29.08 Shared Decision-making for Unilateral Breast Cancer Patients Choosing between CPM and UM

Posted on January 31, 2019

J. Huang1, A. Chagpar1  1Yale University School Of Medicine,New Haven, CT, USA

Introduction:

Choosing between contralateral prophylactic mastectomy (CPM) and unilateral mastectomy (UM) is a personal decision, but the patient’s surgeon may influence this. We sought to determine how different patient-doctor communication strategies play into the decision-making process.

Methods:

Female unilateral breast cancer patients who had a mastectomy at a large academic institution were approached to participate in a survey regarding patient-physician communication and their decision between CPM and UM. Patient satisfaction with decision was measured using the 5-point Satisfaction with Decision (SWD) scale (higher = more satisfied). Non-parametric statistics were performed using SPSS version 24.

Results:

100 (91.7%) of 109 patients approached completed the survey and were included in this cohort; the median age was 49.5 years (range 29-82). 54 patients chose to undergo CPM (54%). 33 patients (33%) reported being recommended UM, 6 patients (6%) reported being recommended CPM, and 61 patients (61%) reported that their doctors employed shared decision-making (SDM), i.e., made no strong recommendation either way. The majority of patients who stated their doctors recommended UM chose UM (78.8%); similarly, 83.3% of those who stated their doctors recommended CPM chose CPM. Of the 39 patients whose doctors recommended a surgery, 8 patients (20.5%) did not follow their doctor’s advice. These patients were equally as satisfied with their decision as those who did follow their doctor’s advice (p=0.441). Compared to patients that followed their doctor’s advice, patients who did not tended to use a 2nd physician’s opinion (38.7% vs. 0%, p=0.042) as well as photos of cosmetic results (37% vs. 6.5%, p=0.049) in their decision-making process. There was no difference in age, race, education, insurance type, or income between patients who followed their doctor’s advice versus those who did not (p>0.05). Patients who reported engaging in SDM tended to choose CPM (68.3% vs. 30.8%, p<0.001). The mean SWD score of the entire cohort was 4.80 out of 5.00 (range 3.17-5.00). Patients who did not engage in SDM were similarly satisfied with their decision as those who did engage in SDM (mean SWD score 4.77 vs. 4.83, p=0.286). In terms of patient reported preferences for patient-physician communication, 12 patients (12%) preferred the doctor to provide a recommendation, 7 (7%) preferred to make the decision on their own, and 81 (81%) preferred to engage in SDM. Race, education, insurance type, income, and age did not differ between types of preferred communication strategies (p>0.05).

Conclusion:

When the physician provides an initial recommendation between UM and CPM, patients tend to follow it, while patients who engaged in SDM tend to choose CPM. While most patients state that they prefer to have physicians engage in SDM, patients were equally as satisfied with their surgical decision whether they engaged in SDM or not.

29.07 Increased APOBEC3C-H Gene Expression is Associated with Improved Outcome in Breast Cancer

Posted on January 31, 2019

M. Asaoka1,2, S. K. Patnaik1, A. L. Butash1, E. Katsuta1, T. Ishikawa2, K. Takabe1,2  1Roswell Park Cancer Institute,Surgical Oncology,Buffalo, NY, USA 2Tokyo Medical University,Department Of Breast Surgery And Oncology,Shinjuku, Tokyo, Japan

Introduction:

APOBEC enzymes are known as strong mutagenic factors, particularly in breast cancer. APOBEC3B (A3B) gene expression is significantly increased in breast cancer and associated with tumor mutation load and intra-tumor heterogeneity. The relevance of the other APOBEC3s (A3A, C-H) is not yet clear in breast cancer. Therefore, we analyzed these genes, looking at their association with survival, mutations, and immune activity.

Methods:

We collected gene expression data for primary tumors (1091) and adjacent normal tissues (113) from The Cancer Genome Atlas (TCGA). Patients were divided into 3 equal groups by gene expression to compare high and low expressors. Tumor immune features like cytolytic activity and T cell receptor (TCR) diversity were quantified from gene expression data. Data for some of these features, mutation-related aspects, and survival were obtained from TCGA publications. Gene expression data for 55 breast cancer cell-lines was from Cancer Cell Line Encyclopedia. Cox regression and Spearman methods were used for survival and correlation analyses, resp. Welch t test was used for group comparison, with P <0.05 deemed significant. Hallmark gene-sets were used for enrichment analysis.

Results:

A3B and A3C represented 91% of A3 gene expression in breast cancer cell-lines. In patients, expression of A3B was higher in tumors compared to normal tissue (4.5x), while that of A3C was lower (0.5x). A3B or A3A levels had no effect on overall (OS) or disease-specific survival (DSS). But, higher expression for each of A3C-H was significantly associated with improved OS (HR, 0.45-0.66) or DSS (0.43-0.61). The prognostic value of high A3C-H expression was validated in two gene expression meta-datasets (KMPlot and SurvExpress). A3A and A3B expression levels correlated with both tumor mutation burden and neoantigen load (ρ = 0.28-0.34), which resp. were 2.0-2.9x more in high expressors. There was no association of tumor mutation burden or neoantigen load with A3C-H. A3C-H expression levels correlated positively with both total immune cell and lymphocyte populations in tumor (ρ = 0.29-0.70 & 0.20-0.50, resp.), whereas the correlations were poor for A3B (0.10 & -0.01, resp.). Expression of genes related to immune function like interferon response and complement activation was enriched in high A3C-H expressors, which also had significantly more CD4 and CD8 T cells, and TCR diversity (2.3-4.0x, 2.1-5.4x & 1.3-2.1x, resp.). Concordantly, for each of A3C-H, expression correlated with tumor immune cytolytic activity (ρ = 0.31-0.79), which was increased 3.1-7.9x in high expressors.

Conclusion:

APOBEC3s are DNA mutators. However, unlike A3B, whose expression is associated with poor survival, increased expression of A3C-H confers a survival benefit. Further studies are warranted to explore if the increased A3C-H expression reflects a heightened anti-cancer immune response, and if A3C-H can be used as prognostic biomarkers.

29.06 Prepectoral Direct-to-Implant Breast Reconstruction: Safety Outcomes and Delineation of Risk Factors

Posted on January 31, 2019

K. P. Nealon1, R. E. Weitzman1, N. Sobti1, A. S. Colwell1, W. G. Austen1, E. C. Liao1  1Massachusetts General Hospital,Division Of Plastic And Reconstructive Surgery,Boston, MA, USA

Introduction:  Breast cancer is among the most common cancers diagnosed in women, affecting 1 in 8 women per year. Immediate implant-based breast reconstruction is the leading technique for post-mastectomy reconstruction, trending toward direct to implant (DTI) as the preferred method when compared to the traditional tissue expander method. Although implants are generally placed beneath the pectoralis major muscle, recent developments have allowed for implant placement above the muscle in a pre-pectoral plane. This study compares the safety endpoints and risk factors in prepectoral vs. subpectoral DTI breast reconstruction cohorts. We hypothesize that prepectoral DTI breast reconstruction is a safe alternative to subpectoral DTI breast reconstruction.

