22.10 Fluorescence Guided Surgery May Enhance Localization of Residual Disease During NSCLC Surgery

Posted on January 25, 2017

J. D. Predina1, A. Newton1, J. Keating1, O. Venegas1, S. Singhal1  1University Of Pennsylvania,Philadelphia, PA, USA

Introduction:  Postoperative cancer recurrences occur in 40% of patients undergoing resection for NSCLC.  Such recurrence rates are partially attributable to limitations in intraoperative tools that assist the surgeon in disease identification.  We hypothesized that intraoperative imaging using a fluorescent imaging agent, 5-aminolevulinic acid (5-ALA), could enhance intraoperative identification of NSCLC cancer deposits that would otherwise be missed.

Methods:  The murine NSCLC line, TC1, was cultured in vitro and exposed 1 mM concentrations of 5-ALA for 0 to 24 hours.  Fluorescence was assessed with flow cytometry.  Next, in vivo feasibility of systemic 5-ALA was tested using a small animal model of NSCLC cancer surgery (n=25).   

Results: In vitro, TC1 exhibited high levels of fluorescence after 2 hours of exposure to 5-ALA.  Additional exposure did not augment signal.  Involving in vivo studies, systemic administration of 5-ALA helped identify the presence of residual tumor cells after surgery in 17/25 resections.  The mean residual tumor size was 1.8mm, with a mean fluorescence signal-to-background ratio (SBR) of 4.1.  Of note, minimal fluorescence was noted within thoracic structures including lung parenchyma, pericardium and pleura.  

Conclusion: Systemic 5-ALA reliably accumulates in murine models of NSCLC and helps identify residual disease deposits.  This data supports additional pre-clinical studies which will set the basis for a Human Trial utilizing 5-ALA to enhance NSCLC resection.  

 

22.09 Effect of collagen substrates on oxidative stress-induced changes in SMC phenotype in BAV aortopathy.

Posted on January 25, 2017

P. G. Chan1, M. Billaud1, J. Phillippi1, T. Gleason1  1University Of Pittsburgh,Cardiothoracic Surgery,Pittsburgh, PA, USA

Introduction: Bicuspid aortic valve (BAV) is the most common congenital cardiac malformation and is associated with ascending aortopathy in form of dissection or aneurysmal disease which involve extensive collagen remodeling.  Our prior studies have uncovered that the smooth muscle cells (SMC) from the ascending aorta of BAV specimens have decreased oxidative stress defense compared to SMCs from patients with tricuspid aortic valve (TAV).  We hypothesize that oxidative stress-induced alterations in SMC phenotype can be alleviated by collagen substrates in TAV SMCs but not in BAV SMCs.

Methods: Aortic tissue was obtained from patients undergoing aortic surgery or heart transplantation with IRB approval and informed patient consent.  SMCs from TAV (n=2) and BAV (n=3) patients were harvested using previously established protocols.  SMCs were seeded at a density of 2.8×103/cm2 on tissue culture polystyrene (TCP) in the presence or absence of type 1 collagen substrate.  SMCs were cultured in the presence or absence of 20µM tert-butyl hydroperoxide (tBHP) and monitored in three random fields per well using phase contrast time-lapse microscopy in a stage-top incubation chamber for up to 12 hours.  Post-imaging, the aspect ratios (long axis/short axis) of cells (>17 cells/well) were measured at period time points as a means to assess SMC phenotype.

Results: Aspect ratio for SMCs isolated decreased in the presence of tBHP from 4.4±0.81 to 2.4±0.17 (p<0.001) for TAV SMCs and from 5.0±0.46 to 2.3±0.52 (p<0.001) for BAV SMCs.  When cultured on Type 1 collagen coated wells, TAV SMCs maintained a high aspect ratio despite exposure to oxidative stress.  TAV SMCs increased in aspect ratio on collagen to 6.2±1.55 which was relatively maintained at 4.8±0.83 (p=0.557) when exposed to tBHP.  The aspect ratio of BAV SMCs cultured on collagen was found to be decreased under tBHP treatment (3.88±0.38 vs. 2.1±0.32, p<.001) (Figure 1).

