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

Posted on December 22, 2014

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

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

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

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

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

 

60.09 MiR-155 Dysregulation is Associated with Increased Monocyte RhoA Activity in Patients with AAA

Posted on December 22, 2014

A. T. Nguyen1, K. M. Samadzadeh1, K. C. Chun1, A. E. Rona1, A. M. Afkhami1, P. Ghosh3,4, E. S. Lee1,2  3University Of California – Davis,Urology,Sacramento, CA, USA 4VA Northern California Health Care System,Mather, CA, USA 1VA Northern California Health Care System,Surgery,Mather, CA, USA 2University Of California – Davis,Surgery,Sacramento, CA, USA

Introduction:
MiR-155 has been reported to inhibit ras homology member A (RhoA) in multiple cell lines. RhoA acts as a molecular switch in a signaling cascade that induces cytoskeletal reorganization within cells. Several studies suggest increased RhoA activity is linked with enhanced cellular transmigration through the endothelium. Increased monocyte activity was found to be correlated to abdominal aortic aneurysm (AAA) disease. However, the relationship between RhoA and AAA disease is not fully understood. This study examines whether miR-155 is associated with higher RhoA activity in patients with AAA.

Methods:
Monocytes were isolated from patient blood and lysed for Rhotekin-RBD Assay and western blotting. BCA assay measured protein concentration and 5ug of protein was loaded onto SDS PAGE gels. The subsequent PVDF membrane was incubated with a rabbit RhoA antibody and developed using ECL substrate. Images were scanned and processed using Image J software. MicroRNAs were isolated from serum and quantified using Agilent BioAnalyzer 2100. MiR-155 levels were assessed using quantitative PCR with Exiqon LNA primers and normalized using the 2-ΔΔCT method. Student’s t-test was used to compare AAA and control groups for statistical analysis.

Results:
Patients with large AAAs (>5.5cm in diameter, lanes 1, 3, and 4 ) displayed higher RhoA activity (n=3) versus small AAAs (<5cm in diameter, lane 2, 5, and 6) (n=3) (Figure 1a). Lane 7 was THP-1 used for data normalization. Rho-GTP activity was then quantified using Image J software, values are represented as relative change in expression (*p<.05) (Figure 1b). Q-PCR results showed miR-155 was down-regulated in serum, displaying a 3-fold decrease (*p< .05) in AAA subjects (n=5) versus non-AAA controls (n=4) (Figure 1c).

Conclusion:
Patients with large AAA have increased RhoA activity compared to small AAA. Additionally, decreased serum miR-155 is associated with greater RhoA activity in patients with large AAA. Pharmacological regulation of these microRNAs may attenuate monocyte RhoA activity in patients with AAA.
 

60.10 Disturbed Laminar Flow Elicits Sustained ERK5 Activation in HUVECs

Posted on December 22, 2014

S. Y. Shalaby1, G. Chitragari1, B. J. Sumpio1, J. Kurita1, B. Sumpio1  1Yale University School Of Medicine,Vascular Surgery,New Haven, CT, USA

Introduction:  Extracellular signal regulated kinase 5 (ERK5) has been reported to regulate endothelial integrity and protect from vascular dysfunction under laminar flow. Atherosclerotic lesions predominate in areas of disturbed flow but the mechanism of this phenomenon is not well defined. Previously reported research indicate ERK5 activation under laminar flow induces ERK5 activation and production of atheroprotective molecules. However, the characterization of ERK5 activation under different flow patterns has not been investigated.    

Methods:  Confluent HUVECs were serum starved for 2 hours with 1% Fetal Bovine Serum (FBS) then seeded on fibronectin coated glass slides. HUVECs were exposed to CLF, TFF, or PFF in a parallel plate flow chamber controlled by a computerized pump for up to 2 or 4 hours at 37° C in a humidified CO2 environment. HUVECs incubated in 1% FBS, 20% FBS, or hydrogen peroxide culture medium under static conditions served as control and positive stimulant, respectively. At the end of experimentation, cell lysates were prepared and immunoblotted with antibodies to phospho-ERK5 and total ERK5. ERK5 activity was assessed by the levels of phosphorylated ERK5. The densitometric mean ± SEM is calculated and analyzed by student t-test. p < 0.05 is considered significant.  

Results: Levels of ERK5 decreased with all flow conditions as previously reported. TFF and CLF exhibited sustained ERK5 phosphorylation in HUVECs stimulated for up to 4 hours (Figure. 1). PFF had transient phosphorylation of ERK5 at 2 hours, which returned to baseline levels at 4 hours of exposure to flow. 

Conclusion: Exposure of HUVEC to different types of shear stress results in varying patterns of activation of ERK5. Activation of ERK5 with TFF suggests a role in the pathogenesis of atherosclerosis and vascular remodeling under disturbed flow conditions.       

 

60.11 Disturbed Flow Causes Degradation of YAP via Non-proteasomal Pathway

Posted on December 22, 2014

G. Chitragari1, S. Y. Shalaby1, B. J. Sumpio1, J. Kurita1, B. E. Sumpio1  1Yale University School Of Medicine,Section Of Vascular Surgery, Department Of Surgery,New Haven, CT, USA

Introduction:
Yes Associated Protein (YAP) regulates cell growth and proliferation based on cell shape and matrix stiffness and has been implicated in the pathogenesis of atherosclerosis. Previous experiments showed that the level of YAP decreases significantly when exposed to disturbed flow (TFF). We hypothesized that the decrease in YAP is due to ubiqutination dependent proteasomal degradation.  The aim of this study was to elucidate the mechanism of decrease in the level of YAP under TFF.