Methods:  Retrospective chart review at a tertiary academic medical institution identified 107 patients who underwent prepectoral DTI reconstruction and 158 patients who underwent subpectoral DTI reconstruction. Univariate analysis was performed to compare patient characteristics between both cohorts. A penalized logistic regression identified relationships between postoperative complications and covariate variables in each group.

Results: A binomial regression model revealed that prepectoral DTI breast reconstruction is associated with lower risk of surgical site infection (p = 0.011) and lower risk of revision (p = 0.015) when compared to subpectoral DTI breast reconstruction. Prepectoral DTI breast reconstruction is also associated with lower risk of capsular contracture, trending towards significance (p = 0.064). Rates of overall complication, explant, skin necrosis and hematoma were comparable between groups.

Conclusion: This study compares the safety outcomes and risk factors in prepectoral versus subpectoral DTI breast reconstruction cases. Prepectoral DTI breast reconstruction is associated with lower rates of surgical site infection, revision, and capsular contracture. It is speculated that the significant difference in surgical site infection may be due to decreased procedure time of the prepectoral procedure, or less dissection and devascularization of the soft tissue surrounding the implant. Fewer overall complications in the prepectoral group also resulted in a decreased number of revisions. Due to lack of manipulation of the pectoralis major muscle, prepectoral implant placement reported decreased rates of capsular contracture. These results demonstrate that prepectoral DTI reconstruction is a safe alternative when compared to subpectoral DTI reconstruction. 

 

29.05 Non-Discrimination Laws and Medical Necessity Criteria for Gender Affirmation Surgery

Posted on January 31, 2019

A. Almazan1, O. Ganor1, E. Boskey2  1Harvard Medical School,Boston, MA, USA 2Boston Children’s Hospital,Department Of Plastic And Oral Surgery,Boston, MA, USA

Introduction:  A recent movement to pass state-based non-discrimination laws has led to mandatory coverage for gender-affirming surgical care in many private insurance plans. However, it is unclear whether these laws have improved access to treatment for transgender patients. While more plans may cover gender affirmation surgery, restrictive medical necessity criteria used by insurers to decide reimbursement can still delay treatment or preclude access to surgery for many patients. In this study, we examine how coverage and medical necessity criteria for gender affirmation surgeries vary between states that do and do not have laws protecting coverage of gender-affirming medical care in private insurance.

 

Methods: Medical coverage guidelines for surgical treatment of gender dysphoria were taken from the websites of the largest insurer in each state by market share.

Expansiveness of coverage for each insurer was assessed by examining whether each plan offered any of the following procedures: FTM phalloplasty, MTF vaginoplasty, FTM mastectomy, MTF augmentation mammoplasty, electrolysis, and laryngoplasty.

Restrictiveness of medical necessity criteria was assessed by examining whether each plan mandated any of the following prerequisites for gender affirmation chest surgeries: proof of social transition, documentation of hormone therapy for FTM mastectomy and MTF augmentation mammoplasty, documentation of legal name change, and proof of employment/schooling/community involvement.

Each state was labeled according to whether it had a law protecting insurance coverage of gender affirmation surgery. The number of covered procedures and number of chest surgery prerequisites were compared between states that did and did not have transition-related protections.

 

Results: The mean number of procedures covered in states with legal protections is 4.81, compared to 3.92 in states without protections (t=-2.32, p=0.013). The mean number of pre-requisites for chest reconstruction surgeries in states with legal protections is 0.88, compared to 0.92 in states without protections (t=0.10, p=0.46).

 

Conclusion: Insurers in states with non-discrimination laws protecting transition-related insurance coverage tend to cover more gender-affirming surgical procedures. However, the number of requirements that must be fulfilled to deem a chest reconstruction medically necessary is effectively identical between insurers in states that do and do not have non-discrimination laws. Transition-related insurance protections are associated with enhanced coverage of gender affirmation surgery. However, they are not associated with improvements in the restrictiveness of medical necessity criteria, which may still act as a barrier to care even when coverage is nominally increased in accordance with these laws.
 

29.04 Radiation Following Autologous Breast Reconstruction – Is It Safe Practice?

Posted on January 31, 2019

L. A. Gamble1, S. Sha2, J. L. Kelly1, L. A. Jarvis4, G. L. Freed3, K. M. Rosenkranz1, C. V. Angeles1  2Stony Brook University,School Of Medicine,Stony Brook, NEW YORK, USA 3Dartmouth-Hitchcock Medical Center,Plastic Surgery,Lebanon, NEW HAMPSHIRE, USA 4Dartmouth-Hitchcock Medical Center,Radiation Oncology,Lebanon, NEW HAMPSHIRE, USA 1Dartmouth-Hitchcock Medical Center,General Surgery,Lebanon, NEW HAMPSHIRE, USA

Introduction:  The incidence of immediate breast reconstruction (IBR) following mastectomy for breast cancer has steadily been on the rise while the indications for post mastectomy radiation therapy (PMRT) have broadened. Current literature demonstrates conflicting data regarding surgical complications and timing of PMRT, while the safety of PMRT following autologous breast reconstruction (ABR) is still considered controversial. We sought to investigate the safety of PMRT in breast cancer patients who undergo ABR.

Methods:  Retrospective chart review was performed on all patients treated with mastectomy between 2000-2006 at a single, academic institution. Data collected included patient demographics, PMRT, and postoperative complications including seroma, infection, fat necrosis, and contracture documented from the time of surgery until one year post surgery. Median follow-up was 6.19 years. Chi-square analysis was performed with significance set at p <0.05.

Results: 592 patients underwent mastectomy for breast cancer treatment or prophylaxis. Only half of these patients (49%; 292/592) underwent reconstruction. The majority (83%; 240/292) received ABR, and 95% (228/240) were done at the time of mastectomy (IBR). The most common flap performed was the transverse rectus abdominis (TRAM) flap at 72.1%, followed by 24.2% latissmus dorsi (LD), and 3.7% other flaps (including superior and inferior gluteal artery perforators, and transverse upper gracilis). Of the immediate ABR patients, 57/228 received PMRT. Of these, 54% (31/57) suffered any surgical complication and 23% (13/57) were classified as Clavien-Dindo grade IIIb (CD IIIb). Comparatively, 171/228 patients did not receive PMRT with almost half (84/171) having complications, but only 26% (45/171) were classified as CD IIIb. There was no statistically significant difference in overall complication rate or CD IIIb complications between ABR patients with or without PMRT (p=0.742 and p=0.357, respectively). Additionally, we found no significant difference in overall complication rate in patients who underwent PMRT when comparing between those who had no reconstruction versus those who had ABR (p=0.0623).

Conclusion: Our data shows no statistically significant difference in the complication rate between breast cancer mastectomy patients who received PMRT after ABR versus no reconstruction.  Additionally, there is no difference in the rate of complications between ABR patients who did or did not receive PMRT. This study supports the idea that it is safe to radiate a reconstructed breast.

