Imaging Sub-Cellular Methionine and Insulin Interplay in Triple Negative Breast Cancer Lipid Droplet Metabolism.

Triple negative breast cancer (TNBC) is a particularly aggressive cancer subtype that is difficult to diagnose due to its discriminating epidemiology and obscure metabolome. For the first time, 3D spatial and chemometric analyses uncover the unique lipid metabolome of TNBC under the tandem modulation of two key metabolites - insulin and methionine - using non-invasive optical techniques. By conjugating heavy water (D2O) probed Raman scattering with label-free two-photon fluorescence (TPF) microscopy, we observed altered de novo lipogenesis, 3D lipid droplet morphology, and lipid peroxidation under various methionine and insulin concentrations. Quantitative interrogation of both spatial and chemometric lipid metabolism under tandem metabolite modulation confirms significant interaction of insulin and methionine, which may prove to be critical therapeutic targets, and proposes a powerful optical imaging platform with subcellular resolution for metabolic and cancer research.

A high-throughput technique to map cell images to cell positions using a 3D imaging flow cytometer.

We develop a high-throughput technique to relate positions of individual cells to their three-dimensional (3D) imaging features with single-cell resolution. The technique is particularly suitable for nonadherent cells where existing spatial biology methodologies relating cell properties to their positions in a solid tissue do not apply. Our design consists of two parts, as follows: recording 3D cell images at high throughput (500 to 1,000 cells/s) using a custom 3D imaging flow cytometer (3D-IFC) and dispensing cells in a first-in-first-out (FIFO) manner using a robotic cell placement platform (CPP). To prevent errors due to violations of the FIFO principle, we invented a method that uses marker beads and DNA sequencing software to detect errors. Experiments with human cancer cell lines demonstrate the feasibility of mapping 3D side scattering and fluorescent images, as well as two-dimensional (2D) transmission images of cells to their locations on the membrane filter for around 100,000 cells in less than 10 min. While the current work uses our specially designed 3D imaging flow cytometer to produce 3D cell images, our methodology can support other imaging modalities. The technology and method form a bridge between single-cell image analysis and single-cell molecular analysis.

Advances in stimulated Raman scattering imaging for tissues and animals.

Stimulated Raman scattering (SRS) microscopy has emerged in the last decade as a powerful optical imaging technology with high chemical selectivity, speed, and subcellular resolution. Since the invention of SRS microscopy, it has been extensively employed in life science to study composition, structure, metabolism, development, and disease in biological systems. Applications of SRS in research and the clinic have generated new insights in many fields including neurobiology, tumor biology, developmental biology, metabolomics, pharmacokinetics, and more. Herein we review the advances and applications of SRS microscopy imaging in tissues and animals, as well as envision future applications and development of SRS imaging in life science and medicine.

Visualizing Cancer Cell Metabolic Dynamics Regulated With Aromatic Amino Acids Using DO-SRS and 2PEF Microscopy.

Oxidative imbalance plays an essential role in the progression of many diseases that include cancer and neurodegenerative diseases. Aromatic amino acids (AAA) such as phenylalanine and tryptophan have the capability of escalating oxidative stress because of their involvement in the production of Reactive Oxygen Species (ROS). Here, we use D2O (heavy water) probed stimulated Raman scattering microscopy (DO-SRS) and two Photon Excitation Fluorescence (2PEF) microscopy as a multimodal imaging approach to visualize metabolic changes in HeLa cells under excess AAA such as phenylalanine or trytophan in culture media. The cellular spatial distribution of de novo lipogenesis, new protein synthesis, NADH, Flavin, unsaturated lipids, and saturated lipids were all imaged and quantified in this experiment. Our studies reveal ∼10% increase in de novo lipogenesis and the ratio of NADH to flavin, and ∼50% increase of the ratio of unsaturated lipids to saturated lipid in cells treated with excess phenylalanine or trytophan. In contrast, these cells exhibited a decrease in the protein synthesis rate by ∼10% under these AAA treatments. The cellular metabolic activities of these biomolecules are indicators of elevated oxidative stress and mitochondrial dysfunction. Furthermore, 3D reconstruction images of lipid droplets were acquired and quantified to observe their spatial distribution around cells' nuceli under different AAA culture media. We observed a higher number of lipid droplets in excess AAA conditions. Our study showcases that DO-SRS imaging can be used to quantitatively study how excess AAA regulates metabolic activities of cells with subcellular resolution in situ.

