permeability over controls.  Supplemental, enteral IAP significantly decreased intestinal permeability.  Evaluation of ileal intestinal homogenates showed a significant decrease messenger RNA levels for CL-1, 2, and 3 in IAP fed rats. For Western Blot analysis, ZO-1 and CL-1 displayed a significant increase in the IAP fed group while CL-2 showed a slight decrease in protein expression.  In the IAP gene knockout rats, CL-1 showed a significant increase mRNA expression compared to wild-type.  In the FHs 74 cell line, Cl-1 expression was significantly decreased in the IAP group while the other claudins didn’t have noticeable expression in either group. 

Conclusion: Neonatal intestinal permeability is increased by formula feeding and LPS and may increase the risk of NEC and sepsis. These data suggest IAP has a direct effect on expression and stability of CL-1 in the neonatal intestinal epithelial cell that is a novel mechanism by which IAP may function to protect newborns from NEC. .  

 

3.13 Development of an Endoluminal Intestinal Lengthening Device: Geometric Intestinal Attachment

Posted on December 22, 2014

F. R. Demehri1, J. Freeman1, Y. Fukatsu1, D. H. Teitelbaum1  1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA

Introduction:   Distraction-mediated enterogenesis, whereby longitudinal force applied to small bowel leads to functional lengthening, may provide a novel therapy for short bowel syndrome (SBS).  Previously described methods have relied upon isolated small bowel segments or transmural fixation, requiring multiple operations and subsequent morbidity.  Our work consisted of two objectives: First, to develop a fully endoluminal distraction-mediated enterogenesis device; Second, to develop a novel approach utilizing geometric coupling between a tapering device and the mesenteric curvature to allow intestinal attachment and trans-stomal distraction-mediated enterogenesis.                          

Methods:   A tapering catheter device was designed to provide endoluminal intestinal attachment via geometric coupling between the rigid device and mesenteric curvature.  This consisted of a high-rigidity 24-Fr malleable catheter with a 30 cm taper to a low-rigidity 8-Fr latex tip to prevent perforation (Fig A).  Yorkshire pigs underwent jejunal Roux limb creation with placement of the device via jejunostomy.  Intestinal attachment and distraction was achieved without significant reduction in bowel perfusion as measured by laser Doppler (0.95 ± 0.03 P.U. vs 1.03 ± 0.04 P.U. with inflation vs deflation; p=0.14).  An external clamp was secured at the stoma to provide external fixation of the device (Fig B).  The device was manually advanced 1cm/day for 7 days before explant.  A second group of pigs underwent extended implant, with explant at day 14.  Results (mean±SEM) were analyzed for significance by t-test.

Results:   After 7 days of trans-stomal, externally-applied endoluminal distraction, the distracted segment achieved a 32.4±4.6% increase in length versus fed, nondistracted bowel, corresponding to an absolute gain of 10.6±1.2cm.  After 14 days, the Roux limb achieved an 80.3±1.4% increase in length versus fed control bowel, corresponding to an absolute gain of 16.7±3.4cm.  The device fixation site demonstrated occasional epithelial sloughing with intact submucosa.  No device-related perforation or stoma-related complication occurred (Fig C). 

Conclusion:  A novel catheter device with tapering rigidity allows for fully endoluminal intestinal attachment via geometric coupling, with successful externally-applied distraction-mediated enterogenesis.  This approach may allow development of clinically applicable technology for the treatment of patients with SBS.

3.14 Safety of a Collagen-Targeted Peptide Amphiphile Nanofiber for Intravascular Use

Posted on December 22, 2014

W. Jiang1,2, E. M. Bahnson1, M. R. Kibbe1  1Feinberg School Of Medicine – Northwestern University,Vascular Surgery,Chicago, IL, USA 2Northwestern University,McCormick School Of Engineering,Chicago, IL, USA

Introduction: We developed, characterized, and validated a collagen-binding peptide amphiphile (PA) nanofiber as a novel targeted delivery vehicle that specifically binds to and releases a therapeutic agent directly at the site of vascular injury and prevents neointimal hyperplasia following systemic administration. However, in order to translate this therapy to the clinical arena, the safety of this nanofiber as a drug delivery tool must be evaluated. Thus, the goal of this current project is to evaluate the safety of this systemically administered targeted nanofiber. We hypothesize that a systemically injected targeted nanofiber will be safe following in vivo administration.