 

29.03 Adhering to Surgical and Oncologic Standards Improves Survival in Breast Cancer Cohorts

Posted on January 31, 2019

B. Zhao1, C. Tsai1, K. K. Hunt2, S. L. Blair1  2University Of Texas MD Anderson Cancer Center,Breast Surgical Oncology,Houston, TX, USA 1University Of California – San Diego,Surgery,San Diego, CA, USA

Introduction:
The American College of Surgeons Clinical Research Program published evidence-based surgical and oncologic standards for breast cancer in the Operative Standards for Cancer Surgery.  Recommended standards include surgical resection with negative margins, removal of all sentinel lymph nodes (SLN) and removal of >10 lymph nodes (LN) for complete axillary dissection (ALND), and the use of adjuvant therapy after surgical resection. However, the rates of adherence to these standards nationwide is unknown. 

Methods:
Using the National Cancer Database from 2004-2015, we selected distinct cohorts of breast cancer patients who underwent surgical resection: clinical T1N0M0 under age 70 (CT1), clinical T2N0M0 or T3N0M0 (CT2/3), and clinical M0, pathologic N2 or N3 (PN2/3). For CT1 and CT2/3 patients, we considered patients with negative margins, any form of adjuvant therapy, and ³2 LNs examined as meeting standards. For PN2/3 patients, we considered those with negative margins, any form of adjuvant therapy, and ³10 LNs examined as meeting standards. We compared outcomes of those who met standards versus those who did not for all cohorts. We performed Kaplan-Meier analysis with log-rank test to compare survival for patients based on achieving standards and Cox proportional hazards model for individual predictors of improved survival while controlling for patient comorbidities. 

Results:
We identified 318,853 (65.0%) CT1 patients, 164,593 (67.3%) CT2/3 patients, and 77,626 (67.7%) PN2/3 patients who met surgical and oncologic standards. Survival data is shown in the table. For PN2/3 patients, the median survival for those who met standards was significantly longer than those who did not meet standards (109.34 months versus 72.97, p<0.001). Patients were significantly more likely to meet standards if they were treated at an academic center (p<0.001 for all cohorts). For CT1 and CT2/3 patients, ³2 LNs examined, endocrine therapy, radiation therapy, and negative margins were predictors of improved survival. For CT1 patients, chemotherapy was a predictor of worse survival, but was a predictor of improved survival in CT2/3 patients. For PN2/3 patients, ³10 LNs examined, endocrine therapy, chemotherapy, radiation therapy, and negative margins were predictors of improved survival. 

Conclusion:
Approximately a third of patients are not receiving evidence-based minimal standards as part of their surgical and oncologic treatment for breast cancer.  Adhering to surgical and oncologic standards improves survival in CT1, CT2/3, and PN2/3 breast cancer patients.  Efforts to improve knowledge of, and adherence to, these surgical and oncologic standards should be emphasized. 
 

29.02 Underinsurance and Healthcare Utilization among Working-Age Breast Cancer Patients

Posted on January 31, 2019

S. Obeng-Gyasi1, L. Timsina1, O. Bhattacharyya3, S. E. Severance1, C. S. Fisher1, D. A. Haggstrom2  1Indiana University School Of Medicine,Department Of Surgery,Indianapolis, IN, USA 2VA HSR&D Center for Health Information and Communication,Indianapolis, IN, USA 3Indiana University Purdue University,Department Of Economics,Indianapolis, IN, USA

Introduction: Breast cancer is the most common female cancer in the United States. For working-age patients, a cancer diagnosis can be financially devastating secondary to disease related reduction in work productivity, loss of employment, and subsequent increased economic burden. The objective of this study is to understand out-of-pocket costs (OOP), health care utilization costs (outpatient visits, office-based visits, ambulatory care, prescription medication cost), and the rate of underinsurance among working age breast cancer patients. 

Methods: The study data was obtained using the Medical Expenditure Panel Survey data from 2008-2012.  Self-responding patients ages 18-64 with an age at diagnosis of breast cancer within two years of the survey interviews were included. The data was divided into three groups based on insurance:  private, Medicaid, and other public.  The other public includes patients with Non-Medicaid state or local insurance or other federal programs. Bivariate intergroup analysis was conducted. A multivariable logistic regression model tested variables associated with underinsurance. Underinsurance was defined as spending at least 10% of the household income on breast cancer related OOP.

Results:The study cohort included 14,586 patients. The groups differed significantly by marital status (p=0.004), race/ethnicity (p=0.0002), education (P <0.0001), percent below the poverty level (p<0.0001), family income (P <0.0001) and employment (P<0.0001).   Mean total annual OOP costs were $2006.0 (95% CI 1705.5, 2305.5) for the privately insured, $991.0 (95% CI -160.1, 2142.3) for Medicaid, and $7420.0 (95% CI 1722.8, 13117) for other public insurance. The majority of OOP cost were on prescriptions, $706.0 (95% CI 557.7, 854.6), and office-based visits, $779. 0(95% CI 641.7, 916.3). Patients with other public insurance spent the most OOP costs on prescriptions $3258.0 (95% CI 2047.2, 4467.8) and office-based visits $3258.0 (95% CI 2047.2, 4467,8). Being divorced (OR 5.6, p=0.029), living in the Midwest (OR 18.6, p=0.001) or South (OR 7.49 p=0.015) compared to the Northwest and having other public insurance (OR 12.2, p=0.012) were all associated with an increased rate of underinsurance. Conversely, employment (OR 0.21, p=0.011) and having Medicaid (OR 0.09, p= 0.006) were associated with a reduced rate of underinsurance.

Conclusion:Breast cancer patients spend most of their OOP costs on prescriptions and office-based visits. Since Medicaid was protective against underinsurance and higher OOP costs, future longitudinal studies should monitor whether Medicaid policy changes continue to reduce the economic vulnerability among cancer patients. Fifteen states in the South and Midwest have not expanded Medicaid, and this public policy decision appears to expose breast cancer patients to substantially greater financial burdens. Medicaid expansion should be considered to mitigate financial burden among working age women with breast cancer.

 

29.01 Impact of Global Migration on Asian Breast Cancer: A Comparison between US and Taiwan

Posted on January 31, 2019

J. Wu1,2, Y. Hung2, S. M. Stapleton2, Y. Hsu2, S. T. Oseni2, C. Huang1, D. C. Chang2  1National Taiwan University Hospital,Surgery,Taipei, Taiwan 2Massachusetts General Hospital,Surgery,Boston, MA, USA

Introduction:

More than half of the Asian Americans with breast cancer were born in Asia, however it is unknown whether their disease patterns are different from Asians born in the US. We hypothesize that nativity status may have an impact on the onset and the presentation of breast cancer in the Asian population.

Methods:

A retrospective analysis was performed for Asian females³ 20 years old in the US Surveillance, Epidemiology, and End Results (SEER) Program database, and as a convenience sample from Asia, in the Taiwan Cancer Registry (TCR) for the years 2004-2010. The primary end point was proportion of patients who had early-onset breast cancer, defined as breast cancer age at onset before 50. Secondary outcome was the proportion of advanced breast cancers, defined by American Joint Committee on Cancer staging III to IV. Three groups of patients were compared: Native Asian in Taiwan (TW), Asia-born Asian American (AAA), and US-born Asian American (USAA).