Mammalian cell and tissue imaging using Raman and coherent Raman microscopy.

Direct imaging of metabolism in cells or multicellular organisms is important for understanding many biological processes. Raman scattering (RS) microscopy, particularly, coherent Raman scattering (CRS) such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), has emerged as a powerful platform for cellular imaging due to its high chemical selectivity, sensitivity, and imaging speed. RS microscopy has been extensively used for the identification of subcellular structures, metabolic observation, and phenotypic characterization. Conjugating RS modalities with other techniques such as fluorescence or infrared (IR) spectroscopy, flow cytometry, and RNA-sequencing can further extend the applications of RS imaging in microbiology, system biology, neurology, tumor biology and more. Here we overview RS modalities and techniques for mammalian cell and tissue imaging, with a focus on the advances and applications of CARS and SRS microscopy, for a better understanding of the metabolism and dynamics of lipids, protein, glucose, and nucleic acids in mammalian cells and tissues. This article is categorized under: Laboratory Methods and Technologies > Imaging Biological Mechanisms > Metabolism Analytical and Computational Methods > Analytical Methods.

A Novel Approach Using Computed Tomography Angiograms to Predict Sternotomy or Complicated Anastomosis in Patients Undergoing Robotically Assisted Minimally Invasive Direct Coronary Artery Bypass.

OBJECTIVE: Robotically assisted minimally invasive direct coronary artery bypass is an alternative to sternotomy-based surgery in properly selected patients. Identifying the left anterior descending artery when it is deep in the epicardial fat can be particularly challenging through a 5- to 6-cm mini-thoracotomy incision. The objective of this study was to evaluate a technique for predicting conversion to sternotomy or complicated left anterior descending artery anastomosis using preoperative cardiac-gated computed tomography angiograms. METHODS: Retrospective review of 75 patients who underwent robotically assisted minimally invasive direct coronary artery bypass for whom a preoperative computed tomography angiogram was available. The distance from the left anterior descending artery to the myocardium was measured on a standardized "5-chamber" axial computed tomography view. The relative risk of sternotomy or complicated anastomosis was compared between patients whose left anterior descending artery was resting directly on the myocardium (left anterior descending artery to the myocardium distance = 0 mm) with those whose left anterior descending artery was resting above (left anterior descending artery to the myocardium distance > 0 mm). RESULTS: The average left anterior descending artery to the myocardium distance was 3.2 ± 2.6 mm (range = 0-11.5 mm). Fourteen patients (18.7%) had an left anterior descending artery to the myocardium distance of 0 mm. Of the entire group of 75 patients, 6 (8.0%) required conversion to sternotomy. Four others (5.3%) were reported to have a complication with the anastomosis intraoperatively. For patients with left anterior descending artery to the myocardium distance of 0 mm, the relative risk of sternotomy or complicated anastomosis was 18.0 (95% confidence interval = 4.3-75.6, P = 0.0001). CONCLUSIONS: In our experience, patients with left anterior descending artery to the myocardium distance of 0 mm were at significantly higher risk of either conversion to sternotomy or technically challenging anastomosis, with 8 (57.1%) of 14 patients in this group experiencing either end point. This novel measurement may be useful to identify patients who may have anatomy, which is not well suited to the robotically assisted minimally invasive direct coronary artery bypass approach.

Surgical Versus Percutaneous Coronary Revascularization in Patients With Diabetes and Acute Coronary Syndromes.

