Citations

Abstract Background: Many loci have been found to be associated with risk of epithelial ovarian cancer (EOC). However, although there is considerable variation in progression-free survival (PFS), no loci have been found to be associated with outcome at genome-wide levels of significance. Methods: We carried out a genome-wide association study (GWAS) of PFS in 2,352 women with EOC who had undergone cytoreductive surgery and standard carboplatin/paclitaxel chemotherapy. Results: We found seven SNPs at 12q24.33 associated with PFS (P < 5 × 10–8), the top SNP being rs10794418 (HR = 1.24; 95% CI, 1.15–1.34; P = 1.47 × 10–8). High expression of a nearby gene, ULK1, is associated with shorter PFS in EOC, and with poor prognosis in other cancers. SNP rs10794418 is also associated with expression of ULK1 in ovarian tumors, with the allele associated with shorter PFS being associated with higher expression, and chromatin interactions were detected between the ULK1 promoter and associated SNPs in serous and endometrioid EOC cell lines. ULK1 knockout ovarian cancer cell lines showed significantly increased sensitivity to carboplatin in vitro. Conclusions: The locus at 12q24.33 represents one of the first genome-wide significant loci for survival for any cancer. ULK1 is a plausible candidate for the target of this association. Impact: This finding provides insight into genetic markers associated with EOC outcome and potential treatment options. See related commentary by Peres and Monteiro, p. 1604

Introduction: Von Willebrand factor (VWF) is a key component of acute ischemic stroke (AIS) thrombi. The aim of our prospective study was to investigate the immunohistochemical expression of VWF in clots and to evaluate whether VWF is associated with certain subtypes of AIS. Methods: VWF immunostaining was performed on 79 thrombi collected as part of the multi-center Stroke Thromboembolism Registry of Imaging and Pathology (STRIP) registry. The cases were classified according to TOAST criteria. The VWF expression was quantified using Orbit Image Analysis (www.Orbit.bio) machine learning software. IBM SPSS statistics 25 was used to assess the relationship between the VWF levels and different etiology subtypes. Results: A cardioembolic stroke was defined in 39 cases (49.4%) whereas an atherosclerotic origin was identified in 13 patients (16.5%). Other causes accounted for 12 cases (15.1%). Unknown etiology was reported in 15 cases (19%). The mean VWF content in the clots was 12.8%. According to the Mann-Whitney U-test, the level of VWF was significantly higher in the cases with unknown etiology compared to cardioembolic origin (p=0.044). We found also that patients with unknown etiology of stroke had higher VWF expression as compared to the other two subtypes, although this difference was not statistically significant. Conclusions: Among the patients with ischemic stroke included in this study, the VWF expression was significantly increased in those with unknown etiology compared to the group with cardioembolic stroke. Our finding provides new insights into clot composition in cryptogenic stroke and may influence the treatment and secondary prevention in these cases.

Introduction: Previous studies have successfully created blood clot analogues for In-vitro testing using animal blood. Blood components vary greatly among species and thus, creating clot analogues with human blood is likely a more accurate representation of thrombi formed in the human vasculature. We present a novel method of creating clot analogues from human blood and platelets that mimic the process by which clots form In-vivo . Methods: Following IRB approval from Mayo Clinic, human whole blood and platelets donations were obtained from the Blood Transfusion service. The whole blood was centrifuged at 1,200RPM for 20 minutes to separate it into its constituents. Plasma was removed and the remaining Red Blood Cells and Buffy Coat were mixed together by inverting. A total of 12 clot analogues were created with varying concentrations of components; Red Blood cells/Buffy Coat, Plasma and Platelets. Thrombin was added first to stimulate platelets activation for a total of 5 mins whilst continuously mixing by inversion. The RBC/WBC mixture was added next followed by CaCl2. The mixture was then quickly drawn into a 3cc syringe and spun overnight at 20RPM at room temperature to mimic dynamic flow conditions. Macro-photographs were taken to display the variation in texture and color between different clot analogue types. The clots were then fixed in 10% neutral buffered formalin for 24 hours prior to being processed. Histopathological analysis was performed using Hematoxylin and Eosin (H&E) and Martius Scarlet Blue (MSB) staining to confirm clot composition. Results: Red Blood cell-rich, Fibrin-rich, Platelet-rich and mixed clot analogues that accurately mimic clots retrieved from Acute Ischemic Stroke Patients were created. The range of histopathological compositions of the clot analogues is similar to that of the clinical samples. Conclusions: The addition and activation of platelets is key to creating accurate clot analogues for In-vitro testing. Spinning the clots is important to prevent natural sedimentation and mimic the In-vivo situation.