Methods: Male Sprague-Dawley rats received a single dose of a fluorescently labeled collagen-binding PA (2.5mg, N=3). Liver, spleen, and kidney were harvested at different time points (24, 48, and 72h) for H&E staining and immunohistochemical evaluation for macrophage infiltration (ED1 antibody). Blood samples were collected for coagulation studies. C3a levels in plasma was assessed by ELISA. Functional complement activation was assessed by a hemolysis assay following incubation of human serum with PA (1 and 0.5mg/ml) for 1h. Platelet activation was evaluated by assessing adherence ex vivo after incubation of rat platelet rich plasma with PA (1 and 0.5mg/ml) for 30 min.

Result: Assessment of liver, spleen, and kidney histologically revealed the presence of fluorescence at 24, 48, and 72h, suggesting the presence of the targeted nanofiber. However, no change in organ architecture was observed in H&E stained slides and there was no difference in macrophage infiltration in the liver, kidney, and spleen of nanofiber-treated animals compared to control animals. The serum dose-response curves for the complement activation assay showed no difference between treated and untreated serum. At a 1:15 serum dilution, the percentage of residual complement for different concentrations of the PA (1 and 0.5mg/ml) was 99.8 +/- 7.7% and 96.4 +/- 8.8%, respectively vs. 100.7 +/- 9.9% for untreated serum, indicating no significant activation of complement. Similarly, C3a levels in rat plasma did not differ between control and PA-treated rats. Regarding the coagulation studies, the PT was increased to 22 +/- 4s at 24h (p=0.027) and 21 +/- 0.1s at 48h (p=0.044) after PA administration compared to 15 +/- 2s for control, but returned to normal at 72h (14 +/- 0.2s). PTT, fibrinogen and platelet count showed no statistically significant change after treatment. Finally, PA did not cause platelet activation ex vivo.

Conclusion: This data suggested that systemic administration of our novel targeted intravascular nanotherapy is safe, as organ morphology, inflammation, and complement and platelet aggravation were not impacted following administration in vivo. While there was a transient rise in the PTT, this returned to baseline within 3 days. Thus, no major safety concerns were found in our study.

3.15 Pharmacokinetic Model for Supramolecular Nanoscale Carriers Targeted to the Injured Vasculature

Posted on December 22, 2014

E. M. Bahnson1, H. Kassam1, K. T. Nennig3, M. J. Avram2,3, M. R. Kibbe1  1Feinberg School Of Medicine – Northwestern University,Vascular Surgery,Chicago, IL, USA 2Feinberg School Of Medicine – Northwestern University,Anesthesiology,Chicago, IL, USA 3Northwestern University,Donnelley Clinical Pharmacology Core,Chicago, IL, USA

Introduction: Nanoscale carriers that incorporate targeting information are emerging as a promising modality of delivering a therapeutic load with high efficacy and minimal toxicity to the vasculature. However there is little information about the pharmacokinetic properties of such compounds. To prevent neointimal hyperplasia, we developed a novel targeted therapy capable of delivering a therapeutic agent to the site of vascular injury via systemic administration, using a highly customizable peptide amphiphile (PA). The goal of this project is to study the disposition of the collagen-targeted PA and develop a pharmacokinetic model for it. We hypothesize that the targeted nanofibers will exhibit a pharmacokinetic behavior distinct from that of classic small molecule drugs.