Methods:
HUVECs seeded on fibronectin coated glass slides were grown to confluence in culture medium containing 20% fetal bovine serum (FBS). Thereafter, they were serum starved overnight in medium containing 5% FBS and exposed to TFF utilizing a parallel plate flow chamber system controlled by a computerized pump for 4 hours at 37°  C in a humidified CO2 environment in the presence or absence of 7.5µM MG132 (a proteasomal inhibitor). HUVECs under static conditions served as control. At the end of experimentation, cell lysates were prepared and immunoblotted with anti-YAP and anti-Ubiquitin antibodies. Percent change compared to static control at 0 hours was calculated and statistical analysis was performed using t-test.  ‘p’ value of less than 0.05 was considered statistically significant.

Results:
The addition of MG132 increased ubiquitinated products as shown in Figure 1A. Exposure of HUVECs to TFF for 4 hours significantly decreased the level of YAP to 79.4± 3.7% compared to static control at 0 hours and compared to static control at 4 hours (124.1± 3.7%; p=0.001).  However, addition of MG132 failed to significantly inhibit the decrease in the level of YAP when exposed to TFF for 4 hours (84.7± 7.3; p=0.54)

Conclusion:
Exposure of HUVECs to TFF decreased the level of YAP. Since addition of MG132 which blocked proteasomal degradation of ubiquitinated YAP did not lead to accumulation of YAP under TFF, it can be concluded that the observed decrease in the level of YAP is due to degradation via a proteasomal independent mechanism. Further studies have to be done to elucidate the details of this unknown mechanism of degradation and to better understand its role in the pathogenesis of atherosclerosis.
 

60.12 The Absence of MATURE CD4+ T-lymphocytes CompromiseS Arteriovenous Fistula Maturation in Rats

Posted on December 22, 2014

R. I. Vazquez-Padron1, J. C. Duque1, M. Tabbara1, L. H. Salman1, R. I. Vazquez-Padron1  1University Of Miami,Vascular Surgery,Miami, FL, USA

Introduction: 


The role of immune cells in arteriovenous (A-V) fistulae maturation is poorly understood and has received, until quite recently, little attention. This study examines the role of T-lymphocytes in A-V fistula vascular remodeling and maturation. 



Methods: 


Experimental A-V fistulae were created in two groups of athymic nude rats lacking mature T-lymphocytes, and euthymic control animals by anastomosing the left superior epigastric vein to the nearby femoral artery. Histopathogical analysis and morphometric assessment of venous wall thickness and real luminal area was performed at 30 days post surgery. Flow measurements were recorded using a Transonic Flowmeter system.



Results:


The absence of T-lymphocytes compromised blood flow and increased failure rate. Immunodeficiency caused a constrictive vascular remodeling characterized by a reduced lumen, a thick muscular layer and a low number of inflammatory cells. The adoptive transfer of CD4+ T-lymphocytes from euthymic rats prior and after fistula creation in athymic animals prevented negative remodeling



Conclusion:


These results point at the key role of CD4+  T-lymphocytes in the proper remodeling of the  A-V fistula vascular wall

60.13 Feasibility of Arteriovenous Fistula Creation After Previous Radial Artery Harvesting for Aortocoronary Bypass

Posted on December 22, 2014

C. Rivera1, N. J. Gargiulo1  1North Shore University And Long Island Jewish Medical Center,Manhasset, NY, USA

Introduction:   Arteriovenous fistula (AVF) formation remains the procedure of choice in patients requring hemodialysis. The feasibility of AVF creation in the setting of prior radial artery harvesting after aortocoronary bypass remains unknown. This investigation elucidates which patients might be candidates for AVF creation despite prior radial artery harvesting.

Methods:   A retrospective review was performed on 2,100 patients undergoing hemodialysis access procedures from 2003 to 2010. Of these patients, 11 (0.5%) were identified as having prior radial artery harvesting for aortocoronary bypass. Pre/Post-operative vein mapping, arterial duplex, digital plethysmography, selective angiography, and sestamibi scanning was performed to evaluate the ulnary artery and palmar arch. Patients with evidence suggesting an intact ulnar artery circulation then underwent AVF creation.

Results:  All 11 patients had an adequate preoperative work up.  Seven (64%) of the 11 patients had digital plethysmography suggesting an intact ulnar artery/palmar arch and underwent successful AVF creation.  Three (27%) of the patients had a variety of findings precluding successful AVF creation.  One (9%) patient with normal preoperative plethysmography developed a steal syndrome requiring revision of the arteriovenous fistula.

Conclusion:  Successful AVF creation is feasible in patients with prior radial artery harvesting for aortocoronary bypass. The use of preoperative digital plethysmography, selective ulnar artery/palmar arch arteriography and sestamibi scanning to evaluate forearm muscle perfusion may be used as adjuncts to guide a successful intervention.

 

60.14 Time-dependent PKC-ε Modulation Prevents Resistin-induced Vascular Smooth Muscle Cell Dysfunction

Posted on December 22, 2014

M. C. Zuniga2, G. Raghuraman2, W. Zhou1,2  1Stanford University,Vascular Surgery,Palo Alto, CA, USA 2VA Palo Alto Healthcare Systems,Surgical Services,Palo Alto, CA, USA

Introduction:  Dysfunction of vascular smooth muscle cells (VSMCs) is a key process in vascular restenosis and atherosclerotic plaque formation. VSMCs are known to transform from a differentiated contractile state to a dedifferentiated synthetic state, characterized by increased proliferative and migratory capacity. Adipokine resistin, found elevated in obese and diabetic patients, has been suggested as a risk factor for cardiovascular complications, and we have shown that PKC-epsilon (PKC-ε) is involved in resistin-induced VSMC migration. In this study we examined the role of PKC-ε and its time-dependent effects on resistin-induced VSMC dysfunction. 

