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.

 

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.

 

59.13 Novel Use of Porcine Extracellular Matrix with Basement Membrane for Pilonidal Wound Care in Children

Posted on December 22, 2014

R. M. Dorman1, K. D. Bass1,2  1State University Of New York At Buffalo,Department Of Surgery,Buffalo, NY, USA 2Women And Children’s Hospital Of Buffalo,Department Of Pediatric Surgery,Buffalo, NY, USA

Introduction:   Extracellular matrix is used in various applications. We sought to use a new device that incorporates a porcine extracellular matrix with a basement membrane (ACELL)  for adolescent pilonidal disease.  Pilonidal disease is characterized by deep sinus tracts that result in large soft tissue abscesses.  Current treatment includes debridement with the following options: primary closure, saline dressings, or negative pressure dressings. Primary closure results in a high rate of recurrence therefore wound care is favored in our practice. Saline dressings are the most inexpensive method, but are associated with the most pain and longest recovery time. Negative pressure dressings are more expensive but reduce the number of dressing changes, and accelerate wound healing reducing duration of wound care and pain. The goals for children with pilonidal disease are accelerating wound healing while minimizing painful dressing changes, in order to return to baseline function. Experience with ACELL in other wound applications prompted our investigation with children.

Methods:   A series of 4 patients with pilonidal abscess were debrided.  Three occurred in the gluteal cleft, and the fourth in the umbilicus. In the first patient, the wound deficit was filled with ACELL powder and a sheet of 2-ply ACELL was placed to close the wound. In the second patient, two sinus tracts were debrided, packed with ACELL, and a sheet of 5-ply ACELL was applied.  In the third patient, powder and a 6-ply ACELL was applied as a roll filling the dead space.  In the last patient, an umbilical sinus 3 cm deep was packed with ACELL powder followed by a roll of 2 ply ACELL sheet. Patients were evaluated weekly postoperatively and more powder and sheet ACELL material was added if their wound deficit was still present.  Measurements were taken in three dimensions to characterize wounds.

Results: Resolution of wound deficit was graphed vs. time. Pain was assessed by scoring 0-10.  The graph depicts rapid wound healing by both depth and maximum diameter.  The rate of wound healing increased as the volume or thickness (ply) of ACELL material in the wound deficit increased. Two of the patients had failed wound healing with saline dressing changes prior to ACELL application. These 2 patients specifically were highly satisfied with minimal pain scores in their first week postoperative compared to their time with saline dressings.  All 4 patients had no pain after 1 week when bolster sutures were removed.

Conclusion: The use of ACELL in adolescent pilonidal disease was extremely well tolerated with decreased pain compared to saline dressings.  The rate of wound healing was accelerated by the volume of ACELL material used.  The patient satisfaction with the device was high.  This experience supports a prospective study.

59.14 Receptor-interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing

Posted on December 22, 2014

A. J. Godwin1, W. Yang1,2, A. Sharma2, J. Nicastro1, G. F. Coppa1, P. Wang1,2  1Hofstra North Shore-LIJ School Of Medicine,Surgery,Manhasset, NY, USA 2The Feinstein Institute For Medical Research,Manhasset, NY, USA

Introduction:  Acute cutaneous wounds from trauma can become chronic non-healing wounds, resulting in a significant morbidity and mortality to the patients. Wound healing consists of a complex, dynamic process which involves three overlapping processes, namely; inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular levels is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3) has been identified to involve in controlling programmed necrosis in response to TNF-α during inflammation. We then hypothesized that RIPK3 regulated the progress of cutaneous wound healing.

Methods:  Full-thickness 2.0-cm diameter circular wounds were created on the dorsum of male wild-type (WT) and RIPK3-knockout (KO) mice on C57BL/6 background. Wound area was measured daily until day 14 post-wound and the pixel of the traced area was analyzed by NIH ImageJ. Skin tissues were collected from the wound sites at various days for histological evaluation and gene expression analysis by qPCR. Mouse embryonic fibroblasts (MEFs) were isolated from WT and RIPK3-KO mice for a transwell migration assay.