Methods: PAs were synthesized using solid-phase peptide synthesis, and purified by preparative reverse-phase HPLC. Male Sprague Dawley rats received a single dose (2.5 mg) of PA via tail vein injection. Blood was collected for analysis at various times over three days (0.1, 0.2, 0.25, 0.5, 0.45, 1, 1.5, 2, 3, 4, 6, 8, 16, 24, 48, and 72 h; n=3/time point). After addition of a PA internal standard, samples were reduced with 5 mM tris(2-carboxyethyl)phosphine. Proteins were precipitated with acetonitrile before HPLC injection.  Concentrations of the PA were determined by HPLC with mass spectrometry detection (Lower Limit of Quantification = 1 ng/ml). A multicompartmental pharmacokinetic model was then developed for the PA nanofibers.

Results: A 3-compartment pharmacokinetic model fit yielded the parameters shown in Figure 1. Estimate of the central volume is consistent with the volume of plasma and interstitial space in rats. The concentration of the nanofibers in plasma decreased two orders of magnitude in the first hour. The first fast exponential phase generally corresponds to distribution clearance of rapidly equilibrating tissues. However, for the PA nanofibers, this first exponential phase corresponded to elimination clearance, that is a process that removes the nanofibers from plasma irreversibly within the time frame of the study. The ex-vivo stability of the nanofibers in plasma revealed that this rapid process is not due to rapid degradation by plasma components. The very fast elimination clearance could be due to organ sequestration or irreversible conjugation.

Conclusions: We describe an unusual 3-compartment pharmacokinetic model of a targeted nanofiber that clearly differs from classic small molecule pharmacokinetics. This information becomes essential when trying to understand the differential properties of nanocarrier-based drugs. Further work is required in order to elucidate the mechanism responsible for the very fast elimination clearance.

3.16 Implantable Hemofilter: An Overview of the Pre-clinical Canine Surgical Model

Posted on December 22, 2014

C. Kensinger1, J. Groszek2, S. Karp1, D. Laneve1, P. Williams1, R. Kant3, T. Yeager3, S. Roy3, W. Fissell2  1Vanderbilt University Medical Center,Department Of General Surgery,Nashville, TN, USA 2Vanderbilt University Medical Center,Department Of Medicine,Nashville, TN, USA 3University Of California – San Francisco,Department Of Bioengineering And Therapeutic Sciences,San Francisco, CA, USA

Introduction:  Patents with end-stage renal disease (ESRD) have high mortality and morbidity rates on dialysis.  Transplantation offers the best treatment option but is limited by organ availability.  An implantable artificial kidney using silicon nanoporous membranes is in development to address this problem.   A key challenge is the long-term blood patency of the hemofilter with respect to thrombosis formation and membrane fouling from plasma protein adsorption. These experiments assess the long-term biocompatibility of an implantable artificial kidney.

Methods:  Single-channel, polycarbonate parallel-plate hemofilters (membrane area 0.72 cm2) are designed using computational fluid dynamics to minimize turbulence and stasis through the device flow path.   Two ultrathin membranes with highly controlled pores made by sacrificial silicon oxide techniques, which allows size selective sieving, define the parallel plates along the flow path.  Hemofiltration results from the hydrostatic pressure gradient across the membrane. 

Seven millimeter polytetrafluoroethylene grafts are anastomosed to the common iliac artery and vein to be used as inflow and outflow conduits to the hemofiltration device.  Heparin is administered intra-operatively.  Effluent collection reservoirs are placed in the upper abdomen.   Access ports are connected to the reservoirs and brought through the abdominal wall into the subcutaneous tissue.  This allows post-operative monitoring of hemofilter function and filtration rates.  Following the operation, the animals are housed without restrictions.   The grafts are serially assessed post-operatively with a doppler ultrasound to ensure patent flow through the devise.   

Results: Fourteen pre-clinical canine surgeries have been performed. Hemofilter patency rates were 67% (8/12) prior the initiation of post-operative Warfarin treatment. With adequate systemic anticoagulation, patency rates have been 100% (2/2). Intra-operative filtration rates have averaged 0.41 microliters/minute with an expected filtration rate of 0.42 microliters/min based on membrane characteristics.  Blood flow through the device has averaged 300cc/minute at the time of hemofilter implant and explant. 

Conclusion: These experiments highlight successful surgical technique and manufacturing biocompatibility providing the foundation for further preclinical experiments aimed towards the future realization of an implantable artificial kidney.
 