Ischemic stroke results from thrombus becoming lodged in a vessel in the cerebrovascular circulation. The constituents of the thrombus can vary widely and have direct effects on the mechanical properties of the thrombus. Thrombectomy procedures could potentially be improved if the mechanical properties of the thrombus could be determined prior to treatment. In the study, we developed a new non-contact method associated with Rayleigh wave propagations for evaluating mechanical properties of human clotted blood, referred to as ultrasound-mediated optical coherence elastography (UOCE). Due to clot mechanical properties being associated with fibrin and platelet content, eight different clot analogues with varying fibrin/platelets concentrations, from 0.6% (control group) to 19.0%, were produced, and the clot analog in each concentration was repeatedly measured five times. A highly focused transducer with a center frequency of 7.5 MHz was used to generate acoustic radiation forces exerted on the surface of clotted blood to induce Rayleigh wave propagations. The transducer was driven by a 50 dB radio frequency amplifier a 4 ms pulse which was repeated at a rate of 19 Hz. The optical coherence tomography (OCT) acquisition was set to 50 lateral positions and 4000 A-scans for each lateral position at a 76 kHz scan rate for total acquisition time of 52.6 ms. The results demonstrated that the Rayleigh wave group velocities positively correlated with the concentrations of fibrin/platelets. The group velocities ranged from 0.78 ± 0.02 m/s for 0.6% fibrin/platelets and increased to 2.42 ± 0.06 m/s at a fibrin/platelets concentration of 19.0%. There are statistically significant difference ( p <0.05) between control group and all clot analogues with a fibrin/platelets concentration of greater than 2.3%. In conclusion, we propose UOCE as a novel approach capable of evaluating mechanical properties of human blood clots based on Rayleigh wave propagation. This sensitive method can be used for the evaluation of the mechanical properties of human blood clot analogues and thrombectomy samples.

Introduction: Characterization of acute ischemic stroke thrombi has typically focused on the two dimensional (2D) histological characterization of the thrombus. Little is known about the three dimensional (3D) structure of these occlusive thrombi. Electron Microscopy (EM) can be used to obtain 3D information about the organization of clot components. Individual structures can be segmented and qualitative and quantitative information obtained from the clots. Methods: Following IRB approval from Mayo Clinic, acute ischemic stroke clot samples were collected from 10 patients treated at Mayo Clinic. Samples were immediately fixed in fresh modified Trumps fixative solution. A representative fragment of clot was taken and split longitudinally into two pieces; one piece was sent for 3D-EM analysis and the other for histological processing and analysis. Hematoxylin and eosin stain (H&E) and Martius Scarlett Blue (MSB) histological stains were used for conventional histopathology evaluation and components analysis. Immunohistochemical (IHC) staining using anti-CD42b (Platelets) was applied to confirm the presence of platelets in each clot. Serial block-face scanning electron microscopy was used to generate high-resolution three-dimensional images. Results: The 2D morphological features of the clot were identified using H&E, MSB and anti-CD42b staining. The 3D organization of thrombi varies greatly, both within and between clots depending on the constituents. Red Blood Cell-rich areas are mainly composed of Red Blood Cells packaged tightly together, with a small number of Fibrin strands interwoven between the cells. Fibrin-rich areas are characterized by dense networks of fibrin strands along with platelets and White Blood Cells with some sparse Red Blood Cells present. Platelet-rich areas are composed of large volumes of de-granulated platelets, some fibrin stands and white blood cells and an absence of Red Blood Cells. Conclusions: 3D electron microscopy can provide important information on the organization of components within thrombi. This could provide key information on the age and etiology of the thrombus. In addition, it may help to inform thrombolytic treatment and medical device design.