Conclusion: Oxidative stress was found to alter SMC phenotype by decreasing aspect ratio. Culture on collagen prevented oxidative stress induced alteration for TAV SMCs, but not for BAV SMCs.  These results provide further support that BAV SMCs have impaired basal oxidative stress response compared to TAV SMCs despite modulating the extracellular milieu.  Ongoing work is focused on how the extracellular matrix interplays with oxidative stress mediated effects on SMC function in BAV aortopathy.  Ultimately, with this knowledge, we hope to develop therapeutics for patients who are at risk for aneurysmal progression, delineate the molecular mechanisms associated with BAV aortopathy, and improve rupture risk predictions and recommendations for surgical or medical intervention.

22.08 Role of Cardiac Progenitor Cells in Macrophage Polarization During Fetal Cardiac Regeneration post-MI

Posted on January 25, 2017

C. Zgheib1, M. H. Hodges1, M. Allukian2, J. Xu1, K. L. Spiller3, J. H. Gorman2, R. C. Gorman2, K. W. Liechty1  1Laboratory For Fetal And Regenerative Biology, Department Of Surgery, School Of Medicine, University Of Colorado Denver – Anschutz Medical Campus And Colorado Children’s Hospital,Aurora, COLORADO, USA 2The University Of Pennsylvania School Of Medicine,Surgery,Philadelphia, PA, USA 3Drexel University, School Of Biomedical Engineering,Philadelphia, PA, USA

Introduction: Myocardial infarction (MI) remains the leading cause of death in the US. In contrast to the fibrotic response observed in adults, we have previously shown that the fetal response to MI is regenerative and is associated with increased angiogenesis and decreased inflammation. This cardiac regeneration requires both recruitment of cardiac progenitor cells (CPC) to replace lost myocardium as well as restoration of blood supply to support these recruited cells. Macrophages are also known to contribute to this repair process; however, the contribution of macrophages to tissue repair depends on their phenotype: M1 macrophages are pro-inflammatory and initiate angiogenesis; M2a macrophages are pro-fibrotic and contribute to blood vessel maturation; and M2c macrophages are pro-angiogenic, anti-inflammatory, and contribute to tissue remodeling. However, the relationship between CPCs and macrophages during the regenerative process has yet to be defined. Thus, we propose that CPCs promote fetal cardiac regeneration after MI by regulating the polarization of macrophages.

Methods: To test this hypothesis, 20% apical infarcts were created in fetal sheep by LAD ligation. Subsets of these infarcts were injected with a lentivirus containing the SDF-1α inhibitor (SDFi) transgene or an empty vector control. Hearts were harvested 3 or 30 days following infarction. Real time PCR was used to analyze the expression of macrophage phenotypes markers: IL-1β (M10, CD206 (M2a), and CD163 (M2c). 

Results: Our results revealed a significant upregulation of IL-1β, CD206, and CD163 gene expression in the fetal hearts 3 days post-MI; this upregulation returned to baseline at 30 days. In contrast, fetal hearts treated with SDFi demonstrated a significant increase in the expression of all macrophage markers 30 days post-MI, when compared to both untreated fetal hearts and treated hearts 3 days post-MI. These results suggest an early increase in the expression of M1, M2a, and M2c macrophage markers in the fetal hearts 3 days post-MI, with reversal of this upregulation following myocardial regeneration 30 days post-MI. Blocking CPC recruitment by inhibition of SDF-1α was associated with a persistent elevation in M1, M2a, and M2c gene expression, consistent with prolonged inflammation, fibrosis, and remodeling.

Conclusion: Our results suggest that following MI, CPCs promote fetal cardiac regeneration via the modulation of macrophage phenotype. Strategies to manipulate macrophage phenotype may represent a therapeutic target to promote regeneration in adult hearts after MI.