Results: The wound healing rate in RIPK3-KO mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in RIPK3-KO mice was 53% larger than WT mice (n=7/group, P < 0.05). H&E and Masson-Trichrome staining indicated that RIPK3-KO wounds had a worse quality of wound closure and less collagen deposition in comparison with WT wounds. The number of Gr-1-positive cells for indicating neutrophils infiltrating in RIPK3-KO wounds was much less than WT wounds at day 1. The expression of growth factors (VEGF and TGF-β) and proinflammatory cytokines (TNF-α and IL-1β) were less and delayed in RIPK3-KO wounds, compared to WT wounds (Table). Furthermore, the numbers of migrated MEFs from RIPK3-KO mice toward PDGF and TGF-β, as chemoattractants, were 62% and 73%, respectively, lower than those of MEFs from WT mice. 

Conclusion: RIPK3-KO mice exhibit impairment of wound healing in association with reduced and delayed production of growth factors and proinflammatory cytokines. The chemotactic activity of RIPK3-deficient fibroblasts to growth factors is suppressed. Thus, RIPK3 is an important molecule required for normal progression of wound closure.

 

59.15 Can exposure to a healthy systemic circulation improve fracture healing in diabetic mice?

Posted on December 22, 2014

R. Tevlin1,2, A. Mc Ardle1,2, E. Seo2, K. Senarath-Yapa1,2, C. Duldulao1, T. Wearda1,2, O. Marecic1,2, E. R. Zielins1, D. Atashroo1, M. S. Hu1, G. G. Walmsley1,2, S. Li1, Z. Maan1, D. C. Wan1, C. K. Chan2, G. C. Gurtner1, M. T. Longaker1  1Stanford University,Division Of Plastic Surgery,Palo Alto, CA, USA 2Stanford University,Institute For Stem Cell Biology And Regenerative Medicine,Palo Alto, CA, USA

Introduction:
Diabetes mellitus results in a decline in the regenerative capacity of bone and is associated with increased incidence of malunion, non-union and delayed union of fractures. Our previous studies have illustrated that this phenotype of impaired fracture healing is preserved in a mouse model of diabetes and that reduced angiogenesis occurs in the diabetic fracture callus. As osteogenesis and angiogenesis are intrinsically linked, we hypothesize that exposure to a healthy, wild-type (Wt) circulation will ameliorate the impairment of fracture healing in diabetic (Db) mice and that this effect may arise from improved systemic response to the ischemic injury of a fracture. 

Methods:
Parabiosis was surgically established between Db and Wt mice, and experimental chimeric controls were created consisting of homogenous Wt/Wt pairs and Db/Db pairs. The creation of a chimeric circulation between Db and Wt mice allowed for evaluation of the role of the skeletal niche in promoting a diabetic phenotype during normal homeostasis and in response to injury. Four weeks post parabiosis, after confirmation of chimeric circulation, a stabilized midshaft femoral fracture was created in one mouse of each chimeric pair. Fracture healing was assessed by weekly radiography and femoral mechanical strength testing was performed at 4 weeks post injury. Protein immunoblot of the circulating serum was performed prior to harvest at 4 weeks post fracture to delineate the effect of parabiosis on the systemic angiogenic response, quantifying angiokines known to be of importance in response to fracture healing

Results:
Diabetes is associated with impaired fracture healing. Heterogenous parabiosis (Db/Wt) accelerates both the development of the diabetic callus as seen radiographically and increases resistance to failure upon mechanical strength testing. Furthermore, exposure of the injured Db mouse to a healthy Wt circulation results in a significant increase in systemic angiokines, such as stromal derived factor-1, matrix metalloproteinases and serpin F1, in response to fracture injury in comparison to Db/Db control chimeric pairs (p<0.05). 

Conclusion:
Skeletal regeneration is intrinsically linked to angiogenesis, a process known to be dysfunctional in the presence of diabetes. Following exposure to a healthy wild-type circulation, we note improvement in indices of fracture healing in a mouse model of diabetes, with concomitant significant systemic upregulation of angiokines, known to play an instrumental role in fracture healing. 
 