Methods:  Primary human coronary artery smooth muscle cells were treated with resistin at pathological plasma concentration, 40 ng/mL, for 0–7 days, and selective PKC-ε inhibitor (εV1-2) and activator (ψεRACK) were used at a 1 µM concentration. Migration was quantified with the Boyden chamber assay. Dedifferentiation was evaluated by examining protein expression of the VMSC lineage markers, smoothelin and SM22α, and the structural markers, α-actin and calponin. Cell proliferation was determined by computing VSMCs growth over a period of 7 days. Data was analyzed with one-way ANOVA, and a p-value of <0.05 was considered significant.

Results: Resistin induced more than a 2-fold increase in VSMC migration as compared to the untreated control (Figure 1 c). Significant increase in VSMC proliferation and significant decreased expression of all VSMC markers was observed after 3 days of treatment (Figure 1 a-b). Timelines for ψεRACK showed that short pre-treatment (30 min) had a protective effect in resistin-induced VSMC migration whereas prolonged pre-treatment (≥ 6 h) augmented resistin-mediated effects. On the other hand, εV1-2 required longer treatment times (≥ 12 h) to mitigate resistin-induced VSMC migration. Combined exposures (ψεRACK for 30 min; then εV1-2 during assay time) significantly decreased resistin-induced migration and proliferative growth to similar levels of untreated controls (Figure 1 b-c).

Conclusion: Resistin promoted VSMC migration, proliferation, and loss of lineage and structural markers. We demonstrated that acute PKC-ε activation followed by prolonged PKC-ε inhibition may provide an optimal therapeutic strategy for resistin-induced VSMC restenosis.

 

6.15 The Impact of Obesity on Operative Time in Elective Colorectal Surgery Procedures

Posted on December 22, 2014

H. Saiganesh1, D. Stein1, J. L. Poggio1  1Drexel University College Of Medicine,Department Of Surgery, Division Of Colorectal Surgery,Philadelphia, Pa, USA

Introduction: Obesity currently affects more than a third of the US population and is associated with increased surgical complications. Compared to all other subspecialties, colorectal surgery is the most affected by the increasing trend in obese surgical patients. Operative time has been found to have the greatest impact on hospital costs and physician workload. This study was conducted to determine whether obesity has a direct impact on operative time in elective colorectal surgical procedures using a high-powered, nationally representative patient sample.

Methods: A retrospective analysis was conducted on 45,362 patients who underwent open and laparoscopic ileocolic resections, partial colectomies, and low pelvic anastomoses using ACS-NSQIP data from 2005 to 2009. Operative time (in minutes) was the main outcome variable, while body mass index (in kg/m^2) was the main independent variable. Body mass index was divided into three classes: normal (<25), overweight/obese (25-35), and morbidly obese (>35). A univariate linear model was used to analyze the relationship while controlling for confounding factors such as demographics and preoperative conditions. Statistical significance was established at p < 0.05.

Results: Morbidly obese patients were found to have longer operative times than did normal patients across each individual colorectal surgical procedure (p < 0.001), ranging from a mean difference of 17.8 minutes for open ileocolic resections to 56.6 minutes for laparoscopic low pelvic anastomoses with colostomies.

Conclusions: BMI, as an objective measure of obesity, is a direct, statistically significant independent predictor of operative time across elective colorectal surgery procedures. We suggest future studies to further discuss the modification of surgical reimbursement to account for the greater procedural and temporal costs in treating obese patients.

 

6.16 Continous Non-Invasive Assessment of Hemoglobin and Fluid Responsiveness in Obesity and Laparoscopy

Posted on December 22, 2014

M. D. DeBarros1, M. W. Causey1, P. Chesley1, M. Martin1,2  1Madigan Army Medical Center,Department Of General Surgery,Tacoma, WA, USA 2Oregon Health And Science University,Department Of Trauma And Critical Care,Portland, OR, USA

Introduction: During surgery, proper fluid resuscitation and hemostatic control is critical.  Pleth variability index (PVI) is advocated as an accurate means of optimizing intraoperative fluid resuscitation.  PVI is a measure of dynamic change in perfusion index during a complete respiratory cycle.  Non-invasive monitoring of total hemoglobin could provide an accurate means to determine the need for transfusion. We analyzed the impact of insufflation and obesity on non-invasive measurements of hemoglobin and PVI in laparoscopic procedures to validate its usefulness in assessing fluid responsiveness and hemoglobin levels.

Methods: A non-invasive hemoglobin and PVI monitoring device was prospectively analyzed in patients undergoing abdominal operations.   Patients were stratified by open and laparoscopic approach and obesity (BMI≥35).  PVI and hemoglobin values were assessed before, during, and after abdominal insufflation and compared to control patients undergoing open surgery.

Results: 63 patients were enrolled (mean age 42 years; 71% female; mean BMI 36) with 24 patients being laparoscopic non-obese (LN), 20 being laparoscopic obese (LO), and 19 undergoing open operations.  There was no significant blood loss in any case.  Hemoglobin did not change significantly before or after insufflation (Figure 1a). There was false elevation of PVI with insufflation and more pronounced in obese patients (Figure 1b).

Conclusion: Insufflation or obesity was not associated with significant variations in hemoglobin.  Non-invasive monitoring of hemoglobin is useful in laparoscopic procedures in obese and non-obese patients. PVI values should be used cautiously during laparoscopic procedures, particularly in obese patients.