Aim Th1 cells have potent pro‐inflammatory properties and have been shown to play an important role in the development and progression of acute renal injury to chronic renal disease. The aim of this study is to test the hypothesis that chronic renal damage in mice with renal artery stenosis (RAS) would be ameliorated in mice with homozygous deletion of the Th1 transcription factor T‐bet. Methods C57BL6/J (WT) and C57BL6/J mice with homozygous deletion of T‐bet (KO) were obtained from Jackson Laboratory. Renal artery stenosis surgery (RAS) was performed by placement of a polytetrafluoroethylene cuff on the right renal artery of KO (KO RAS group, n =5) and WT mice (WT RAS group, n =6). Sham (SH) surgery was performed with manipulation of the right renal artery without cuff placement in KO (KO SH group, n =4) and WT mice (WT SH group, n =5). Renal blood perfusion and oxygenation (R2*) were measured using 16.4T MRI. Blood pressure measurements were done using tail‐cuff method. Kidneys were harvested at 28 days after RAS for assessment of histology. Results Baseline intra‐renal perfusion in both KO RAS and WT RAS mice was reduced to a similar extent following cuff placement. In KO RAS group, baseline intra‐renal perfusion (795 ± 57 ml/100g/min) was reduced to 234 ± 22 ml/100g/min ( p <0.0001 vs baseline, mean reduction 71%) and in WT RAS group, baseline intra‐renal perfusion (836 ± 87 ml/100g/min) was reduced to 220 ± 21 ml/100g/min ( p =0.002 vs baseline, mean reduction 74%). Both KO RAS and WT RAS mice developed hypertension, which persisted throughout the 4 week study. WT RAS group showed significant elevation in cortical R2*, a measure of hypoxia, following RAS compared to their baseline ( p =0.002). KO RAS mice showed slightly higher R2* value, which was not significantly different from their baseline values. The weight of the stenotic kidney of the KO RAS group (105 ± 11.6 mg) was significantly lower than the KO SH mice (178 ± 14.1 mg, p =0.0021) but significantly higher than the WT RAS group (51.7 ± 7.9 mg, p =0.0010). On histologic and immune‐histochemical analysis, we observed that the stenotic kidney of the KO RAS group had significantly lower (%) cortical tubular atrophy (15% ± 6.7, p <0.0001) compared to WT RAS mice (81.7% ± 7). KO RAS group had significantly lower macrophage (MQ) ( p =0.0016) and T cell influx ( p =0.0052) compared to the WT RAS, as assessed by the % of cortical surface areas staining positive for F4/80 (MQ) and CD3 (T cell). When compared to their respective shams, KO RAS showed no significant differences in the % cortical tubular atrophy, MQ and CD3+ T cell influx. Conclusion We conclude that deficiency of Th1 transcription factor T‐bet, protects against chronic renal injury as evidenced by the less cortical atrophy in KO RAS compared to WT RAS group, despite a similar elevation in blood pressure and reduction in perfusion. This model may provide a novel therapeutic target to prevent renal disease progression in patients with renovascular hypertension. Support or Funding Information National Institutes of Health Grant AI 100911 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Background Stem cells undergo significant death after transplantation, but the mechanisms underlying this deleterious response remain unclear. Increased oxidative stress has been shown to be present in states of hypoxia or cellular stress. Thus, we hypothesize that oxidative stress blockade would ameliorate the increased cell death when exposed to hypoxia. Methods Cardiac stem cells were exposed to hypoxic conditions in cell culture (1% O 2 for 24 hours). Oxidative status (by measuring reactive oxygen species‐ROS‐) and cell survival (by the MTT assay) were measured. In a separate group of cells, oxidative stress was blocked using a superoxide dismutase mimetic (Tempol, 1‐10µm/L). Results Hypoxia resulted in increased expression of the oxidative stress enzyme NAD(P)H oxidase (subfraction 47 phox ) and in the amount of ROS (increased in the conversion of dihydroethidium ‐DHE‐ and dichlorofluorescein ‐DCF‐) what led to a decrease in stem cell viability. Pre‐conditioning of stem cells with antioxidants (Tempol) decreased the amount of cellular ROS and improved stem cell survival. The increased expression of NAD(P)H p47 phox seen in hypoxia was unaltered by oxidative stress blockade. Conclusion Increased oxidative stress plays a role in the early cell death after hypoxia. Understanding of the mechanisms underlying the fate of stem cells will be critical for the advancement of the field of stem cell therapy.