Results:

We identified 13,404 patients (2,743 USAA & 10,661 AAA) in SEER and 49,322 TW in TCR. TW presented at an earlier age than AAA (median age 51vs 56) and USAA (median ­­­­­­61). TW had the highest proportion of early-onset breast cancer (44.3% vs 31.7% AAA and 23.7% USAA, p < 0.001). In addition, both TW and AAA had significantly higher rates of advanced cancer at presentation than USAA (22.8% and 17.2% vs 13.8%, respectively, p < 0.001).

Conclusions:

Recent immigrants to the US may be at increased risk of earlier and more aggressive breast cancers. Future studies should determine whether these differences are due to biomedical factors, access to healthcare, or poor healthcare quality affecting immigrant communities. The impact of immigration on health and disease remains an under-appreciated but important way through which we can further understand the interaction between social and biomedical factors on disease onset and progression.

28.10 EphB4 regulates mechanical properties of mouse arteriovenous fistulae

Posted on January 31, 2019

S. Lee1, K. Brownson1, R. Khosravi1, K. Goldstein1, T. Isaji1, J. Humphrey1, A. Dardik1  1Yale University School Of Medicine,New Haven, CT, USA

Introduction:
Veins are typically thin-walled and compliant at low pressures, optimal for their dual roles as conduits for blood returning to the heart and reservoirs for holding most of the blood volume. Surgically connecting a vein to the arterial system as an arteriovenous fistula (AVF) exposes the vein to higher pressure, flow magnitudes and frequencies, triggering a set of molecular pathways that result in venous remodeling, such as dilatation and thickening.  Recent work showed that the venous identity marker EphB4 is upregulated and required for the venous remodeling that occurs during AVF maturation.  However, it is not known how EphB4 function or fistula creation affects the mechanical properties of veins; we hypothesize that increased EphB4 function during fistula remodeling enhances venous compliance.

Methods:
C57BL/6 wild type (WT) and Ephb4+/- (heterozygous) mice were treated with sham surgery or abdominal aortocaval fistulae creation via needle puncture (n=4-6 per group). The thoracic IVC were harvested at post-operative day 21 for uniaxial mechanical testing and subsequent histology. Veins were axially stretched to their in vivo length, then cyclically distended from 1 to ~20 mmHg with phosphate buffered saline while simultaneously recording outer diameter with a side-mounted video camera. Compliance was calculated as the change in cross-sectional luminal area per unit pressure. Veins were sectioned at 5 μm with wall (intima-media) thickness measured manually. Statistical analyses were performed with one-way ANOVA, using the Tukey post-hoc test for multiple comparisons.

Results:
WT AVF distended to a greater maximal diameter compared to WT control veins (1290±35μm vs. 986±55μm; p = 0.04). Similarly, Ephb4+/- AVF distended to a greater diameter compared to Ephb4+/- control veins (1231±75μm vs. 878±77μm, p = 0.007). At physiologic venous pressures (0-5 mmHg), Ephb4+/- veins were less compliant than WT veins (<ΔCWT,EphB4> = -0.05 mm2/mmHg, p = 0.015). Veins became more compliant with fistula creation in both WT (<ΔCWT,F> = 0.17 mm2/mmHg, p = 0.01) and Ephb4+/- (<ΔCEphB4,F> = 0.12 mm2/mmHg, p = 0.006) mice, with Ephb4+/- fistulae remaining less compliant than WT fistulae (<ΔCEphB4F,WTF> = 0.1 mm2/mmHg, p = 0.01). Ephb4+/- veins were significantly thicker than WT veins with (73.2±5.9μm vs 41.9±1.6μm, p = 0.03) or without (81.5±12μm vs 46.3±4.5μm, p = 0.01) fistula creation. 

Conclusion:
Although creation of an AVF results in more distensible and circumferentially compliant veins in both WT and Ephb4+/- mice, veins from Ephb4+/- mice are thicker and stiffer than veins in WT mice both at baseline and after AVF creation.  These results suggest that the structural changes of EphB4-regulated venous remodeling are accompanied by functional changes that support venous adaptation to the fistula environment.

28.09 The Inhibition of Wnt Signaling Attenuates RANKL-induced Osteoclastogenic Macrophage Activation

Posted on January 31, 2019

K. Igari1, M. J. Kelly1, B. Darwich1, D. Yamanouchi1  1University Of Wisconsin,Division Of Vascular Surgery, Department Of Surgery,Madison, WI, USA

Introduction:
We have previously reported the role of osteoclastogenic macrophage activation in abdominal aortic aneurysms (AAAs). Previous reports indicated Wnt signaling has the dual effect of proliferation and differentiation during osteoclatogenesis. Wnt/β-Catenin pathway is a critical regulator of cell pluripotency, cell survival, and cell fate decision in both embryos and adults. The inhibition of β-catenin suppressed proliferation but induced differentiation of osteoclast precursor cells. The aim of this study is to examine the effect of the Wnt signaling inhibitor, ICG-001, under the hypothesis that ICG-001 inhibits osteoclastogenesis through the inhibition of proliferation without induction of differentiation.

Methods:
RAW 264.7 macrophages were stimulated with soluble receptor activator of NF-kB ligand (RANKL) (30ng/ml) to induce the osteoclastogenesis. To examine the effect of the inhibition of the CBP co-activator in Wnt signaling, macrophages were treated with or without ICG-001 (10mM) during RANKL stimulation. The activation and differentiation of macrophages were examined by western blotting, quantitative PCR, and tartrate-resistant acid phosphate (TRAP) staining in vitro. Data are reported as the means ± standard deviation. P values less than 0.05 were accepted as statistically significant.

Results:
The relative expression level of protein of nuclear factor of activated T-cells cytoplasmic 1, a key transcription factor during osteoclastogeneis, was significantly suppressed by ICG-001 treatment compared to the non-treated group (4.46±0.74 versus 7.13±0.50, P < 0.05). Even though there was not a statistically significant difference in TRAP expression between the ICG-001 treated group and non-treated group (1.78±0.55 versus 2.35±0.38, P = 0.24), we showed the trend that ICG-001 decreased TRAP protein expression. Furthermore, the expression of cathepsin K was significantly suppressed in the ICG-001 treated group compared to the non-treated group (4.62±1.60 versus 12.2±1.73, P < 0.05). The relative expression levels of mRNA of TRAP, cathepsin K, and matrix metalloproteinase-9, were significantly lower in the ICG-001 treated group compared to the non-treated group (72.4±5.3 versus 167.1±5.6, P < 0.05, 38.6±7.2 versus 149.3±14.3, P < 0.05, and 175.5±10.5 versus 323.6±70.0, P < 0.05, respectively). Furthermore, TRAP-staining demonstrated the suppressive effect of ICG-001 on osteoclastogenesis. The number of TRAP-positive decreased in the ICG-001 treated group relative to the non-treated group (24.4±7.0 versus 131.2±19.4, P < 0.05).