BACKGROUND: Randomized trial data support the superiority of coronary artery bypass grafting (CABG) surgery over percutaneous coronary intervention (PCI) in diabetic patients with multivessel coronary artery disease (MV-CAD). However, whether this benefit is seen in a real-world population among subjects with stable ischemic heart disease (SIHD) and acute coronary syndromes (ACS) is unknown. OBJECTIVES: The main objective of this study was to assess the generalizability of the FREEDOM (Future REvascularization Evaluation in Patients with Diabetes Mellitus: Optimal Management of Multi-vessel Disease) trial in real-world practice among patients with diabetes mellitus and MV-CAD in residents of British Columbia, Canada. Additionally, the study evaluated the impact of mode of revascularization (CABG vs. PCI with drug-eluting stents) in diabetic patients with ACS and MV-CAD. METHODS: In a large population-based database from British Columbia, this study evaluated major cardiovascular outcomes in all diabetic patients who underwent coronary revascularization between 2007 and 2014 (n = 4,661, 2,947 patients with ACS). The primary endpoint (major adverse cardiac or cerebrovascular events [MACCE]) was a composite of all-cause death, nonfatal myocardial infarction, and nonfatal stroke. The risk of MACCE with CABG or PCI was compared using multivariable adjustment and a propensity score model. RESULTS: At 30-days post-revascularization, for ACS patients the odds ratio for MACCE favored CABG 0.49 (95% confidence interval [CI]: 0.34 to 0.71), whereas among SIHD patients MACCE was not affected by revascularization strategy (odds ratio: 1.46; 95% CI: 0.71 to 3.01; pinteraction <0.01). With a median follow-up of 3.3 years, the late (31-day to 5-year) benefit of CABG over PCI no longer varied by acuity of presentation, with a hazard ratio for MACCE in ACS patients of 0.67 (95% CI: 0.55 to 0.81) and the hazard ratio for SIHD patients of 0.55 (95% CI: 0.40 to 0.74; pinteraction = 0.28). CONCLUSIONS: In diabetic patients with MV-CAD, CABG was associated with a lower rate of long-term MACCE relative to PCI for both ACS and SIHD. A well-powered randomized trial of CABG versus PCI in the ACS population is warranted because these patients have been largely excluded from prior trials.

Longitudinal treatment patterns with ADP receptor inhibitors after myocardial infarction: Insights from the Canadian Observational AntiPlatelet sTudy.

BACKGROUND: After myocardial infarction (MI) treated with percutaneous coronary intervention (PCI), guidelines recommend dual antiplatelet therapy (DAPT) with aspirin and an ADP receptor inhibitor (ADPri) for at least 1year. However, whether real-world Canadian practice patterns reflect this recommendation is unknown. METHODS: We studied 2175 MI patients treated with PCI and discharged from 26 Canadian hospitals between 12/2011 and 05/2013 in the Canadian Observational Antiplatelet sTudy (COAPT). Hierarchical Cox proportional hazard regression modeling was used to determine baseline demographic and clinical factors associated with duration of ADPri therapy post-discharge. RESULTS: At index-hospitalization discharge, 1597 (73%) patients were treated with clopidogrel, 220 (10%) with prasugrel, and 358 (17%) with ticagrelor. ADPri was discontinued prior to 1year in 474 (21.8%) patients; discontinuation rates were lowest for patients discharged on prasugrel (17.7%), compared with clopidogrel (22.5%) or ticagrelor (21.0%), (log rank test, p=0.03). In addition to regional variability, factors associated with shorter ADPri duration included older age, low body weight, Killip III/IV heart failure, atrial fibrillation, ticagrelor on discharge, and bare metal stent use, while longer ADPri duration was associated with history of prior MI. CONCLUSIONS: One in five PCI-treated MI patients did not complete Canadian guideline-recommended 1-year course of ADPri treatment. Premature ADPri discontinuation was most strongly associated with factors that increase the risk of bleeding. Further study is required to assess the clinical implications of premature ADPri discontinuation on patient outcomes.

Clinical characteristics, angiographic findings, and one-year outcome of 101 consecutive stent thrombosis cases in British Columbia.