22.07 Reduced Immunogenicity Observed in Multi-Transgene Heart Xenotransplant Model with Suppressed MCP-1

Posted on January 25, 2017

J. L. Chan1, A. K. Singh1, P. C. Corcoran1, M. L. Thomas2, B. G. Lewis2, K. A. Horvath1, M. M. Mohiuddin1  1National Institutes Of Health,Cardiothoracic Surgery Research Program/National Heart, Lung And Blood Institute,Bethesda, MD, USA 2National Institutes Of Health,Division Of Veterinary Resources/Office Of Research Services,Bethesda, MD, USA

Introduction:
Considerable progress has been achieved in xenotransplantation with genetic engineering and the inclusion of additional humanized transgenes appears to be associated with improved outcomes. Monocyte chemoattractant protein-1 (MCP-1), a potent chemotactic factor, has been implicated in transplant immune interaction and rejection. We report the novel use of six-transgene cardiac xenografts and characterize their impact on MCP-1.

Methods:
Heterotopic cardiac xenotransplantations were performed on baboons with swine donor hearts expressing three-gene modifications (GTKO.CD46.TBM) or six-gene modifications (GTKO.CD46.EPCR.DAF with TFPI.CD47 or CD39.TBM). Standard immunosuppressive therapy was administered. MCP-1 levels were measured by fluorescence intensity (FI) using Luminex assay.

Results:
No significant difference in MCP-1 levels were identified preoperatively. Within 48 hours of transplantation, recipients with six-gene modifications (n=3) had a significant reduction in MCP-1 compared to the three-gene cohort (n=3) (1376FI vs. 1928FI, p=0.03). The six-gene group continued to demonstrate decreased MCP-1 in the immediate 30-day postoperative period (1403FI vs. 1916FI, p=0.02) and remained sustained after three months (1159FI vs. 1761FI, p=0.004). Analysis of additional cytokines (IFNγ, TNFα, IL-6) and transmyocardial biopsy histology paralleled findings of a reduced inflammatory response in six-gene recipients. Elevations of MCP-1 to >10% above baseline were associated with transplant rejection within 30 days (odds ratio: 7.40, p=0.01).

Conclusion:
Expression of additional humanized factors in the six-gene cardiac xenografts, including DAF (complement inhibition), CD47 (cellular immune suppression), and CD39 (effector immune modulation), is associated with suppression of MCP-1 production. Decreased MCP-1 levels observed with use of these expanded multi-transgenic donors may reflect superior inflammatory regulation, reduced immunogenicity, and improved xenotransplantation outcomes.

22.05 Calpain Inhibition Decreases Oxidative Stress Via Mitochondrial Pathways in Ischemic Myocardium

Posted on January 25, 2017

B. A. Potz1, L. A. Scrimgeour1, R. T. Clements1, F. W. Sellke1  1Brown University School Of Medicine,Cardiothoracic Surgery Research,Providence, RI, USA

Introduction: Calpain is an intracellular calcium depedent protease whose activity gets overexpressed in cells during times of stress.  Calpain overexpression has been linked to myocardial ischemic disease and organ dysfunction in patients with metabolic syndrome.  Calpain overexpression is implicated in mitochondrial damage leading to tissue oxidative stress.  The purpose of this study was to investigate the potential ability of calpain inhibition on mitochondrial impairment and oxidative stress in a swine model of chronic coronary ischemia in the setting of metabolic syndrome. 

Methods:   Yorkshire swine were divided into 3 groups, fed a high cholesterol diet for 4 weeks, then underwent surgical placement of an ameroid constrictor to their left circumflex artery. Three weeks later animals received either: no drug, high cholesterol control group (CON; n= 8); a low dose calpain inhibitor (0.12 mg/kg; LCI, n= 9); or high dose calpain inhibitor (0.25 mg/kg; HCI, n= 8).  The diets and CI was continued for 5 weeks then the heart was harvested for analysis.  OxyBlot which measures protein carbonyl content (Billerica, MA) was used to determine oxidative stress.  Western Blot was used to identify protein expression (all data was normalized to GAPDH for loading control).