Conclusion:
The inhibition of Wnt signaling pathway via ICG-001 suppressed osteoclastogenic macrophage activation. Our previous studies showed the importance of osteoclastogenic macrophage activation in AAA, therefore, further studies to examine the therapeutic potential of ICG-001 on AAA are warranted.
 

28.08 The Pro-Resolving Lipid Mediator Maresin 1 (MaR1) Attenuates Abdominal Aortic Aneurysm Formation

Posted on January 31, 2019

C. T. Elder1, G. Lu1, G. Su1, A. K. Sharma1, A. Mast1, G. R. Upchurch1  1University Of Florida,Department Of Surgery,Gainesville, FL, USA

Introduction:  Formation of abdominal aortic aneurysms (AAA) is a multifactorial process and is characterized by inflammation of the aortic wall. Maresin 1 (MaR1) is an endogenous pro-resolving lipid mediator derived from docosahexanoic acid, an ω-3 polyunsaturated fatty acid, and is involved in the resolution phase of acute inflammation. We hypothesized that treatment with exogenous MaR1 would attenuate experimental murine AAA formation.

Methods:  Abdominal aortic aneurysms were induced in C57BL/6 (wild-type; WT) mice (n=4 per group) using an established topical elastase model. Mice were treated with MaR1 (100 ng/mouse for each dose) or vehicle via intraperitoneal injection on days 1, 3, 5, and 7 post AAA induction. On day 14, abdominal aortas were harvested for phenotypic evaluation. 

Results:  Mean abdominal aortic dilation was 131% ± 16 for vehicle treated mice as compared to 88% ± 7 for MaR1 treated mice (p = 0.003). Decreased inflammatory changes were noted on gross phenotypic examination for MaR1 treated mice as compared to vehicle treated mice. 

Conclusion:  The present results demonstrate that systemic administration of MaR1 attenuates abdominal aortic aneurysm formation in mice. Decreased inflammatory phenotypic changes suggest this attenuation is mediated through the pro-resolving effects of MaR1 and represent a potential future target in the treatment of AAA. 

 

28.07 CCR2 Targeted PET Imaging of Rodent Abdominal Aortic Aneurysms Stimulated to Rupture

Posted on January 31, 2019

S. E. Sastriques Dunlop1, S. J. English1  1Washington University,Surgery,St. Louis, MO, USA

Introduction:  Abdominal aortic aneurysm (AAA) development is common in the aging population, and AAA rupture is associated with high mortality. Indications for surgical intervention and AAA rupture prediction rely on antiquated aortic diameter criteria. AAA development is characterized by destructive remodeling of the aortic extra cellular matrix, in part by mononuclear phagocytes. Previous work suggests that the monocyte chemoattractant protein (MCP-1)/C-C chemokine receptor type 2 (CCR2) axis plays an important role in AAA development. We sought to evaluate if increased rat AAA uptake of 64Cu-DOTA-ECL1i, a radiolabeled CCR2 binding peptide under current clinical evaluation, by positron emission tomography (PET) imaging, is predictive of subsequent AAA rupture.

Methods:  ECL1i was conjugated to a DOTA chelator and radiolabeled with 64Cu. 64Cu-DOTA-ECL1i biodistribution analysis was performed in male Sprague-Dawley (SD) rats (N = 4). Utilizing the porcine pancreatic elastase (PPE) exposure model, infrarenal abdominal aortas of male SD rats were exposed to active PPE to induce AAA formation, and each animal received b-aminopropionitrile (BAPN, N = 8) to stimulate AAA rupture. PET imaging was performed at 6 days post AAA induction, and AAA uptake was quantified by mean standardized uptake values (SUVmean). AAA radiotracer uptake was confirmed by ex vivo autoradiography after PET imaging. Immunohistochemistry and RT-PCR were also utilized to identify CCR2 in AAA tissue.  

Results: Biodistribution analysis demonstrated rapid renal clearance of 64Cu-DOTA-ECL1i, with less than 0.5% ID/g in blood 1 hour post intravenous via tail vein injection. PET imaging demonstrated tracer uptake by AAAs that subsequently ruptured (RAAA, N = 5), as well as those AAAs that did not rupture (NRAAA, N = 3). See Figure. RAAAs demonstrated greater 64Cu-DOTA-ECL1i uptake (SUVmean 0.75±0.05) than NRAAAs (SUV mean 0.67 ± 0.05) (p = 0.07). Ex vivo autoradiography confirmed 64Cu-DOTA-ECL1i uptake in each case; while immunohistochemistry and RT-PCR demonstrated upregulation of the CCR2 receptor in each case as well.

Conclusion: CCR2 targeted 64Cu-DOTA-ECL1i PET imaging demonstrated inflammation associated with rat AAA development. Increased radiotracer uptake by AAAs that subsequently ruptured warrants further study to determine the ability of 64Cu-DOTA-ECL1i PET imaging to aid in assessing AAA rupture potential associated with our model, as well as for patients.

 

28.06 Nitric oxide in extracorporeal circulation preserves platelet function as measured by microparticles

Posted on January 31, 2019

T. R. Bellomo1, M. Jeakle1, T. Major1, M. Meyerhoff2, R. H. Bartlett1  1University Of Michigan,Department Of Surgery,Ann Arbor, MI, USA 2University Of Michigan,Department Of Chemistry,Ann Arbor, MI, USA

Background: Clotting, anticoagulation, platelet consumption, and poor platelet function are major factors in clinical extracorporeal circulation (ECC). We have shown that Nitric Oxide releasing coatings (NOReL) prevents thrombosis in a rabbit model of ECC without systemic anticoagulation. NOReL prevents platelet adhesion and activation, resulting in preserved platelet count and function. Previous work has shown that activated platelets form platelet-derived microparticles (PMPs). These experiments were designed to determine if PMPs can identify platelet function during ECC.

Objective: The objective of this study is to investigate the effects of NOReL on platelet activation and PMP formation during ECC.

Methods: Uncoated and NOReL coated ECCs were tested in a 4-hour rabbit thrombogenicity model without systemic anticoagulation. Before and after ECC exposure, platelets were stimulated with collagen and PMPs were measured using flow cytometry. 

Results: The uncoated ECCs clotted within the first hour, while the NOReL coated ECCs circulated for 4 hours. Pre ECC exposure, platelets stimulated with collagen produced PMPs. Post ECC exposure, platelets from uncoated circuits generated less PMPs than baseline (186 ± 123 uncoated baseline vs.-131 ± 311 uncoated post circuit) when stimulated with collagen.  However, platelets from the NOReL circuit generated the same amount of PMPs as baseline values (74 ± 61 NOReL baseline vs. 56 ± 118 NOReL post circuit).

Conclusions: Blood exposure during ECC results in platelet activation and clotting. The remaining circulating platelets have lost function, as demonstrated by the low PMP formation in response to collagen. NOReL coated ECCs prevent platelet activation and clotting. In addition, function of the circulating platelets was preserved, as demonstrated by PMP formation in response to collagen. These results indicate that PMPs may be an important measure of platelet activation during ECC. PMPs may provide a simplified way to measure platelet function during clinical ECC.