59.16 Simplified Prenatal Coverage of Fetal Myelomeningocele with Biosurgical Sealant in Sheep Model

Posted on December 22, 2014

J. L. Peiro1,2, J. Orellana2, S. G. Keswani1, M. Marotta1,2, M. Aguirre2, F. Soldado2, C. G. Fontecha2  1Cincinnati Children’s Hospital Medical Center,The Center For Fetal, Cellular And Molecular Therapy – Pediatric Surgery Division,Cincinnati, OH, USA 2Vall D’Hebron Research Institute (VHIR),Bioengineering, Orthopedics And Pediatric Surgery Group,Barcelona, BARCELONA, Spain

Introduction:  

Myelomeningocele (MMC) is the most frequent non-lethal congenital malformation of the central nervous system, appearing in 1 in every 2000 babies. It is characterized by a deficit of closure of the posterior part of the spine, causing neural tissue exposure to the intrauterine environment, which causes a loss of cerebrospinal fluid and a mechanical-chemical injury on the neural tissue exposed. The closure of the defect by intrauterine open fetal surgery repair has shown better results than the postnatal treatment. Simplified techniques are seeking to enable this repair by fetoscopic approach. Our aim is to assess whether a simple technique of coverage with a biosurgical sealant achieves complete closure and produces the same neural protection as the classic technique of fetal MMC repair.

Methods:

Twenty fetal lambs underwent a surgically like-myelomeningocele lumbar defect on the 75th day of gestation. Four of them were non-repaired (NR). Eight of them underwent immediate coverage with a synthetic biosurgical sealant  (IC). The other 8 underwent delayed coverage with same sealant on the 95th day (DC). Animals were obtained every week, from 1st to 7th postoperative week by c-section for histological analysis.

Results:

All NR animals showed a wide defect of closure, continuous leakage of cerebrospinal fluid, and histological neural damage. All IC and DC animals showed partial coverage of the defect (30 to 100%), with regeneration of dura-mater, muscle and skin only in the covered segment.

Conclusion:

Synthetic bio-surgical sealant applied  over the MMC spinal defect produces partial closure of the neural tube defect without complete healing after its reabsorption in 4 weeks in the fetal lamb model.
 

59.17 Inhibition of Heterotopic Ossification by Cox-2 Inhibitors Is Independent of BMP Receptor Signaling

Posted on December 22, 2014

S. Agarwal1, J. Peterson1, S. Loder1, O. Eboda1, C. Brownley1, K. Ranganathan1, D. Fine1, K. Stettnichs1, A. Mohedas2, P. Yu2, S. Wang1, S. Buchman1, P. Cederna1, B. Levi1  1University Of Michigan,Surgery,Ann Arbor, MI, USA 2Brigham And Women’s Hospital,Boston, MA, USA

Introduction:  Treatment options for heterotopic ossification (HO) including surgical excision and radiation cause tissue damage and result in recurrence. Prophylactic treatment with non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin and celecoxib has been reported to decrease ectopic bone formation in patients after orthopedic procedures although the pathway remains unexplored. Here we demonstrate that administration of celecoxib, a Cox-2 specific inhibitor, decreases HO formation following trauma independent of bone morphogenetic protein receptor (BMPR) function.

Methods:  For our HO model, male C57BL/6 mice underwent Achilles tenotomy of the left hindlimb with 30% total body surface area partial-thickness burn over the dorsum. Mice were administered intraperitoneal celecoxib or carrier daily.  HO was quantified by micro-CT imaging at 2 week intervals up to 9 weeks after trauma (threshold Hounsfield units 1250). Wound-healing was assessed by daily imaging.  To analyze BMP signaling, we used the BRE-luc reporter in C2C12 cells in vitro. C2C12 cells were administered indomethacin (cox-1/cox-2 inhibitor) in the presence of BMP2, BMP4, BMP6, or BMP9 followed by quantification of luciferase activity.  

Results: Administration of celecoxib resulted in an 80 percent decrease in HO formation at 7 weeks (5.06 mm3 v. 1.30 mm3, p<0.05) and 77 percent decrease at 9 weeks (5.61 mm3 v. 1.55 mm3) after trauma. In contrast to the carrier-treated group, HO formation was undetectable for the first 3 weeks in the celecoxib-treated group. All Achilles tenotomy incision and dorsal burn sites showed grossly normal healing. Mesenchymal stem cells from burned mice treated with 1 uM celecoxib in vitro demonstrated 60% less alkaline phosphatase staining and 75% less alizarin red staining than untreated cells. Finally, in vitro administration of indomethacin to C2C12 cells with the BRE-luc reporter resulted in no significant decrease in luciferase activity, suggesting that Cox-2 inhibition does not inhibit BMP receptor function. 