BACKGROUND: Stent thrombosis (ST) is rare, but is associated with significant morbidity and mortality. METHODS: We analyzed data from the British Columbia (BC) Registry from April 2011-January 2012. RESULTS: 101 ST cases were reported and verified. Based on timing, ST was considered early (≤30days) in 35.6%, late (>30days-1year) in 17.8% and very late (>1year) in 46.5%. The majority (68.5%) presented with STEMI, and the remaining with non-STEMI (31.5%). Most vessels were functionally occluded (TIM1 flow grade ≤1 in 67.1%). Thrombus burden was high (TIMI thrombus grade ≥4 in 77.2%). Aspiration thrombectomy was performed in 41% of cases. New stents were implanted in 62.4% cases. Intra-coronary imaging was low (11%). At the original stent implantation, STEMI was the clinical presentation in 39.6%, the lesion was complex in 62.1%, and thrombus was visualized in 23.0%. Prognosis after ST was unfavorable with high mortality (11.9% at 30days and 16.8% at one year), and further revascularization (5.0% repeat PCI and 6.9% coronary artery bypass graft surgery). Early ST was associated with worse clinical outcome compared to late/very late ST: 30-day mortality at 22.2% versus 6.2% (p=0.02), and 1-year mortality at 27.8% versus 10.8% (p=0.05). CONCLUSIONS: In this prospective registry from BC, all ST presented with myocardial infarction, and the majority was treated with emergency PCI. Additional stents were commonly implanted with infrequent use of intracoronary imaging. Mortality rate was higher for early ST in comparison with late/very late ST. A comprehensive approach should be developed to treat this difficult complication.

Angiographic appearance of spontaneous coronary artery dissection with intramural hematoma proven on intracoronary imaging.

BACKGROUND: The pathognomonic appearance of multiple radiolucent lumen on angiography is used to diagnose spontaneous coronary artery dissection (SCAD). However, this finding is absent in >70% of SCAD, in which case optical coherence tomography (OCT) or intravascular ultrasound (IVUS) is useful to assess arterial wall integrity. METHODS: We report the angiographic appearance of SCAD that were proven on intracoronary imaging with OCT or IVUS. Our angiographic classification and algorithm for SCAD diagnosis was previously reported. Patients with type 1 SCAD (multiple radiolucent lumen) do not require OCT/IVUS, whereas, it was recommended for those with suspected type 2 (diffuse stenosis) or 3 (mimic atherosclerosis) SCAD. RESULTS: Twenty-two consecutive patients with non-type 1 angiographic SCAD in 25 coronary arteries (22 OCT and 4 IVUS) were studied. Mean age was 52.9 ± 9.9 years, 89.5% were women, and 16/22 (72.7%) had underlying fibromuscular dysplasia. Sixteen SCAD arteries were type 2 SCAD, and nine were type 3. All 25 SCAD arteries had intramural hematoma and intimomedial membrane separation with double lumen on OCT or IVUS. The mean visual angiographic stenosis was 74.6 ± 17.5% (range 40-100%). Dissected segments were long with mean qualitative coronary analysis (QCA) length 45.2 ± 29.2 mm, especially in patients with type 2 SCAD (mean QCA length 58.3 ± 29.0 mm). The mean QCA length in type 3 SCAD lesions was 22.1 ± 5.7 mm. CONCLUSIONS: Intracoronary imaging confirms that SCAD may appear angiographically without multiple radiolucent lumen. Angiographers should be familiar with angiographic SCAD variants to improve SCAD diagnosis, and utilize intracoronary imaging when the diagnosis is uncertain.

Embolic protection device use and its association with procedural safety and long-term outcomes following saphenous vein graft intervention: An analysis from the British Columbia Cardiac registry.

BACKGROUND: Embolic protection devices (EPDs) have been designed and introduced to reduce distal embolization and peri-procedural myocardial infarction during saphenous vein graft (SVG) intervention. Current guidelines give a class I recommendation to EPD use during SVG intervention when technically feasible. However, the routine use of these devices has recently been debated. METHODS: We analyzed 1,359 patients undergoing isolated SVG intervention between 2008 and 2013 in the British Columbia Cardiac Registry. We analyzed (a) post-procedural TIMI flow; and (b) target vessel revascularization (TVR) and mortality at 1 and 2 years. RESULTS: EPD use was an independent predictor of post-procedural TIMI 2/3 flow (OR = 2.38, 95% CI: 1.51-3.74, P < 0.001). At 1 year, EPD use was an independent predictor for lower TVR (HR = 0.35, 95% CI: 0.14-0.85, P = 0.021) and a trend towards lower mortality (HR = 0.45, 95% CI: 0.18-1.10, P = 0.082). These associations were lost at 2 years where EPD use was not predictive of mortality (HR = 0.62, 95% CI: 0.33-1.17, P = 0.144) or TVR (HR = 0.70, 95% CI: 0.41-1.17, P = 0.176). These findings were confirmed in propensity-matched and inverse probability treatment weighted analyses. CONCLUSIONS: In this analysis of patients undergoing SVG intervention, EPD use was a strong predictor for improved post-procedural TIMI flow. Whilst EPD use was associated with lower TVR and a trend for lower mortality at 1 year, these associations were lost at 2 years. These findings would appear to support the use of EPD for SVG intervention. © 2015 Wiley Periodicals, Inc.