28.05 Rapamycin Improves Adaptive Venous Remodeling and Decreases Arteriovenous Fistula Wall Thickening

Posted on January 31, 2019

A. Fereydooni1,2, X. Guo2,3, H. Hu2, T. Isaji2, N. Nassiri4, L. Zhang3, A. Dardik2,4  1Howard Hughes Medical Institute,Chevy Chase, MD, USA 2Vascular Biology And Therapeutics Program,Yale School Of Medicine,NEW HAVEN, CT, USA 3Renji Hospital, Shanghai Jiaotong University,Department Of Vascular Surgery,Shanghai, SHANGHAI, China 4Yale University School Of Medicine,Department Of Surgery, Section Of Vascular And Endovascular Surgery,New Haven, CT, USA

Introduction: Arteriovenous fistulae (AVF) continue to be the most common access created for hemodialysis, but up to 50% of AVFs fail to mature, suggesting a need to improve AVF maturation. In a mouse model, Akt1 expression increases during AVF maturation and reduced Akt1 expression in vivo reduces fistula wall thickness and diameter and improves long-term patency.  Mammalian target of rapamycin (mTOR) is a key regulatory protein that integrates signals from the Akt pathway to coordinate cell growth and proliferation. We hypothesized that inhibition of the Akt1-mTORC1 axis reduces pathologic venous remodeling that is associated with failure of AVF maturation.

Methods:  A C57BL6/J mouse aortocaval fistula model was used (male, 9–12 weeks). Mice were injected with 0 or 100 μg of rapamycin (intraperitoneal) daily.  The AVF (venous limb) of control- and rapamycin-injected mice were harvested at days 0, 3, 7 and 21 and for comparison analysis.  Post-operative vessel remodeling was assessed using serial ultrasound measurements of the AVF diameter and computer morphometry to measure vessel wall thickness.  AVF were compared for leukocyte, M1 and M2 macrophage surface markers and expression level of Akt1 signaling proteins using Western blot and immunofluorescence (IF) intensity.

Results: Rapamycin reduced AVF wall thickness (day 3, 4.4 μm vs 7.6 μm; day 7, 4.7 μm vs 17.8 μm; day 21, 6.2 μm vs 42.2 μm; p<0.01; n=4), without any change in AVF diameter (1-11% reduction in relative diameter; p>0.5 for day 21; n=6).  Rapamycin decreased PCNA expression (day 3 and 7, p< 0.05; n=3), but did not increase cleaved caspase-3 expression (day 3, 7, and 21 p>0.05; n=3) in AVF.  Deposition of collagen I, collagen III and fibronectin also decreased in AVF of rapamycin-treated mice, compared to control mice (41-63% reduction in IF intensity of all three markers at day 21, p< 0.05 for collagen I and III day 7 and 21; n=4; p< 0.01 for fibronectin day 3, 7 and 21; n=5).  Rapamycin treatment was associated with diminished phosphorylation of the mTORC1 pathway: Akt1, 4EBP1 and p70S6K (p<0.001; n=5-7), but not of the mTORC2 pathway: PKC-α  and SGK1 (p>0.4; n=4).  Both leukocyte CD45+ and macrophage CD68+ protein expression increased in AVF compared to sham-operated vein (days 3, 7 and 21; p<0.05).  Macrophage depletion with clodronate liposomes reduced AVF wall thickness compared to control veins (p< 0.01, day 21; n=3). Rapamycin also reduced macrophage CD68+ protein expression as well as both M1 and M2 macrophage activity in AVF (iNOS, TNF-α, IL-10 and CD206, day 7, p<0.04; n=4).

Conclusion: Rapamycin reduces inflammation and wall thickening during AVF maturation through the Akt1-mTORC1 signaling pathway.  Rapamycin may be a translational strategy to improve AVF patency.

 

28.04 The Critical Role of Autophagy and Mitochondrial Remodeling in Vascular Tissue Engineering

Posted on January 31, 2019

K. E. Hekman1, C. He2, J. A. Wertheim1  1Feinberg School Of Medicine – Northwestern University,Department Of Surgery,Chicago, IL, USA 2Feinberg School Of Medicine – Northwestern University,Department Of Cell And Molecular Biology,Chicago, IL, USA

Introduction:
Vascular tissue derived from patient-specific induced pluripotent stem cells (iPSCs) suffers from premature replicative senescence, creating a significant barrier to the advancement of organ and tissue bio-engineering. Viral overexpression of longevity gene sirtuin1 as well as nutrient starvation each attenuate this premature senescence. Both are known mediators of autophagy, which is the process of cellular self-digestion that recycles intracellular components. Selective autophagy of the mitochondria, i.e., mitophagy, regulates accumulation of dysfunctional mitochondria which contribute to endothelial cell (EC) senescence. The mechanisms of premature senescence in reprogrammed iPSC-derived ECs are currently unknown.

Methods:
The iPSC lines ACS1028 and Y6 were subjected to directed differentiation and acquired endothelial lineage markers over 6 days. Cells were then purified by positive selection for VE-cadherin. Autophagy activity was monitored by quantifying the key autophagy protein, microtubule-associated proteins 1A/1B light chain 3B (LC3), through Western blotting. Mitochondrial mass, morphology and membrane potential were evaluated with MitoTracker staining. Endothelial cell function was assessed through light microscopy and quantification of nitric oxide production using the 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate substrate.

Results:
During directed differentiation from iPSCs to ECs, mitochondrial morphology evolved from globular to filamentous, and the membrane potential per mass decreased >50% (p=0.09 ACS1028, p<0.05 Y6). LC3 expression decreased by >40% (p<0.05 Y6), which correlated with increased mitochondrial mass on day 3. Mature iPSC-derived ECs had minimal autophagy activity within 5 days after purification, over which time nitric oxide production also declined by 60% (p<0.05). These iPSC-derived ECs senesced by day 13 after purification.

Conclusion:
The evolution of mitochondrial morphology and membrane potential represent remodeling during EC differentiation from iPSCs. The inverse correlation between mitochondrial mass and autophagy activity suggests this process is mediated by mitophagy. Mature iPSC-derived ECs demonstrate a significant decline in autophagy activity that correlates with the loss of mature EC function, evidenced by decreased nitric oxide synthesis, which precedes the onset of premature replicative senescence. This supports the role of mitophagy in mediating cellular senescence of iPSC-derived ECs, and renders autophagy induction a target for attenuating senescence. Overcoming this premature senescence would enable a more durable supply of patient-specific ECs for diagnostic and therapeutic applications in a wide range of disease states.
 

28.03 Targeted Spatiotemporal Delivery of Peptide Amphiphile to the Vasculature

Posted on January 31, 2019

H. A. Kassam1, C. Gillis1, N. Tsihlis1, S. Stupp2, M. Kibbe1  1University Of North Carolina At Chapel Hill,Surgery,Chapel Hill, NC, USA 2Northwestern University,Chicago, IL, USA

Introduction:   The aim of this study is to develop and evaluate a novel, systemically delivered, targeted therapy that will prevent restenosis following all cardiovascular interventions.  Increased fractalkine (CX3CL1) levels have been seen in atherosclerotic arteries; however, limited studies have demonstrated presence in injured vessels.  Recently, our lab discovered that fractalkine levels are increased after arterial injury.  Thus, for this study we identified a unique peptide sequence that binds to fractalkine with the goal of synthesizing a self-assembled peptide amphiphile (PA) nanofiber targeted to fractalkine after arterial injury.  In addition, our goal was to determine the optimal dose of the fractalkine-targeted nanofiber to bind to the site of injury, along with binding duration of the targeted nanofiber to the site of arterial injury.