 

6.17 Fluorescent Cholangiography during Robotic Cholecystectomy: Early Experiences at an Urban Hospital

Posted on December 22, 2014

L. Policastro1, R. Huang1, P. J. Chung2, H. Patel2, A. Schwartzman2, R. Lee2, G. Sugiyama2  1SUNY Downstate College Of Medicine,Brooklyn, NY, USA 2SUNY Downstate Medical Center,Department Of Surgery,Brooklyn, NY, USA

Introduction:
Intraoperative cholangiography using contrast fluid is traditionally a useful visual aid during laparoscopic cholecystectomy, but its disadvantages include increased operative time and exposure to radiation. Recently, near-infrared fluorescent cholangiography using indocyanine green (ICG) dye became available to users of the da Vinci surgical platform. It provides a real-time, 3D endoscopic view highlighting the bile ducts. ICG imaging has been utilized safely and effectively during robotic cholecystectomy. Adequate visualization of biliary structures is paramount in patients presenting with advanced gallbladder disease. This study compares operative results in robotic cholecystectomy with and without the use of ICG at an inner-city academic medical center.

Methods:
Eighty-six patients underwent robotic cholecystectomy between June 2013 and July 2014. Use of fluorescent cholangiography began in November 2013 after FDA approval was obtained. Patients were administered 5 mg of ICG on induction of anesthesia. If there was a failure to visualize biliary anatomy, another 2.5 mg was administered. Both single-site and multiport techniques were used. Patient records were compiled retrospectively.

Results:
There were 46 patients in the non-ICG group and 40 in the ICG group. The groups were similar, except that single-site was utilized more frequently with ICG (73% vs. 48%; p=.02), and chronic cholecystitis was slightly more prevalent in the ICG group (88% vs. 83%; p=.07). Overall, the mean BMI was 30.4 ± 6.5, and the rate of acute cholecystitis was 21%. Use of ICG neither lengthened nor shortened operation time (120 min with, 133 min without; p=.22) or robot time (58 min with, 57 min without; p=.85). The rate of conversion to either open or laparoscopic cholecystectomy was favorable (3% with, 13% without), but to qualify this result, logistic regression was used to account for age, sex, BMI, use of single-site, acute and chronic cholecystitis, tobacco use, and alcohol use. While marginally significant (p=.079), the use of ICG was associated with fewer conversions (OR 0.10, 95% CI 0.008−1.30). There was no difference in post-op length of stay (p=.91).

Conclusion:
ICG fluorescent cholangiography during robotic cholecystectomy enables confident identification of biliary structures without impacting operation time. It is helpful in the training of residents and fellows. It is safe for routine use and may reduce conversions, though its overall cost-effectiveness cannot be concretely established without a large randomized study.
 

6.18 The ‘Inside-out’ Technique for Ventral Hernia Repair with Mesh Underlay

Posted on December 22, 2014

A. E. Berhanu1,2, S. G. Talbot1,2  1Brigham And Women’s Hospital,Division Of Plastic Surgery,Boston, MA, USA 2Harvard School Of Medicine,Boston, MA, USA

Introduction: Techniques for mesh installation in hernia repair are varied and can be improved by preventing recurrent herniation, reducing intraoperative visceral injury, and increasing procedural efficiency. We present a method of securing mesh (either prosthetic or biologic) as an underlay concurrent with component separation, with pre-placed sutures on the material, to the overlying fascia through an ‘inside-out’ technique using a Carter-Thomason suture passer. The Carter-Thomason is a sharp, narrow trocar (3mm diameter) with the ability to grasp a suture at the distal end. It was originally designed to aid with closure of laparoscopic ports by facilitating the placement of sutures through fascia around port sites.

Methods:  The ‘inside-out’ technique was performed on 23 patients at a single tertiary academic medical center from November, 2011 to February, 2014.  We have followed these patients for a median of 12.5 months to assess for post-operative complications and hernia recurrence.  The innovative steps in this technique include (1) the preplacement of sutures on the mesh and removal of needles, (2) placement of mesh into the abdomen, and (3) retrieval of each end of the sutures with the Carter-Thomason for safe passage through the fascia from within the abdomen under direct visualization (Figure).

Results: There have been two recurrences (2/23=8.7%), one in a patient at 383 days post-operatively and the other at 311 days post-operatively.  The former recurrence occurred in a patient who underwent repair for a recurrent ventral hernia and the latter patient had significant loss of domain requiring an inlay mesh.  There were no hernia recurrences in patients who underwent repair of a primary ventral hernia with an underlay technique.

Conclusion: The ‘inside-out’ technique for ventral hernia repair with a mesh underlay after component separation using a Carter-Thomason suture passer is easy, safe, and reliable.  We have observed no hernia recurrence in patients who underwent repair for a primary ventral hernia with an underlay technique.  By pre-placing the sutures in the mesh, these can be evenly spread, giving the surgeon better control over the distribution of tension across the repair.  The technique described here seemingly reduces the risk of bowel injury by allowing direct visualization of the entire path of the sharp instrument, the tip of the Carter-Thomason, as it passes through the rectus. Additionally, pre-placed sutures lie between the mesh and posterior rectus fascia, allowing the mesh to shield the viscera from the path of the Carter-Thomason.