3.17 Changes in Liver Lobe Function After Portal Vein Ligation Determined by Selective Biliary Drainage

Posted on December 22, 2014

A. Szijarto1, A. Fulop1, A. Budai1, G. Lotz2, A. Kiss2, L. Harsanyi1  1Semmelweis University,1st Department Of Surgery,Budapest, BUDAPEST, Hungary 2Semmelweis University,2nd Department Of Pathology,Budapest, BUDAPEST, Hungary

Introduction: Portal vein ligation (PVL) and embolization (PVE) are techniques used before extended hepatic resections to prevent posthepatectomy liver failure. These therapies redirect portal blood to liver lobes that will remain after surgery resulting in hypertrophy, while the portal deprived lobes undergo atrophy. Although, the effect of PVL on liver volume is well-documented, the parallel alterations in lobar liver function are still the subject of controversy. Therefore, the aim of the present study was to evaluate the morphological, hemodynamic and functional alterations caused by the selective occlusion of the portal vein in a well-established rat model.

 

Methods:  Male Wistar rats (n=84) underwent PVL by the ligation of the portal veins feeding the median, left lateral and caudate lobes (approximately 80% of total liver mass). Before PVL, as well as 24-, 48-, 72-, 120-, 168 hours after PVL, liver morphology (liver weight; mitotic activity; necrotic-, apoptotic cell death and lobular area), hepatic microcirculation (laser Doppler flowmetry), global liver function (laboratory blood test; total hepatic bile flow; plasma disappearance rate of indocyanine-green (PDR); the percentage of biliary ICG excretion to the administered ICG during the first 20 minutes (ICG%20min)) as well as hepatic lobar function (lobar bile flow and lobar biliary ICG excretion) were examined.

Results: PVL induced atrophy of ligated lobes (from 0.35±0.037 to 0.09±0.018g/BWkg) and hypertrophy of non-ligated lobes (from 0.1±0.102 to 0.31±0.019g/BWkg), while the total liver weight remained unchanged. The microcirculation of ligated lobes impaired, while the microcirculatory blood flow of non-ligated lobes significantly increased with the peak response at 48th postoperative hours. Serum albumin-, total bilirubin levels and total hepatic bile flow did not changed significantly throughout the entire experiment. PDR and ICG%20min significantly decreased after PVL, with the lowest value at postoperative 48th hours and returned near to the baseline at 168 hours after the operation. The bile flow and biliary ICG excretion of ligated lobes decreased significantly after PVL (from 81.99±8.6 to 17.62±3.9g/BWkg and from 1.16±0.03 to 0.1±0.03g/BWkg, respectively), while bile flow and biliary ICG excretion of non-ligated lobes showed a significant increase (from 23.65±2.75 to 88.8±4.67g/BWkg and from 0.34±0.03 to 1.4±0.03g/BWkg, respectively).

 

Conclusion: PVL induced a temporary impairment in global liver function, followed by a rapid recovery mainly caused by the increase in the function of non-ligated liver lobes. In the non-ligated lobes, the functional increase was more pronounced than suggested by the degree of hypertrophy. Consequently, the functional capacity of the liver was shifted towards the regenerating lobes in a greater extent than would be expected according to the volumetric alterations.

3.18 Remote Ischemic Preconditioning's effect on Fat Graft volume

Posted on December 22, 2014

A. A. Gassman1, M. Lewis2, J. C. Lee1  1University Of California – Los Angeles,Surgery/ Plastic Surgery,Los Angeles, CA, USA 2West Los Angeles VA,Pathology,Los Angeles, CA, USA

Introduction:
Fat grafting has become a useful adjunct in the reconstructive surgeon’s treatment armamentarium. Inconsistencies in transfer and local ischemia prior to the development of recipient circulation all contribute to highly variable long-term results associated with fat grafting. Remote Ischemic Preconditioning (RIPC) is a cheap non-invasive technique that has been used in several animal models and multicenter clinical trials to protect several organ systems. The specific aim of this project was to analyze the volume retention of lipoaspirate transferred in the setting of either donor or recipient RIPC.  