Conclusion: We demonstrate that Cox-2 inhibition decreases HO volume in a burn/trauma model. Decreased mineral deposition also occurs in the in vitro setting with mesenchymal stem cells, suggesting a direct effect on the cells responsible for bone formation. Furthermore, the BRE-luc reporter assay demonstrates that Cox-2 inhibition likely does not impart its effect through the BMP pathway.  Our findings suggest that therapeutic targets for HO need not be limited to the BMP pathway, and that the Cox-2 enzyme deserves further attention.  Patients at risk for HO following trauma may benefit from early celecoxib treatment with minimal impact on wound healing. 

59.18 Hesperidin Accelerates Closure of Splinted Cutaneous Excisional Wounds in Mice

Posted on December 22, 2014

A. A. Wick1, T. Lecy1, T. W. King1  1University Of Wisconsin,Plastic Surgery,Madison, WI, USA

Introduction:
Every year in the United States, more than 6.5 million patients suffer from wound-related complications, and treatment is estimated to cost over $25 billion. After an injury, keratinocytes from the wounded edge must proliferate, migrate across the wound bed, and differentiate in order to restore normal barrier function. We are interested in discovering new methods by which to enhance proliferation, migration, and differentiation in order to improve the wound healing process. Hesperidin, a natural flavonoid found in citrus fruits and honey, has been shown to improve epidermal barrier function. In this study, we investigated the effects of hesperidin on the rate of wound healing in murine splinted cutaneous excisional wounds, a model shown to simulate healing in human tissue. 

Methods:
Six week old, male mice (n=14) were anesthetized, shaved, and two full-thickness 6 mm wounds were created on their backs under aseptic conditions. A 12 mm silicone stent was secured around each wound using cyanoacrylate glue and interrupted 5-0 nylon sutures in order to prevent healing by contraction and to promote healing by formation of granulation tissue. A sterile non-adherent dressing and transparent occlusive dressing were placed over the wound. Twenty-four hours after injury, hesperidin (10 μM) or vehicle  (0.01% DMSO) was applied topically to each wound and was repeated daily. Digital photographs were taken of the wounds every day at each dressing change and treatment application. Wound closure was defined by gross visualization of resurfacing epithelia and calculated as a percent area of the original wound size. Wounds were quantified using ImageJ software (NIH) and expressed as a ratio of wound area to stent area, with scaling normalized to the inner diameter of the splint.

Results:
The wound sizes were similar in both groups at the beginning of treatment.  Addition of hesperidin (10 μM) significantly accelerated the rate of wound closure compared to DMSO control on day 4 (% open, 44±3 vs 53±2 respectively, p<0.05) and on day 5 (% open, 23±3 vs 33±3 respectively, p<0.05).  Wound closure for both groups was complete by day 8.  No negative side effects were noted in the mice.

Conclusion:
Hesperidin accelerates cutaneous wound closure in our in vivo model. Based on this novel finding, further studies should evaluate the mechanisms by which hesperidin accelerates the wound healing process, possibly leading to the development of new and effective therapeutics for wound healing in patients.
 

59.19 Raman Spectroscopy Provides a Rapid, Non-invasive Method for Identifying Calciphylaxis

Posted on December 22, 2014

S. Agarwal1, B. Lloyd1, S. Nigwekar2, S. Loder1, K. Ranganathan1, P. Cederna1, S. Fagan2, J. Goverman2, M. Morris1, B. Levi1  1University Of Michigan,Surgery,Ann Arbor, MI, USA 2Massachusetts General Hospital,Boston, MA, USA

Introduction:  Calciphylaxis is a painful and debilitating condition which creates large open wounds most frequently in patients with renal failure. Calciphylactic lesions are characterized by the precipitation of calcium deposits in the skin and soft tissues, resulting in vessel thrombosis and tissue necrosis. Diagnosis of calciphylaxis traditionally occurs via histologic evaluation of biopsy specimens. However, incisional biopsy of the affected site may result in further local inflammation leading to a cycle of further calcium deposition. We set out to develop a non-invasive diagnostic method which can identify calciphylaxis lesions and avoids creating local inflammatory trauma. 