6.19 Robotic TAPP Inguinal Hernia Repair: Early Experiences at an Inner-City Academic Medical Center

Posted on December 22, 2014

R. Huang1, L. Policastro1, P. J. Chung2, H. Patel2, A. Schwartzman2, R. Lee2, G. Sugiyama2  1SUNY Downstate College Of Medicine,Brooklyn, NEW YORK, USA 2SUNY Downstate Medical Center,Department Of Surgery,Brooklyn, NEW YORK, USA

Introduction: Laparoscopic inguinal hernia repair was introduced in the 1990s and has been widely adopted. Many studies have shown that the transabdominal pre-peritoneal (TAPP) and totally extra-peritoneal (TEP) approaches to inguinal hernia repair have low recurrence rates and allow for early return to activity due to decreased pain. The robotic surgical platform improves upon standard laparoscopic approaches, providing enhanced dexterity and 3D viewing. Robotic inguinal hernia repair has been previously reported en-passant with prostatectomy, but has not been studied as a stand-alone procedure. We report one of the first case series of stand-alone robotic TAPP inguinal hernia repair at an inner-city academic medical center.

Methods: From December 2013 to June 2014, 26 robotic TAPP inguinal hernia repairs were performed at a single institution. We employed a three port approach, with a camera port at the umbilicus and two 8 mm lateral ports roughly 2 cm above the umbilicus. Patient characteristics, operative times, complications, and hernia recurrence were assessed. Data were compiled retrospectively.

Results: Twenty-six robotic TAPP inguinal hernia repairs were performed, of which seven were bilateral. Twenty-four patients were male and two were female. The mean age of the patients was 59.6 ± 11.2 years. The mean BMI of the patients was 26.3 ± 3.4. The mean incision-to-closure time was 127 ± 42 minutes. The mean robotic operation time was 82 ± 37 minutes. The mean robot docking time was 6 ± 3 minutes. Seven patients had scrotal hernias, three presented with incarcerated inguinal hernias, and an additional two had large hernia sacs. On follow-up, 4 of 26 patients (15%) had postoperative hematomas, while 3 patients (12%) had postoperative urinary retention. One patient (4%) returned with a recurrent hernia. No scrotal hernia patients presented with recurrence.

Conclusion: We present a case series of robotic TAPP inguinal hernia repairs at an inner-city institution and conclude that the procedure is feasible and safe for both routine herniorrhaphy and in advanced scrotal cases. Recurrence and morbidity were low during the early learning curve. Large scrotal and incarcerated inguinal hernias were repairable using the robotic approach. Further prospective study is needed to determine whether robotic inguinal hernia repair should be broadly adopted.
 

6.20 Difficult Laparoscopic Cholecystectomy: Conversion to Open versus Laparoscopic Subtotal Cholecystectomy

Posted on December 22, 2014

K. Leick1, D. Ring1, A. Bhama1, H. Chong1  1Universisty Of Iowa,Gastrointestinal-Minimally Invasive Surgery,Iowa City, IA, USA

Introduction: As laparoscopic cholecystectomy is now the gold standard for removal of the gallbladder, trainees and newly graduated surgeons are more experienced and comfortable with laparoscopic cholecystectomy (LC) as compared to open cholecystectomy (OC).  Laparoscopic subtotal cholecystectomy (LSC) is a safe alternative to conversion to OC (COC) in difficult cholecystectomy cases. To our knowledge, no current literature exists comparing complications between these two procedures.

Methods: A single-institution retrospective chart review was performed on patients who underwent attempted LC over a 4-year period from July 2009 to July 2013. LC patients with common bile duct injury prior to conversion to open cholecystectomy were excluded from the study. Demographic characteristics and postoperative outcomes were analyzed. Statistics were performed using paired t-tests and χ2 in Microsoft Excel®, and a p value of <0.05 was considered significant.

Results: A total of 2646 patients underwent LC. Of these, 95% (n=2518) were completed laparoscopically,  2% (n=47) underwent COC and 3% (n=81) underwent LSC.  Demographic characteristics were equivalent between COC and LSC group, except for age, as the COC group was significant older than the LSC group (58 ±17 vs. 49 ± 18, p=0.009). Intraoperative records demonstrated longer operative time with COC as compared to LSC (160 ± 90 minutes vs. 130 ± 37 minutes, p < 0.001). There was a significantly longer length of hospital stay in the COC group as compared to the LSC group (5.3 ± 2.3 days vs. 2.1 ± 2.5 days, p < 0.05). Overall, total complication rate was significantly higher with COC as compared to LSC (22.2% vs. 45.8%, p<0.02). These complications included: bile leak, retained common bile duct stone, acute kidney injury, urinary complications surgical site infection, small bowel obstruction or ileus, and postoperative arrhythmias. There was no significant difference in need for postoperative ERCP between the two groups.

Conclusion: LSC is associated with shorter length of hospital stay, shorter operative time, fewer complications and lower morbidity when compared to COC. Additionally, LSC is associated with decreased morbidity when compared to COC. In situations where standard laparoscopic cholecystectomy is not possible, performing LSC instead of COC should be considered as the next alternative.

 

60.01 Calpain Inhibition Decreases Apoptosis In A Swine Model Of Chronic Myocardial Ischemia

Posted on December 22, 2014

B. A. Potz1, A. A. Sabe1, N. Y. Elmadhun1, J. Feng1, Y. Lui1, H. Williams1, F. W. Sellke1  1Brown University School Of Medicine,Division Of Cardiothoracic Surgery, Department Of Surgery,Providence, RI, USA

Introduction: Calpain is a family of cysteine proteases that has an important role in the initiation, regulation and execution of cell death.   Our recent studies using a hypercholesterolemic swine model demonstrated that in the setting of metabolic syndrome calpain inhibition improved collateral dependent perfusion, and increased expression of proteins implicated in angiogenesis and vasodilation.   In this study, we hypothesized that calpain inhibition (CI) would decrease myocardial apoptosis in the same model.