Methods:
We obtained subcutaneous adipose tissue from FVB mice transgenically engineered to express eGFP and Luciferase. These samples were obtained either with or without the use of temporary hindlimb tourniquet time prior to harvest. The samples were excised and passed to through serially smaller lipoaspiration cannulas (16 to 19 gauge), centrifuged (500g for 2 min), and decanted. The treatment and control fat was injected into the dorsal skin folds of genetically identical FVB mice that did not express GFP or Luciferase. The viability and volume of the transferred tissue was examined over a 28-day time period by bioluminescence after intraperitoneal injection of Luciferin using a Maestro IVIS optical small animal scanner. Additionally, after experimental completion the tissue transferred was explanted and examined histologically. The specimens were stained with H&E, CD31, CD34, and GFP.

Results:
Bioluminescence was able to non-invasively track the presence of transferred lipoaspirate tissue over a 28 day time period.  There was a significant difference in bioluminescence and calculated graft volume at Day 0 and 28.  The RIPC group demonstrated approximately 700% and 400% increase over control at each time point, respectively.  Histological analysis at 28 days confirmed the presence of donor adipocytes, and that they were gradually replaced by recipient inflammation and scar tissue.  However the amount of interstitial fibrosis was substantially less in the RIPC group.  Additionally, the RIPC group retained a substantially greater amount of GFP suggesting retention of donor cells. The control tissue demonstrated increased CD31 and CD34 suggesting increased vascularity. 

Conclusion:
This work has achieved two goals. Firstly, It demonstrates that the bioluminescence of adipocytes transferred from a luciferase expressing donor may be used to non-invasively monitor tissue viability and volume over a prolonged period of time. Secondly, RIPC has the ability to increase the viability of donor adipocytes when transferred via liposuction cannula, and the transferred tissue is less likely to undergo interstitial fibrosis.
 

3.19 Hemoglobin Based Oxygen Carriers Exacerbate Hyperfibrinolysis Independent of Plasmin

Posted on December 22, 2014

A. P. Morton3, H. B. Moore3, E. Gonzalez3, G. Wiener3, P. Lawson2, M. Chapman3, C. Silliman3, E. Peltz1,3, A. Banerjee3, E. E. Moore2,3  1University Of Colorado Hospital,Surgery,Aurora, CO, USA 2Denver Health Medical Center,Surgery,Denver, CO, USA 3University Of Colorado School Of Medicine,Surgery,Aurora, CO, USA

Introduction: Hyperfibrinolysis plays an integral role in the genesis of trauma induced coagulopathy (TIC). Recent data demonstrates that red blood cell (RBC) lysis promotes fibrinolysis; however, the mechanism is unclear.  Hemoglobin-based oxygen carriers (HBOC) have been developed for resuscitation and have been associated with coagulopathy.  We hypothesize that replacement of whole blood (WB) using an HBOC results in a coagulopathy due to the presence of free hemoglobin.

Methods: Whole blood was sampled from healthy donors (n=6).  The clotting profile of each citrated sample was evaluated using native thromboelastography (TEG).  Serial titrations were performed using both HBOC (Polyheme) and normal saline (NS) (5%, 25%, and 50%) and evaluated both with and without a 75 ng/microliter tissue-plasminogen activator (tPA) challenge.  Tranexamic acid (TXA) was added to inhibit plasmin dependent fibrinolysis.  Fibrinolysis was measured and recorded as LY30, the percentage of clot lysis at 30 minutes after maximal clot strength.  Statistics were calculated using SPSS software.  Dilution of WB with NS or HBOC was correlated using LY30 via Spearman Rho coefficients.  Groups were also compared using a Friedman test and post-hoc analysis with a Bonferroni adjustment.