Methods:  Two histology-confirmed human calciphylaxis biopsy specimens and normal surrounding tissue were examined using either a Raman microscope (interrogating a tissue area < 1 mm) or a hand-held Raman spectroscopy probe (interrogating a tissue volume < 1 mm3). Characteristic spectra for each specimen including the normal surrounding tissue and the known calciphylaxis regions were collected and compared to identify common peaks contributed by apatite.  Spectra were pre-processed for removal of cosmic spikes and correction of spectrograph/detector alignment and grating-induced anamorphic magnification (curvature).  Spectra were corrected for the fluorescence background by fitting background to a low order polynomial (Polynomial order = 5). Band heights and areas are measured. Concurrently nano-CT scans were performed to confirm the regions of calcification.

Results: Using nano-CT imaging, we demonstrate large areas of calcification including within the vasculature. Both calciphylaxis specimens exhibited a strong peak at 960 cm-1, consistent with Raman spectrum attributed to apatite and apatitic-like tissue components. Here, this strong peak is attributed to small calcium phosphate precipitates within the tissue. Normal tissue examined from these patients showed no Raman signature of calcium phosphate.   Our results suggest that Raman spectroscopy can differentiate the calcium phosphate deposition of calciphylaxis from normal tissue. 

Conclusion: Here we differentiate calciphylaxis and normal surrounding tissue based on the physical characteristics of the tissue using Raman spectroscopy. Although we have employed this technique in previously excised biopsy specimens, a hand-help, fiber-optic probe can be developed to analyze surface tissue in humans prior to biopsy. In the future, Raman spectroscopy may provide a rapid and non-invasive method for diagnosing calciphylaxis. By avoiding an incisional biopsy, we will be able to avoid exacerbating the cycle of inflammation which precipitates calcium phosphate deposition in these patients.   

59.20 Negative Pressure Wound Therapy in Severe Open Fractures: Complications Related to Length of Therapy

Posted on December 22, 2014

K. A. Rezzadeh1, L. A. SEGOVIA1, A. HOKUGO1, R. Jarrahy1  1University Of California, Los Angeles,Surgery- Plastic,Los Angeles, CA, USA

Introduction: Negative pressure wound therapy (NPWT) is a widely accepted method of temporary wound coverage prior to soft tissue flap reconstruction of severe lower extremity injuries. However, an examination of the effects of NPWT on complications related to limb salvage surgery has yet to be performed. The precise role of NPWT in the perioperative management of patients with complicated lower extremity injuries remains unclear. In this study we examine the impact of NPWT on the outcomes of traumatic lower extremity wounds treated with flap coverage for the purpose of limb salvage. Specifically, we elucidate the effect of NPWT on flap complications and overall outcomes based upon the timing of soft tissue reconstruction relative to initial injury.

Methods: :  An institutional case series was performed consisting of thirty-two consecutive patients receiving lower extremity reconstruction following Gustilo Class IIIB and IIIC open tibial fractures from 1996 to 2001. Demographic, operative, and clinical data were collected retrospectively.  Outcomes of interest included length of hospitalization, number of surgical procedures, extremity amputation, and non-union. 

Results:Among patients reconstructed within all study time periods (i.e., acute, subacute, and chronic), the incidence of overall complications was lower for the group treated with NPWT than for those patients who underwent conventional wet-to-dry dressing changes. Patients operated on in the chronic period and who received conventional dressing changes had the highest rate (83.3%) of complications while those who were reconstructed in the acute period with perioperative NPWT had the lowest incidence (0%). 

Conclusion:We have shown that the use of NPWT therapy in the perioperative management of patients with open distal lower extremity fractures reduces complication rates associated with limb salvage surgery. Based on our results, we conclude that NPWT can be used as a temporizing measure to optimize patients prior to flap surgery, effectively lengthening the window of opportunity for reconstructive surgeons to manage these challenging patients.

 

« Previous Page
Next Page »