Results:  Calpain inhibition was associated with decreased oxidative stress compared to the control group in the ischemic tissue. There was no change in oxidative stress between groups in the nonischemic tissue. [Figure 1A] In the ischemic myocardial tissue, calpain inhibition was associated with increased expression of the following mitochondrial proteins compared to the control group: the mitochondrial antioxidant protein superoxide dismutase 1; the electron transport chain protein succinate:quinone oxidoreductase; and  the citric acid cycle protein pyruvate dehydrogenase. There was no change in expression between groups prohibitin 1 (p=0.13) and cytochrome c (p=0.15).  [Figure 1B]

Conclusions:  In the setting of metabolic syndrome, CI improved oxidative stress in the ischemic myocardial tissue and was associated with increased expression of mitochondrial proteins involved in 1) reducing superoxide radicals and 2) promoting the citric acid cycle and the electron transport chain.  Calpain inhibition had no effect on oxidative stress in the non-ischemic myocardium. The results of the present study demonstrate the potential ability of calpain inhibition to improve myocardial oxidative stress and suggests that the mechanism through which this beneficial effect is taking place is through the mitochondria.  

22.04 Macrophages Differentially Regulate Collagen Expression in Fetal versus Adult Cardiac Fibroblasts

Posted on January 25, 2017

M. M. Hodges1, C. Zgheib1, J. Xu1, J. Hu1, K. W. Liechty1  1University Of Colorado Denver,Department Of General Surgery, Laboratory For Fetal And Regenerative Biology,Aurora, CO, USA

Introduction:  Ischemic heart disease remains the leading cause of death worldwide.  Despite tremendous advances in the medical management of ischemic cardiomyopathy, the 5-year mortality rate following a diagnosis of heart failure exceeds 50%. In our novel, ovine model of myocardial regeneration after myocardial infarction (MI), we have previously shown that the fetal response to MI is regenerative, whereas the adult response to MI is reparative and is associated with decreased angiogenesis, increased inflammation, and increased fibrosis. Macrophages have been shown to play key roles in regulation of angiogenesis and inflammation; however, the impact that macrophages have on the development of fibrosis and scar formation after MI has yet to be described. We hypothesize that macrophages differentially regulate gene expression of collagen, matrix metalloproteinases (MMP-2 and MMP-9), and transforming growth factors (TGFβ-1 and TGFβ-3) in fetal and adult cardiac fibroblasts.

Methods: To test this hypothesis, cardiac fibroblasts were isolated from the left ventricle of adult and fetal sheep. After isolation, these cardiac fibroblasts were co-cultured for 24 hours with macrophages from the RAW 264.7 cell line.  Quantitative polymerase chain reaction was used to quantify the gene expression of collagen 1α2, collagen 3α1, MMP-2, MMP-9, TGFβ-1, and TGFβ-3.

Results: When compared to adult cardiac fibroblasts, fetal cardiac fibroblasts have significantly upregulated expression of both col1α2 and col3α1, as well as significantly upregulated expression of MMP-2, MMP-9, TGFβ-1, and TGFβ-3.  After co-culture with macrophages, adult cardiac fibroblasts demonstrated significant upregulation of collagen 1α2, which was associated with a significant upregulation in TGFβ-3 and downregulation in MMP-9 gene expression. Conversely, after co-culture with macrophages, fetal cardiac fibroblasts demonstrate a significant downregulation in both col1α2 and col3α1, as well as a significant downregulation in MMP-2, MMP-9, TGFβ-1, and TGFβ-3. These results suggest a differential response of fetal and adult cardiac fibroblasts to co-culture with macrophages.  

Conclusion: Our results suggest that macrophages may play an integral role in regulating the differential responses of the fetus and adult following MI. Additionally, our results suggest that fetal cardiac fibroblasts and adult cardiac fibroblasts are characterized by baseline phenotypic differences that may further contribute to the regenerative response observed in the fetus after MI, compared to the reparative response observed in the adult after MI. A more in-depth understanding of both the phenotypic differences between adult and fetal cardiac fibroblasts, as well as the varying responses following co-culture with macrophages will enable a more complete understanding of the role macrophages play in regulating fibrosis after MI.