Methods: Yorkshire swine, fed a high cholesterol diet for four weeks, then underwent placement of an ameroid constrictor on the left circumflex artery. Three weeks later animals received either: no drug, high cholesterol control group (HCC; n= 8); low dose CI (0.12 mg/kg; LCI, n= 9); or high dose CI (0.25 mg/kg; HCI, n= 8). The high cholesterol diet and CI were continued for five weeks, after which the pig was euthanized and the left ventricular myocardium was harvested and analyzed via TUNEL staining, oxiblot analysis and western blots. Data was analyzed via the Kruskal-Wallis test.

Results: The percentage of apoptotic cells to total cells in ischemic myocardial territory was significantly decreased in the LCI and HCI groups compared to the HCC group as shown by TUNEL Staining (p= 0.018).  There was a significant decrease in pro-apoptotic proteins including cleaved caspase 3 (p =0.001), caspase 9 (p = 0.003), cleaved caspase 9 (p=0.004), Bax (p=0.0262) and an insignificant decrease in caspase 3 (LCI 0.973 +/- 0.261 fold, HCI 0.981 +/- 0.290, HCC 1.0 fold, p= 0.737).  There was a significant increase in anti-apoptotic proteins including BCL-2 (p= 0.025) and p-BCL2 (p= 0.004). In the ischemic myocardium there was a significant increase in several pro-angiogenic proteins in the LCI and HCI groups compared to the HCC group including p-AKT (p=0.0001), p-ENOS (p= 0.003) and  ENOS (LCI 1.26 +/- 0.665 fold, HCI 1.87+/- 0.761 fold, HCC 1.0 fold, p=0.006) with an insignificant increase seen in AKT (LCI 1.263+/- 0.613 fold, HCI 1.180+/-0.636 fold, HCC 1.0 fold, p=0.311).  CI significantly decreased tissue oxidative stress in both the LCI  and HCI groups as compared to the HCC group as shown by Oxiblot analysis (p= 0.021).

Conclusion: In the setting of hypercholesterolemia, CI decreases apoptosis and the expression of proteins in the pro apopotic signaling pathway.   CI also increased expression of proteins implicated in angiogenesis and anti apoptotic pathways.

 

60.02 Right Ventricular Recovery Associated With Normalization Of SERCA2a Expression

Posted on December 22, 2014

M. E. Bowen1, X. Liu1, S. H. McKellar1  1University Of Utah,Cardiothoracic Surgery,Salt Lake City, UT, USA

Introduction:  Heart failure affects many Americans and carries a poor prognosis. Chronic systolic heart failure is associated with abnormal excitation-contraction coupling and abnormal intracellular calcium handling.  Previous studies focused on left heart failure, and the purpose of our investigation was to focus on both right heart failure and recovery. We examined the molecular changes occurring with regards to calcium handling proteins in right ventricular failure and recovery. Our aim was to identify changes in molecular gene expression between failed and recovered right ventricles.

Methods:  We performed a targeted gene expression analysis on right ventricular (RV) tissue using our rabbit model of chronic, pressure-overload RV failure and recovery consisting of an adjustable pulmonary artery (PA) band. Fifteen rabbits received PA banding and were assigned to 1 of 3 groups (n= 5 each): RV failure (RVF), RV recovery (RVR), or normal control. qPCR analysis was performed on RV tissue for genes encoding for calcium-handling proteins. These include: phospholamban (PLN), junctophilin-2 (JPH-2), ryanodine receptor-2 (RyR2), and Sarcoplasmic Reticulum calcium ATPase (SERCA2a).

Results: We observed a significant decrease in SERCA2a expression during RVF that improved during RVR, P=0.02. In contrast, minimal changes were observed for the other calcium handling genes: PLN, JPH-2, RyR2. We speculate SERCA2a expression plays a strong role in RV recovery.

Conclusion: We observed abnormal SERCA2a expression in pressure-overload RVF which improved during RV recovery. These data are consistent with previous studies identifying decreased SERCA2a during heart failure but expand our understanding of SERCA2a gene expression during RVR.  Further mechanistic studies evaluating the calcium handling during RVR are warranted.

 

60.03 Slit3 Knockout Mice With Congenital Diaphragmatic Hernia Show Evidence Of Right Ventricular Strain

Posted on December 22, 2014

M. Shah1, M. Phillips1, R. Frye1, S. McLean1  1University Of North Carolina – Chapel Hill,Chapel Hill, NC, USA

Introduction: The Slit3 knockout (KO) mouse reliably produces a congenital diaphragmatic hernia (CDH) phenotype, and is associated with right ventricular (RV) hypertension and pulmonary arterial hypertension (PAH). PAH occurs due to abnormal pulmonary vascular development and pulmonary vascular remodeling due to unknown mechanisms. We hypothesize that long-term exposure to PAH will induce evidence of altered right ventricular (RV) physiology.

Methods: Slit3 KO mice were bred at our institution. At 3 months of age, wild type (WT) and KO mice were harvested. RV pressures were measured by cardiac puncture. The hearts were then harvested for RNA isolation and weighed. They were placed in RNA-later solution at 4 degrees C for 24 hours and then frozen at -80 degrees C. Hearts were then thawed and RNA was harvested using Trizol reagent. Real-time quantitative PCR was performed using TaqMan reagents with probes for brain natriuretic peptide (BNP).

Results: Slit3 KO mice with CDH had elevated RV systolic (18.8 vs 34.6) and mean (10.2 vs 16.4) pressures at 3 months of age. RT-PCR of Slit3 KO and WT mouse showed significantly increased expression of BNP in RNA harvest from murine RVs, with mean relative quantification (RQ) normalized to b-actin of 0.114 vs 0.493, p<0.005. This represents a 4.35 fold increase in RV-BNP expression in Slit3 KO mice with CDH. RV weight in KO mice was also significantly increased when compared as a percentage of total body weight (TBW), (0.53% vs 0.72%), p<0.02.