Results: TPA-provoked fibrinolysis was enhanced by both HBOC (median LY30 at 5%, 25%, 50% titrations: 11%, 21%, 44%; Spearman=0.94; p<0.001) and NS (11%, 28%, 58%; Spearman=0.790; p<0.001). However, HBOC also enhanced fibrinolysis without the addition of tPA (1%,4%,5%; Spearman=0.735; p=0.001) and NS did not (1%,2%,1%; r=0.300; p=0.186; Figure 1).  Moreover, addition of TXA did not alter or inhibit this fibrinolysis (WB vs 50% HBOC: 1.8% vs 5.65%, p=0.04).  There was no significant difference in fibrinolysis of HBOC with or without TXA (50% HBOC vs 50% HBOC +TXA: 5.55% vs 5.65%, p=0.92).  Additionally, the increased fibrinolysis seen with NS was reversed when TXA was present (WB vs 50% NS: 1.8% vs 1.65%, p=1.0).

Conclusion: HBOCs enhance fibrinolysis without the addition of tPA; moreover, this is independent of plasmin as the phenomenon persists in the presence of TXA.  Our findings implicate the hemoglobin molecule or its components in stimulating fibrinolysis

 

3.20 Self-assembly Nanoparticles of PLGA-polyethylenimine (PLGA-PEI) Copolymer for Gene Delivery

Posted on December 22, 2014

J. LU1, Z. Liang1, Q. Yao1, C. Chen1  1Baylor College Of Medicine,Surgical Research/Surgery,Houston, TX, USA

Introduction: Although gene therapy holds great promise, the progress has been slow because the current gene delivery systems are less successful for clinical applications due to their low efficiency and high toxicity. The objective of this study was to develop a better gene delivery system from two biocompatible polymers, poly(lactic-co-glycolic acid) (PLGA) and polyethylenimine (PEI), potentially for clinical applications.

Methods: The PLGA-PEI copolymer was prepared by directly mixing PLGA and PEI in organic solvent. Self-assembly nanoparticles (NPs) of PLGA-PEI copolymer and DNA were prepared by adding the DNA solution to the water solution of PLGA-PEI copolymer. The size and morphology of PLGA-PEI/DNA NPs were determined with dynamic light scattering and scanning electronic microscopy. The cytotoxicity of PLGA-PEI/DNA NPs at different copolymer to DNA ratios was performed in pancreatic cancer cell line (PANC-1) with an MTT assay. For the gene transfection assay in PANC-1 cells, plasmid DNA containing a green or red fluorescence protein (GFP or RFP) gene was used. In vivo toxicity and transfection efficiency of PLGA-PEI PLGA-PEI/DNA NPs were carried in mouse models with the tail vein administration.

Results: PLGA-PEI copolymer was produced in one-step by mixing PEI and PLGA in the tetrahydrofuran solution and PLGA to PEI ratio (0.5:1). Through the analysis of primary amines of PEI before and after its chemical reaction with PLGA, the chemical structure of the PLGA-PEI copolymer was demonstrated.  PLGA was broken down to LGA single units, which were covalently linked to the primary amine groups of PEI; while PEI was intact. PLGA-PEI copolymers spontaneously formed NPs (~100 nm in diameter) with plasmid DNA at the 1.5:1 ratio due to change of the surface charge, achieving 100% DNA loading. The particle size can be controlled by justifying copolymer and DNA ratios.  PLGA-PEI significantly reduced the toxicity of PEI in both PANC-1 cells and mouse models. PLGA-PEI copolymer more efficiently delivered GFP or RFP plasmid into PANC-1 cells compared with commercially available transfection reagents with additional advantages of less toxicity, serum independency and long duration of transgene expression. More importantly, PLGA-PEI/DNA NPs were tested in the mouse model and showed an effective gene delivery to liver, spleen, and pancreas. Direct intratumor administration of PLGA-PEI/DNA NPs also showed a high transfection rate in the nude mouse model.

Conclusions: PLGA-PEI copolymer is a new gene delivery material, which has high DNA loading capacity and low toxicity in vitro and in vivo; and it condenses DNA into small sized NPs. The PLGA-PEI/DNA NPs have a high transfection efficiency in cell cultures and mouse models. The current study demostrates a better gene delivery system, which may have board clinical applications.

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