Necroptosis and parthanatos are involved in remote lung injury after receiving ischemic renal allografts in rats.

Early renal graft injury could result in remote pulmonary injury due to kidney-lung cross talk. Here we studied the possible role of regulated necrosis in remote lung injury in a rat allogeneic transplantation model. In vitro, human lung epithelial cell A549 was challenged with TNF-α and conditioned medium from human kidney proximal tubular cells (HK-2) after hypothermia-hypoxia insults. In vivo, the Brown-Norway rat renal grafts were extracted and stored in 4 °C Soltran preserving solution for up to 24 h and transplanted into Lewis rat recipients, and the lungs were harvested on day 1 and day 4 after grafting for further analysis. Ischemia-reperfusion injury in the renal allograft caused pulmonary injury following engraftment. PARP-1 (marker for parthanatos) and receptor interacting protein kinase 1 (Rip1) and Rip3 (markers for necroptosis) expression was significantly enhanced in the lung. TUNEL assays showed increased cell death of lung cells. This was significantly reduced after treatment with necrostatin-1 (nec-1) or/and 3-aminobenzamide (3-AB). Acute immune rejection exacerbated the remote lung injury and 3-AB or/and Nec-1 combined with cyclosporine A conferred optimal lung protection. Thus, renal graft injury triggered remote lung injury, likely through regulated necrosis. This study could provide the molecular basis for combination therapy targeting both pathways of regulated necrosis to treat such complications after renal transplantation.

The role of interleukin 17 in tumour proliferation, angiogenesis, and metastasis.

With 7.6 million deaths globally, cancer according to the World Health Organisation is still one of the leading causes of death worldwide. Interleukin 17 (IL-17) is a cytokine produced by Th17 cells, a T helper cell subset developed from an activated CD4+ T-cell. Whilst the importance of IL-17 in human autoimmune disease, inflammation, and pathogen defence reactions has already been established, its potential role in cancer progression still needs to be updated. Interestingly studies have demonstrated that IL-17 plays an intricate role in the pathophysiology of cancer, from tumorigenesis, proliferation, angiogenesis, and metastasis, to adapting the tumour in its ability to confer upon itself both immune, and chemotherapy resistance. This review will look into IL-17 and summarise the current information and data on its role in the pathophysiology of cancer as well as its potential application in the overall management of the disease.

Effect of radial versus femoral access on radiation dose and the importance of procedural volume: a substudy of the multicenter randomized RIVAL trial.

OBJECTIVES: The authors sought to compare the radiation dose between radial and femoral access. BACKGROUND: Small trials have shown an increase in the radiation dose with radial compared with femoral access, but many were performed during the operators' learning curve of radial access. METHODS: Patients were randomized to radial or femoral access, as a part of the RIVAL (RadIal Vs. femorAL) trial (N = 7,021). Fluoroscopy time was prospectively collected in 5740 patients and radiation dose quantified as air kerma in 1,445 patients and dose-area product (DAP) in 2,255 patients. RESULTS: Median fluoroscopy time was higher with radial versus femoral access (9.3 vs. 8.0 min, p < 0.001). Median air kerma was nominally higher with radial versus femoral access (1,046 vs. 930 mGy, respectively, p = 0.051). Median DAP was not different between radial and femoral access (52.8 Gy-cm(2) vs. 51.2 Gy·cm(2), p = 0.83). When results are stratified according to procedural volume, air kerma was increased only in the lowest tertile of radial volume centers (low 1,425 vs. 1,045 mGy, p = 0.002; middle 987 vs. 958 mGy, p = 0.597; high 652 vs. 621 mGy, p = 0.403, interaction p = 0.026). Multivariable regression showed procedural volume was the greatest independent predictor of lower air kerma dose (ratio of geometric means 0.55; 95% confidence interval 0.49 to 0.61 for highest-volume radial centers). CONCLUSIONS: Radiation dose as measured by air kerma was nominally higher with radial versus femoral access, but differences were present only in lower-volume centers and operators. High-volume centers have the lowest radiation dose irrespective of which access site approach that they use. (A Trial of Trans-radial Versus Trans-femoral Percutaneous Coronary Intervention (PCI) Access Site Approach in Patients With Unstable Angina or Myocardial Infarction Managed With an Invasive Strategy [RIVAL]; NCT01014273).