Conclusion: Slit3 KO mice develop PAH, right heart strain, and RV hypertrophy when compared to WT mice. This further validates the importance of the Slit3 KO mouse model of PAH in CDH. The use of a validated mouse model of CDH would provide researchers with a novel tool for the study of PAH in CDH. The use of this model to study the mechanisms of PAH development in CDH, may provide temporal and mechanistic targets for therapeutic interventions. 

60.04 Effects of a Pulse Induced by a Continuous-Flow Cardiac Replacement Device on Vascular Permeability

Posted on December 22, 2014

J. Feng1, W. E. Cohn2, S. M. Parnis2, R. R. Clements1, N. R. Sodha1, O. H. Frazier2, F. W. Sellke1  1Brown University School Of Medicine,Surgery,Providence, RI, USA 2Texas Heart Institute,Minimally Invasive Surgical Technology,Houston, TX, USA

Introduction:
We tested the short-term effect of completely nonpulsatile versus pulsatile circulation after ventricular excision and replacement with total implantable pumps in the animal model of calves on peripheral vascular permeability.

Methods:
8 calves underwent cardiac replacement with 2 HeartMate III continuous-flow rotary pumps. In 4 calves, the pump speed was rapidly modulated to impart a low-frequency pulse pressure in the physiologic range (10-25mmHg) at a rate of 30 pulses per minute (PP). The remaining 4 calves were supported with a pulseless systemic circulation and no modulation of pump speed (CP). Skeletal muscle biopsies were obtained before cardiac replacement (baseline) and on postoperative days 1, 7 and 14. Skeletal muscle tissue water content was measured and vascular endothelial (VE)-cadherin (VE), phospho-VE-cadherin (p-VE) and CD31 were analyzed by immuno-histochemistry. 

Results:

There were no significant changes in tissue water content within group or between groups at baseline, postoperative days 1, 7 and 14, respectively. There were no significant alterations in the distribution of VE-cadherin, phospho-VE cadherin and CD31 in skeletal muscle vasculature at baseline, postoperative days 1, 7 and 14 within each group or between the two groups (see Figure), respectively. Even though total CRD  in both pulse and pulseless pumps caused slight increase in the intensity of phospho-VE-cadherin  at postoperative days 7 and 14, it failed to reach statistical significance (See Figure).

 

Conclusion:
There were no significant VE-cadherin degradation and phosphorylation in the calf skeletal muscle microvasculature after constant-flow total CRD, suggesting that short term of constant flow CRD with or without pulse pressure did not cause peripheral endothelial injury and did not increase the peripheral vascular permeability.

60.05 4D MR Imaging of Flow Division in a Model of Aortic Dissection

Posted on December 22, 2014

J. Birjiniuk4, J. M. Ruddy3, E. Iffrig4, T. Henry5, B. G. Leshnower6, J. N. Oshinski4,5, D. N. Ku1, R. K. Veeraswamy2  1Georgia Institute Of Technology,George W. Woodruff School Of Mechanical Engineering,Atlanta, GA, USA 2Emory University School Of Medicine,Division Of Vascular Surgery And Endovascular Therapy, Department Of Surgery,Atlanta, GA, USA 3Medical University Of South Carolina,Division Of Vascular Surgery, Department Of Surgery,Charleston, Sc, USA 4Georgia Institute Of Technology,Wallace H. Coulter Department Of Biomedical Engineering,Atlanta, GA, USA 5Emory University School Of Medicine,Department Of Radiology,Atlanta, GA, USA 6Emory University School Of Medicine,Division Of Cardiothoracic Surgery, Department Of Surgery,Atlanta, GA, USA

Introduction:

Aortic dissection is a tearing of the intimal layer of the aorta leading to two distinct aortic flow lumens with varying degrees of communication between them. This results in a complex biomechanical system with poorly understood fluid dynamics throughout the aorta. Ultimately, these changes may affect tear propagation, intraluminal thrombogenesis, and end-organ perfusion. Developing novel therapies to manage aortic dissection has been limited by the lack of a reproducible system to investigate this pathology.  We present a novel method for studying hemodynamic measurements in a dissected aorta. 

Methods:
 

CT images of several clinical aortic dissections were collected. Rapid prototypes of aortic dissections were created and served as molds for constructing silicone models.  These models have a mobile membrane, which has dimensions and elasticity similar to those of an intimal flap. A positive displacement perfusion pump (COBE, Century HeartLung) with a blood-mimic agent was utilized at a rate of 5 L/min. and magnetic resonance sequences for anatomical data were obtained.  In addition, four dimensional phase-contrast magnetic resonance (4D PCMR) imaging was used to determine velocities in three axes. All images were processed using Siemens 4D Flow (Siemens Medical Solutions USA, Malvern, PA) for particle streams and a custom-made analysis software for flow calculations. Planes perpendicular to aortic wall were chosen at multiple points along the model. In the dissected portion, the two lumens were treated independently after manual segmentation. Luminal regions of interest were drawn manually and used to calculate lumen areas and velocity profiles. Flow parameters were individually measured at each point. Flow rates were calculated using velocity profiles and averaged over several time points in the 4D scan.