Methods:   The fractalkine-targeted PA (C16-VVAASFPELDLENFEYDDSAEA) nanofiber containing a fluorescent TAMRA tag was synthesized using solid-phase peptide synthesis and purified by reversed-phase high-pressure liquid chromatography (HPLC).  Nanofiber formation was assessed via transmission electron microscopy (TEM).  Controls included a PA without the binding sequence (backbone PA, C16-VVAAK), a lower charged fractalkine PA (C16-VVAASFPELDLQNFQYNNSAEA), and injury alone.  Male Sprague Dawley rats (250-300g) underwent the carotid artery balloon injury model followed by intravenous injection of the PA (0.5-5.0 mg) 24 hours after injury.  Arteries were harvested after 5 hours of circulation time (n=3/group).  To assess binding duration, balloon-injured rats received intravenous injections of fractalkine PA (5 mg) 24 hours after injury and arteries were harvested after 5, 24, 48, and 72 hours of circulation time (n=3/group).  Harvested carotid arteries were frozen, cross-sectioned at 5 microns thick, and imaged for fluorescence.

Results:  HPLC confirmed PAs were >95% pure, and TEM showed PAs formed nanofibers.  Animals whose carotid arteries were injured and underwent injection of the fractalkine-targeted PA showed fluorescence at the site of injury in the area of the arterial media.  In contrast, no binding was observed with the backbone PA or lower charged fractalkine PA.  No significant fluorescent signal was detected in the uninjured arteries.  We observed binding of fractalkine-targeted PA at doses as low as 0.5 mg.  Furthermore, PA binding was seen at the injured site for up to 48 hours after injection. 

Conclusion:  We have demonstrated specific binding of our fractalkine-targeted PA nanofiber to the site of arterial injury compared to control PA nanofibers, with binding duration observed up to 48 hours after injection and at doses as low as 0.5 mg.  This research serves as the foundation upon which a targeted, drug-eluting therapy to prevent restenosis, and possibly prevent atherosclerosis, will be evaluated.

 

28.01 Endothelial CEPT1 Promotes Aortic Atherogenesis

Posted on January 31, 2019

C. Yang1,2, L. Belaygorod1, C. Feng3, C. F. Semenkovich3, M. Zayed1  1Washington University,Section Of Vascular Surgery,Department Of Surgery, Washington University School Of Medicine,St. Louis, MO, USA 2Huazhong University Of Science And Technology,Department Of Vascular Surgery,Union Hospital, Tongji Medical College,Wuhan, Hubei, China 3Washington University,Division Of Endocrinology, Metabolism, And Lipid Research, Department Of Medicine,,St. Louis, MO, USA

Introduction: Intracellular lipid metabolism is essential for endothelial cell activation and function. However, it is unknown to what extent this affects atheroprogression. We recently observed that choline ethanolamine phosphotransferase (CEPT1), a central enzyme in phospholipogenesis, is upregulated in peripheral arterial plaque of patients with clinical risk factors such as diabetes. In these patients, plaque phospholipid profiles were also reflective of increased CEPT1 activity. We hypothesize that CEPT1 may be an important EC regulator of plaque progression. To test this, we evaluated aortic atheroprogression in an adult apoe-/- mice following selective conditional endothelial knockdown of cept1.

Methods: Tamoxifen-induced VE-Cadherin driven cre expression and recombination of Loxp (Lp) flanked exon3 of the cept1 was used to induce selective gene knockdown in the endothelium of adult apoe-/- mice (cept1Lp/Lp apoe-/-). Six week old apoe-/- and cept1Lp/Lp apoe-/- mice were maintained on a western diet (42% fat content) for 12 weeks. Aortic atheroprogression was then evaluated and quantified following Oil red O staining. 

Results:Whole mount aortic staining revealed that cept1Lp/Lp apoe-/- mice had markedly reduced aortic plaque volumes, particularly in the areas of high sheer stress such as the aortic arch (p < 0.05; Fig1A) and infrarenal aorta (p < 0.05; Fig1B). Histologic analysis of the aortic root intima also revealed significantly reduced Oil red O staining in cept1Lp/Lp apoe-/- mice (p < 0.05; Fig1C).

Conclusion:Conditional knockdown of cept1 in the endothelium of adult apoe-/- mice is atheroprotective. This novel observation demonstrates the critical role of CEPT1 in plaque progression, and invites further investigation regarding its potential therapeutic targeting to reduce atheroprogression.
 

27.10 TLR4 Inhibition Attenuates TBI-Induced Neuroinflammation and Improves Cognitive Outcomes.

Posted on January 31, 2019

Y. H. Chun1, J. Alonso-Escalante3, B. P. Soares2, W. Fulton1, C. P. Sodhi11, D. J. Hackam1, I. W. Nasr1  1Johns Hopkins University School Of Medicine,Pediatric Surgery,Baltimore, MD, USA 2Johns Hopkins University School Of Medicine,Radiology,Baltimore, MD, USA 3Allegheny General Hospital,General Surgery,Pittsburgh, PA, USA

Introduction:

TBI induces a robust neuroinflammatory response that activates the innate immune system. We sought to investigate the effect of pharmacologically inhibiting the innate immune signaling pathways using a novel TLR4 inhibitor, C34 in a murine TBI model.

Methods:
A murine controlled cortical impact TBI model was used. Three experimental groups: (1)wild-type (WT) control, (2)WT+C34 (1-day treatment), (3)WT+C34 (7-day treatment).  MRI measured lesion volume. Real-time PCR quantified cytokine gene expression. Flow Cytometry assessed microglial and monocyte activation. Behavioral testing was initiated on post-injury day (PID)7 and 28. Statistical analysis: Student’s T-test and One-way ANOVA.

Results:
Brain injury volumes in WT (2.1±1.687mm3, n=11) compared to WT with 7-day C34  treatment (3.2±2mm3, n=7) were not significantly different, However, the 1-day treatment with C34 (4.5±1.1mm3, n=7) had significantly larger lesion volumes on MRI on PID 7.  On PID35, MRI showed no significant difference when C34 was given for 1 day; but lesion size is significantly increased when C34 was given over 7 days (p=0.01). The WT+C34 group had significantly decreased pro-inflammatory cytokines (TNFα, p=0.0010, IL-1, p=0.03) and anti-inflammatory cytokines (IL-10 p=0.0067, TGFβp=0.004) compared to WTs on PID 1.  Apoptosis markers, Caspase (p=0.0001), Bad (p=0.0001) and Bax (p=0.02) were significantly downregulated in the treatment group on PID1; as were necroptosis markers MLKL (p=0.02), RIPK1 (p=0.0001), and RIPK3 (p=0.0001). There was no difference in gene expression on PID7 or PID35. Behavioral testing (water-maze) showed that the 1-day treatment group showed significant improvement in latency during week 1 testing and platform entries on week 4 testing (p=0.026).  Flow Cytometry showed that infiltrating monocytes are significantly increased with C34 1-day treatment on PID 7 (p=0.0007). A difference in monocyte to microglial population ratio was detected only on PID 7 and not on PID 1 nor on 35.