22.03 Role of COX-2 in Microvascular Reactivity of Peripheral Arterioles in Diabetic Patients after CPB

Posted on January 25, 2017

K. Anderson1, J. Feng1, Y. Liu1, A. K. Singh1, A. Ehsan1, F. W. Sellke1  1Rhode Island Hospital,Cardiothoracic Surgery/Surgery/Brown Medical Scholl,Providence, RI, USA

Introduction: Diabetic patients are associated with impaired peripheral microvascular function after cardiopulmonary bypass (CPB) and cardiac surgery.  We hypothesized that upregulation inducible cyclooxygenase 2 (COX-2) contributes to altered microvascular reactivity of peripheral arterioles in diabetic patients undergoing CPB and cardiac surgery.

Methods: Skeletal muscle tissue samples of diabetic (DM) and non-diabetic (ND) patients (n = 5-6/group) undergoing cardiac surgery were harvested before and after CPB.  Peripheral arterioles were dissected from the harvested skeletal muscle tissue samples. The isolated arterioles (80-180µm) were cannulated and pressurized and changes in diameter were measured with video microscopy.  In-vitro relaxation responses of pre-contracted arterioles were examined in the presence of the endothelium-dependent vasodilator bradykinin (10-10 to 10-6M) and in the presence or absence of the selective COX-2 inhibitor NS398 (10-6M). 

Results:The post-CPB protein levels of the inducible COX-2 were increased significantly compared with pre-CPB values in both DM and ND groups (P<0.05), whereas, this increase was higher in DM than that of non-diabetics (P<0.05). In the DM arterioles, not the ND vessels, bradykinin-induced relaxation response was inhibited in the presence of the specific COX-2 inhibitor NS398 at baseline (P<0.05). After CPB, bradykinin-induced relaxation response of the DM and ND arterioles was inhibited in the presence of the specific COX-2 inhibitor NS398, but this effect was more pronounced in the diabetic patients (P<0.05).

Conclusion: Diabetes and CPB are associated with upregulation in COX-2 expression/activation in human peripheral microvasculature. This alteration may lead to altered peripheral microvascular reactivity in diabetic patients undergoing cardiac surgery.

 

22.02 Both HMGB1 and DNA Released from Ischemic Myocardium Are Required to Cause Reperfusion Injury

Posted on January 25, 2017

E. J. Charles1, Y. Tian2, J. H. Mehaffey1, D. Wu1, I. L. Kron1, Z. Yang1  1University Of Virginia,Surgery,Charlottesville, VA, Virgin Islands, U.S. 2Tianjin Medical University General Hospital,Cardiovascular Surgery,Tianjin, , China

Introduction: Damage-associated molecular patterns such as high mobility group box 1 (HMGB1) and mitochondrial DNA (mtDNA) may play critical roles in mediating myocardial ischemia-reperfusion (IR) injury.  We hypothesized that HMGB1 and cell-free mtDNA collectively released from ischemia myocardium would lead to activation of splenic leukocytes and cause reperfusion injury.

Methods: Levels of HMGB1 and cell-free mtDNA were measured in plasma and cardiac perfusate obtained from C57BL6 mice (n=4-8/group) that underwent sham surgery or 10, 20, or 40-minute occlusions of the left coronary artery (LCA) without reperfusion.  Perfusate was obtained via antegrade coronary perfusion with phosphate-buffered saline. Levels were measured using SYTOX® Green florescence and Western blot. Separate C57BL6 mice (n=4-8/group) underwent 20 minutes of LCA occlusion followed by 60 minutes of reperfusion.  Resultant myocardial infarct size was measured with TTC and Phthalo blue staining.  Treated groups received recombinant HMGB1 (0.1μg/g/2μl; rtHMGB1), mtDNA obtained from naive C57BL6 mouse livers (0.5ug/g/2μl; mtDNA), or both rtHMGB1 and mtDNA (H+D) 5 minutes prior to reperfusion via external jugular vein injection.  An additional group of mice underwent splenectomy prior to LCA occlusion and received both rtHMGB1 and mtDNA (H+D+Splx).