Stimulation of T lymphocytes with stromal cell‐derived factor‐1alpha (SDF‐1alpha/CXCL12) results in colocalization of the chemokine receptor, CXCR4, and the T cell receptor (TCR). Internalization of the colocalized complex can initiate prolonged activation of the extracellular signal‐regulated kinases (ERK) ERK1 and ERK2, gene transcription, cytokine expression and migration. The molecular mechanisms behind the internalization of the colocalized complex are incompletely characterized. In this study, we show that there are multiple sites in the CXCR4 C‐terminus that influence the internalization of the CXCR4/TCR complex in Jurkat and JRT3 T cells. A mutation of amino acids 328 and 329 in the CXCR4 C‐terminus, an important beta‐arrestin binding site, significantly reduced internalization in Jurkat cells. Deletion mutations in the CXCR4 C‐terminus also significantly reduced internalization in Jurkat cells. Basal internalization in Jurkat cells was decreased by an element(s) in amino acids 342–352. Basal internalization was induced by an element(s) in amino acids 334–342. Basal internalization in JRT3 cells was increased by an element in amino acids 322–334. SDF‐1alpha induced internalization was similarly regulated by the TCR. Research was supported by the Summer Undergraduate Research Fellowship at Mayo Clinic and Mayo Graduate School.

Background: Guidelines recommend involvement of palliative care (PC) for patients with cardiovascular (CV) disease and their families. Little is known about the current integration of PC and CV services. Research Objectives: Our national survey aimed to describe current practices and to identify benefits, challenges, and barriers with collaboration. Methods: Qualitative content analysis of open-response questions in an investigator-developed electronic survey study of CV and PC providers (MDs, NPs, PAs, and other allied health) using email listservs, social media, and crowdsourcing. Frequencies were tabulated using SAS statistical software. Results: Of 134 respondents, most were physicians (n=56, 57%) or advanced practice RNs or PAs (n=37, 28%), from 52 U.S. institutions, representing 33 states. The most common care for which PC was integrated were mechanical circulatory support, advanced heart failure, and heart transplantation; the least common care for which PC was integrated were TAVR, ECMO, and pulmonary hypertension. Integrated practice (n=80, 61%), PC identification of goals of care (n=20, 15%) and PC aid in patient/family communication (n=15, 11%) were the most commonly identified as positives in current practice. PC provider workforce (n=22, 16%), underutilization of PC referrals (n=20, 15%), late disease PC referral (n=19, 14%), and fundamental knowledge deficit of specialty scope (n=18, 14%) were most frequently identified as challenges. Other barriers specifically noted were underdeveloped health infrastructure and limited allocated resources (n=23,17%) and mis-/negative perception of services (n=15, 11%). Conclusion: PC and CV collaboration occurs for a high proportion of CV patients and is viewed as overwhelming positive, despite the challenges of limited PC specialists availability, underdeveloped healthcare infrastructure, limited understanding of specialty practice, and late referrals. Priorities includes increasing PC provider availability, expanding knowledge of PC services, educating providers on models of collaboration, and improving institutional support.

Introduction: Patient understanding of available therapies for atrial fibrillation (AF) is foundational to shared medical decision making and long term medication adherence. Yet, there is a paucity of data regarding the extent to which patients newly diagnosed with AF in routine community practice understand their options. Hypotheses: 1) Understanding of warfarin, novel oral anticoagulants (NOAC), rhythm control therapy, cardioversion and radio frequency ablation changes little from baseline to 6 months and 2) treatment rates at 6 months are associated with patient understanding of therapies at baseline. Methods: We analyzed survey data from SATELLITE, a substudy of new-onset AF patients enrolled at 56 US sites participating in the ORBIT-AF registry. Patients were surveyed at the baseline and 6 month follow up clinic visit using Likert scales. Agreement between time points was assessed with the McNemar test, and the relationship between understanding and treatment was assessed only for the subset not on treatment at baseline. Results: Of 1000 patients enrolled in SATELLITE, 506 had 6-month survey data (data collection is continuing). Among these, the median age was 69.0 years (IQR 63.0 - 76.0) and 93.7% (474 of 506) were white. There was evidence of improvement in the self-reported understanding of warfarin and NOACs from baseline to 6 months, but not for rhythm control, ablation or cardioversion. The proportion reporting high understanding improved significantly for warfarin (p<.0001) and NOACs (p<.0001) from 47% (223 of 474) and 51% (245 of 481) at baseline to 60% (284 of 474) and 69% (332 of 481) at 6 months respectively (Figure 1). Patients with high understanding of the benefits of ablation (p=0.0005) and options for ablation (p=0.0093) at baseline were more likely to have this therapy at the 6 month follow up (N=590), but improved understanding was not associated with increased use of warfarin/NOACs (N=83) or rhythm control (N=444). Conclusions: Patients with new-onset AF had improved self-reported understanding of some treatment options over the first 6-months from diagnosis; however, factors other than patient understanding may influence AF treatments received at 6 months. Patient understanding of AF treatments remains suboptimal at 6 months, and our results suggest a need for ongoing patient education.