Conclusion:
Our findings demonstrate that TLR4 plays a role in the early pathogenesis of the TBI-induced neuroinflammatory response. Early, short-term pharmacologic inhibition of the TLR4 pathway with C34 attenuates the inflammatory response and leads to improved neurocognitive outcomes compared to the prolonged 7-day treatment.  Moreover, the increase in infiltrated monocyte population may play an important role in neurorecovery after TBI.

27.09 Expression of Regulatory microRNAs in Persistent Injury-Associated Anemia

Posted on January 31, 2019

C. G. Apple1, E. S. Miller1, J. A. Stortz1, J. C. Mira1, M. K. Hollen1, T. J. Loftus1, M. Lopez1, Z. Wang1, K. B. Kannan1, D. C. Nacionales1, C. R. Cogle1, H. K. Parvataneni1, K. K. Sadasivan1, M. Patrick1, J. E. Hagen1, S. Brakenridge1, F. A. Moore1, H. V. Baker1, L. L. Moldawer1, P. A. Efron1, A. M. Mohr1  1University of Florida,Department Of Surgery,Gainesville, FL, USA

Introduction: Previous data has shown that severe traumatic injury is associated with bone marrow dysfunction which manifests as persistent injury-associated anemia. MicroRNAs (miR) have been emerging as critical regulators of hematopoieisis, and several miRs have been specifically linked to the regulation of erythropoiesis. We sought to identify whether the expression of any erythropoiesis-related miRs was altered in trauma patients to determine if these miRs play a role in persistent injury-associated anemia.

Methods: Bone marrow (BM) was collected intraoperatively from severely injured trauma patients [ISS≥15 or hemorrhagic shock (lactate≥2, base deficit≥5, MAP≤65)] who underwent fracture fixation as well as age-matched controls. There were 27 trauma patients and 16 controls analyzed. Total RNA & miR were isolated from CD34 positive cells using the RNeasy Plus Mini kit, and genome-wide and miR expression patterns were calculated with a log2 transformed expression matrix using RMA. Genes with significant expression differences (fold expression changes over age-matched control) were found using BRB Array Tool (p<0.001, t-test). Significance p<0.005 trauma vs controls by two-tailed unpaired t-test.

Results: There were marked differences in expression of 60 miRs in the trauma group when compared to age-matched controls when using a p<0.005. Two of these miR play a role in regulating erythropoiesis. miR 223, associated with suppression of erythrocytic differentiation, was found to be upregulated 1.9 fold in the trauma patients as compared to controls (p<0.0005). miR 150-3p, associated with diversion of stem cells towards megakaryocytes rather than erythrocyte differentiation, was found to be upregulated 1.8 fold in the trauma patients as compared to controls (p<0.0009). Hemoglobin on day of surgery was 10.2±1.89 g/dL in trauma compared to 13.8±2.31 g/dL in controls.

Conclusion: Severe blunt trauma is associated with persistent injury-associated anemia and this study identifies two miRs that were significantly upregulated in their expression when compared to age-matched controls. Further studies are needed to target these miRs and mitigate their downstream effects, thereby improving the anemia seen following severe traumatic injury. 

27.08 Low Blood Flow, not Hypoxemia, Stimulates Systemic TPA Release

Posted on January 31, 2019

N. G. Vigneshwar1, E. E. Moore2, G. R. Stettler1, G. R. Nunns1, M. Fragoso2, J. M. Samuels1, M. G. Bartley1, J. R. Coleman1, A. Sauaia1, A. Banerjee1, C. C. Silliman3  1University Of Colorado Denver,Surgery,Aurora, CO, USA 2Denver Health Medical Center,Surgery,Denver, CO, USA 3Children’s Hospital Colorado,Hematology,Aurora, CO, USA

Introduction: Hyperfibrinolysis, driven by tissue plasminogen activator (tPA) and associated with shock, is a major contributor to trauma-induced coagulopathy. The mechanism by which shock stimulates tPA release is unknown. Shock leads to both decreased flow and O2 carrying capacity to a tissue bed. We hypothesize that maintained flow rates to the liver do not induce early release of tPA in a model of systemic hypoxemia.

Methods: There were four groups of male rats: control (n=4), shock (n=6), 50% plasma replacement (PR) (n=5), and 75% PR (n=2) to simulate a model of decreased oxygen carrying capacity with maintained flow. Rats underwent femoral artery and vein cannulation and mini laparotomy for liver access. An OxyProbe device placed over the liver evaluated blood flow changes. Shock animals were bled to maintain a mean arterial pressure (MAP) of <30mmHg. PR was accomplished by infusing an equivalent volume of banked rat plasma to the blood volume removed. ABGs were obtained at 0, 5, 15, and 30 minutes. Platelet-free plasma (0, 15 and 30 minutes) was used to measure total tPA concentrations.

Results: MAP was maintained in PR groups compared to control at all time points (P>0.05). Compared to control, shock had a lower MAP at all time points (P<0.02). At t=15 both PR groups and shock had lower O2 content vs. control (control: 18.67±1.17, 50% PR: 8.98±1.20, 75% PR: 6.37±0.18, Shock: 13.04±3.29 ml/dL P<0.006). Compared to shock both PR groups had lower O2 content (P<0.05). At t=30, both PR groups and shock had lower O2 content vs. control (control: 16.7±2.1, 50% PR: 7.8±0.28, 75% PR: 5.55±0.24, Shock 3.87±5.62 ml/dL P<0.001). Compared to shock, both PR groups did not have significantly different arterial O2 content (P>0.5). At t=15min, flows, measured by fold-change vs. baseline, were: control -8.50±18.4, shock: -54.7 ±6.55, 50% PR: 27.45±19.8 and 75% PR: 12.93±0.634 (P<0.05). At t=30, control: -5.329±22.4, shock: -74.42±8.49, 50% PR: 15.6±15.9, 75% PR: 21.1±3.03 (P<.05). At t=15 and t=30, shock had lower flow versus control (P.024 and P<.001, respectively). PR groups and control had similar flows at all time points (P>.05). At t=30, tPA release, measured by fold-change vs. baseline, were as follows: control: -0.077±0.195, shock: 3.31±1.51, 50% PR: -0.053±0.998 and 75% PR: 0.03±0.543 (P<0.05). Compared to control, shock showed a trend toward increased release of tPA at 30 min (p=0.06), and PR groups had a similar increase in TPA release (P>0.9) (Figure 1).

Conclusion: Low flow but not hypoxemia alone contributes to tPA release. The low blood flow state is, at least partially, responsible for systemic hyperfibrinolysis following injury while isolated hypoxemia is not a stimulus for tPA release.

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