Intracoronary imaging of coronary fibromuscular dysplasia with OCT and IVUS.

Fibromuscular dysplasia (FMD) is a segmental non-atherosclerotic, non-inflammatory vasculopathy typically of small- to medium-muscular arteries. Coronary FMD (CFMD) is believed to be rare. However, we have found an association between spontaneous coronary artery dissection (SCAD) and FMD as the cause of myocardial infarction in ∼25% of young women age <50 years. It is plausible that pre-existing CFMD predisposed these women to SCAD. Definitive diagnosis of CFMD entails autopsy that is obviously impractical, and prior angiographic description does not differentiate superimposed SCAD from obliterative arteriopathy of CFMD. Adjunctive intravascular ultrasound or optical coherence tomography may aid the diagnosis of CFMD and we report the first of such novel images.

Central nervous system inflammation in disease related conditions: mechanistic prospects.

Inflammation is part of the innate immune response following insults to the body. This inflammatory reaction can spread throughout the systemic circulation and also into the central nervous system (CNS). CNS involvement has been demonstrated following acute peripheral insults including sepsis, surgery, burns and organ injury. It has also been observed in chronic conditions including obesity, diabetes and rheumatoid arthritis. Inflammation within the CNS is part of the pathogenesis of neurodegenerative diseases, in particular Alzheimer's disease, multiple sclerosis and Parkinson's disease. These diseases are prone to exacerbation as a result of increased inflammation within the CNS following peripheral insult. The effect of inflammation within the CNS can also be modulated by other factors including age and also oestrogen, although how pro-inflammatory cytokines within the CNS cause clinical changes remains to be elucidated. The mechanism underlying the passage of inflammation from the periphery into the CNS also remains unclear. Evidence has led to the suggestion of two main pathways: blood brain barrier (BBB) dependent and BBB independent. This uncertainty has led to an increasing body of work exploring the processes involved in both the passage of inflammation into, and the effect of cytokines on, the CNS.

Non-red blood cell transfusion as a risk factor for mortality following percutaneous coronary intervention.

BACKGROUND: Bleeding following percutaneous coronary intervention (PCI) is common and may lead to transfusion and death. Although previous work has examined the effect of red blood cell (RBC) transfusion in patients with coronary disease, no study had investigated whether transfusion of non-RBC components was associated with mortality following PCI. METHODS: All subjects transfused in the 10 days following PCI were identified using the British Columbia Cardiac and Central Transfusion Registries. Patients undergoing cardiac surgery following PCI were excluded as transfusion was assumed to be due to surgical related bleeding. Transfusion products were categorised as RBC and non-RBC comprising platelets, plasma and cryoprecipitate. Blood product use was compared according to thirty day mortality using multivariate regression and propensity adjustment for confounding variables. RESULTS: From a total of 32,580 patients who underwent PCI, 952 patients received at least 1 blood product within 10 days of PCI. Non-RBC transfusion occurred more commonly in the cohort of transfused patients dying within 30 days (p<0.001). After adjustment for baseline risk, transfusion of plasma/cryoprecipitate (HR 5.17; 95% C.I. 2.87-9.32, p<0.001) and platelets (HR 2.13; 95% C.I. 1.10-4.13, p=0.03) was associated with increased 30 day mortality. In a propensity risk adjusted model, transfusion of plasma/cryoprecipitate and RBC transfusion volume remained as significant predictors of 30-day mortality (p<0.001). CONCLUSIONS: Transfusion following PCI appears to be associated with an increased risk of death within 30 days. We now report that transfusion with plasma rich non-RBC products may confer an additional mortality risk to patients undergoing PCI.