Results: Plasma levels of HMGB1 and mtDNA were low and not significantly different between mice undergoing sham or LCA occlusion without reperfusion.  However, cardiac perfusate levels were significantly increased in ischemic hearts after 40 minutes of LCA occlusion (p<0.05 vs. other groups).  Infarct size as a percentage of left ventricular risk region after 40 minutes of LCA occlusion without reperfusion was 23.9±5.4%, compared to 0.0% after 20-minute occlusion (p<0.05). In mice undergoing 20 minutes of LCA occlusion followed by 60 minutes of reperfusion, injection of rtHMGB1 or mtDNA did not exacerbate infarct size compared to control mice (p>0.99).  However, H+D mice had significantly larger infarct size (21.2±4.9%), compared with control, rtHMGB1, and mtDNA (all p<0.01, Figure 1). This increased infarct size was attenuated by splenectomy (H+D+Splx: 5.3±2.1%, p=0.02 vs H+D).  There were no significant differences between groups in size of risk region as a percentage of left ventricular mass.

Conclusion: The release of both HMGB1 and mtDNA together from ischemic myocardium is critical to cause reperfusion injury and leads to increased infarct size.  Blocking the effects HMGB1 and/or mtDNA on splenic leukocyte activation may provide a therapeutic option for attenuating myocardial IR injury.

 

22.01 Divergent Roles of Alveolar Macrophages in Neutrophil-Driven Lung Injury

Posted on January 25, 2017

S. Chiu1, M. Akbarpour1, A. McQuattie-Pimentel2, K. Anekalla2, P. Reyfman2, A. Misharin2, H. Perlman2, G. S. Budinger2, A. Bharat1  1Northwestern University,Surgery,Chicago, IL, USA 2Northwestern University,Medicine,Chicago, IL, USA

Introduction:  Pathogen- and damage- associated molecular patterns (PAMPs and DAMPs) stimulate neutrophil recruitment and initiate injury in the lung. It has been suggested that monocytes recruit neutrophils in response to PAMPs while alveolar macrophages (AM) mediate DAMP-driven lung injury, such as ischemia-reperfusion (IR) following pulmonary transplant. Here, we utilize a novel multi-color flow panel to demonstrate that, contrary to the contemporary paradigm, AM ameliorate neutrophil infiltration following lung transplant, but are responsible for neutrophil recruitment in response to PAMPs. 

Methods:  Intratracheal instillation of lipopolysaccharide (LPS) was used to induce PAMP-driven lung injury. Murine single lung allogeneic transplant utilizing wild-type C57BL/6J and Balb/C was performed to induce DAMP-driven lung injury. Clodronate liposomes were used to deplete alveolar macrophages. EdU was injected at the time of transplant to quantify the number of cells entering S phase and undergoing cell division. Flow cytometry was used to measure cell populations and fluorescence-activated cell sorting to isolate alveolar macrophages. Next Generation RNA Sequencing was utilized to compare the transcriptomes of pre- and post-transplant AM. 

Results: After lung transplant or intratracheal LPS instillation, there was significant neutrophil infiltration at 24 hours (>3 fold increase, *p<0.001, Figure A&B). Following transplantation, donor AM proliferated in vivo, resulting in serial increase in AM cell counts (1.7 fold increase) and proportion of cells that had undergone cell division (1.6-fold increase). The increase in cell count was not due to infiltration by recipient AM, since all AM in the allograft remained of donor origin. Transcriptional profiling revealed that post-transplant AM upregulated genes involved in cell division and proliferation. However, genes responsible for activation of innate immunity, neutrophil recruitment, and pattern recognition receptor response were downregulated (Figure A). Depletion of AM in donor lungs prior to transplant lead to increased neutrophil infiltration following IR (Intact AM: 1.1 x 10^6 vs. Depleted AM: 1.5 x10^6, Figure A). In contrast, the robust neutrophil recruitment after intratracheal LPS was abrogated by depletion of AM (*p<0.001, Figure B).

Conclusion: Alveolar macrophages proliferate following lung transplant and downregulate genes involved in immune activation. Hence, they may play a role in ameliorating IR injury. Contrastingly, they augment the immune response and recruit neutrophils in response to PAMPs, demonstrating their role in pulmonary mucosal immunity.

 

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