Results:

MRI particle streams confirmed flow communicating between the lumens. Flow rate in the true lumen averaged 3.1 +/- 0.8 L/m with 68% of the total flow. The false lumen had a mean flow rate of 1.4 +/- 0.6 L/min (p<0.05). The luminal shapes changed along the length, with a crescent shape proximally, and an elliptical shape distally, for the true lumen. Area in the true lumen decreased along the descending thoracic aorta, while the false lumen increased from 35 to 39%.

Conclusion:

Our model successfully recapitulates the flow behavior in a Type III b dissected thoracic aorta, and our method successfully utilizes 4D PCMR imaging to make relevant hemodynamic measurements. For this particular example, we found that the flow in each lumen was proportional to its share of luminal area.  Future studies will yield detailed hemodynamics with entrance and exit tears.

60.06 Matrix Gla-Protein Regulates Calcification of the Aortic Valve

Posted on December 22, 2014

N. Venardos1, D. T. Bennett1, T. B. Reece1, M. J. Weyant1, X. Meng1, D. A. Fullerton1  1University Of Colorado Denver,Division Of Cardiothoracic Surgery/Department Of Surgery,Aurora, CO, USA

Introduction: The aortic valve interstitial cell (AVIC) has been implicated in the pathogenesis of aortic stenosis. In response to pro-inflammatory stimulation, the AVIC undergoes a phenotypic change from that of a myofibroblast phenotype to that of osteoblast-like cell.  This osteogenic phenotype is characterized by the production of bone forming proteins such as runt-related transcription factor 2 (Runx-2) and osteopontin (Opn). Matrix gla-protein (MGP) has been identified as an important inhibitor of vascular calcification.  We therefore hypothesized that MGP negatively regulates the AVIC osteogenic response, and loss of this protective mechanism contributes to calcification of the aortic valve. Our purpose was to compare MGP expression in normal vs. diseased AVICs and to evaluate the effect of MGP treatment on AVIC osteogenic protein production.

Methods: Human AVICs were isolated from normal aortic valves from explanted hearts (n=4) at the time of heart transplantation. AVICs were also isolated from calcified, diseased valves of patients (n=4) undergoing aortic valve replacement. AVICs were grown in culture until they reached passages 2-6 prior to experimentation. Immunofluorescent staining (IF), RT-PCR, ELISA, and immunoblotting (IB) were used to compare levels of MGP in normal and diseased AVICs. Statistics were by t-test (*p<0.05).

Results: MGP expression was significantly increased in normal AVICs relative to diseased AVICs by IF, RT-PCR, ELISA, and IB (see figure, normalized to β -actin and normal AVICs).

Conclusion: An important anti-calcification defense mechanism is deficient in calcified aortic valves. MGP expression is significantly greater in normal relative to diseased AVICs.  Lack of this important “anti-calcification” protein may contribute to calcification of the aortic valve.

 

6.02 Risk Factors for Conversion of Laparoscopic to Open Cholecystectomy in Acute Cholecystitis

Posted on December 22, 2014

M. Sippey1, A. Mozer1, M. Grzybowski1, M. Manwaring1, J. Pender IV1, W. Chapman1, W. Pofahl1, W. Pories1, K. Spaniolas1  1East Carolina University Brody School Of Medicine,General Surgery,Greenville, NC, USA

Introduction:   Laparoscopic cholecystectomy is one of the most common general surgical procedures performed.  Conversion to an open procedure (CTO) is associated with increased morbidity and length of stay. Patients presenting with acute cholecystitis (AC) are at higher risk for CTO. Studies have attempted to examine risk factors for CTO in patients who undergo laparoscopic cholecystectomy for AC, but are limited by small sample size.  The aim of this study was to identify pre-operative variables that predict higher risk for CTO in patients presenting with AC.

Methods:   Patients undergoing laparoscopic cholecystectomy for AC from 2005 to 2011 were identified from the ACS-NSQIP database. Patients who underwent successful laparoscopic surgery were compared with those who required CTO.  Demographics, co-morbidities, and 30-day outcomes were analyzed. Multivariable logistic regression was used for variables with p-value <0.1, with CTO used as the dependent variable.

Results:  A total of 7,242 patients underwent laparoscopic cholecystectomy for AC.  CTO was reported in 436 (6.0%) patients.  Those who required conversion were older (60.7 ± 16.2 vs 51.6 ± 18.0, p = 0.0001) and mean BMI was greater (30.8 ± 7.55 vs 30.0 ± 7.31, p = 0.028) compared to those whose procedure was completed laparoscopically.  Vascular, cardiac, renal, pulmonary, hepatic disease, diabetes and bleeding disorders were more prevalent in CTO patients.  Mortality (2.1% vs 0.7%, p = 0.001), overall morbidity (21.2% vs 6.0%, p<0.0001), serious morbidity (14.4% vs 3.8%, p<0.0001), reoperation (3.2% vs 1.4%, p = 0.002), and SSI (9.1% vs 1.8%, p<0.0001) rates, as well as LOS (8.52 ± 12.58 vs 3.41 ± 5.60, p<0.0001) were greater in those requiring CTO.  Our model showed the following factors were independently associated with CTO: age (OR 1.01, p = 0.015), male gender (OR 1.77, p = 0.005), BMI (OR 1.04, p<0.0001), pre-operative alkaline phosphatase (OR 1.01, p = 0.0005), WBC count (OR 1.06, p = 0.0001), and albumin (OR 0.52, p = 0.0001).  The overall model had a strong ability to discriminate between patients who did and did not require CTO (c=0.74, p<0.0001).

Conclusion:  CTO for AC remains low, but not clinically negligible.  After controlling for confounding baseline characteristics, for each unit increase in BMI, risk of CTO increases by 4%.  The identified risk factors can potentially guide management and patient selection for delayed intervention for AC.

 

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