Elevated Cardiac Troponin in Clinical Scenarios Beyond Obstructive Coronary Artery Disease.

In this systematic review article, we aim to summarize the most up-to-date evidence regarding elevations of cardiac troponin, especially in clinical scenarios other than obstructive coronary artery disease. The accurate interpretation of raised cardiac troponin is challenging because it relies on unconfirmed postulations and dogmatic knowledge (e.g., the exclusive provenience of cardiac troponin from cardiac myocytes), based on which every troponin elevation is assumed to definitely indicate myocardial damage. Indeed, the investigation of the pathophysiologic mechanism leading to the release in the bloodstream of cardiac biomarkers should be the first step of the diagnostic process to fully understand the clinical significance of the elevated serum levels and identify the best management. A prominent effort should be put in place to identify the contribution of potential confounding factors, both cardiac and non-cardiac in etiology, with the ability to affect synthesis and clearance of cardiac biomarkers. Regardless of the underlying cause, it is well established that cardiovascular biomarkers are increasingly useful to further risk stratification and prognosticate patients. Accordingly, we sought to clarify the meaning and impact of elevated cardiac troponin in those frequently encountered real-world scenarios presenting clinicians with a diagnostic dilemma, with the final goal of facilitating the diagnosis and help optimize individually tailored treatment strategies.

Urocortin Induces Phosphorylation of Distinct Residues of Signal Transducer and Activator of Transcription 3 (STAT3) via Different Signaling Pathways.

BACKGROUND Urocortin (Ucn) is a member of the hypothalamic corticotrophin-releasing factor family and has been shown to reduce cell death in the heart caused by ischemia/reperfusion (I/R) injury. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor known to function as a pro-survival and anti-apoptotic factor, whose activation depends on a variety of cytokines, including IL-6. A recent study demonstrated that urocortin induced IL-6 release from cardiomyocytes in a CRF-R2-dependent manner, suggesting a possible link between CRF-R2 stimulation and STAT3 activation. MATERIAL AND METHODS Experimental work was carried out in HL-1 cardiac myocytes exposed to serum starvation for 16-24 h. RESULTS Ucn stimulation led to IL-6 expression and release from mouse atrial HL-1 cardiomyocytes. Ucn treatment led to rapid phosphorylation of JAK2, which was blocked by the protein synthesis inhibitor cycloheximide or the JAK inhibitor AG490. Urocortin treatment induced STAT3 phosphorylation at Y705 and S727 through transactivation of JAK2 in an IL-6-dependent manner, but had no effect on STAT1 activity. Kinase inhibition experiments revealed that urocortin induces STAT3 S727 phosphorylation through ERK1/2 and Y705 phosphorylation through Src tyrosine kinase. In line with this finding, urocortin failed to induce phosphorylation of Y705 residue in SYF cells bearing null mutation of Src, while phosphorylation of S727 residue was unchanged. CONCLUSIONS Here, we have shown that Ucn induces activation of STAT3 through diverging signaling pathways. Full understanding of these signaling pathways will help fully exploit the cardioprotective properties of endogenous and exogenous Ucn.

Autophagy and Oncosis/Necroptosis Are Enhanced in Cardiomyocytes from Heart Failure Patients.

BACKGROUND Although originally described as a survival mechanism, it is unknown whether and to what extent autophagy is implicated in the terminal stages of heart failure. Here, we studied magnitude and evolution of autophagy in patients with intractable heart failure. MATERIAL AND METHODS Myocardial samples were obtained from 22 patients with ischemic cardiomyopathy and idiopathic dilated cardiomyopathy who were undergoing cardiac transplantation. Hearts from 11 patients who died from non-cardiac causes were used as control samples. Autophagy was evaluated by immunostaining with a monoclonal microtubule associated protein light chain 3 (LC3)-II antibody, while the relationship of autophagy with apoptosis and oncosis was assessed by double staining with TUNEL (terminal deoxynucleotidyl transferase - mediated deoxyuridine triphosphate nick end labeling) assay and complement 9 (C9) immunological staining, respectively. In addition, several necroptotic markers, including RIP1 and RIP3 (receptor interacting protein kinase 1 and 3), anti-C3 (cleaved-caspase-3), and anti-NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) were assessed by immunohistochemistry. RESULTS Anti-LC3-II staining was detected in 8.7±1.6% of the heart failure patient heart samples and in 1.2±0.3% of control patient heart samples. Vacuole formation started at one nuclear pole, before becoming bipolar and involving the cytosol. Subsequently, the autophagic process extended also to the nuclei, which underwent a progressive vacuolization and disintegration, assuming a peculiar "strawberry like appearance". Myocytes with extensive vacuole formation exhibited nuclear degeneration, which was associated with TUNEL, C3, C9, RIP1, and RIP3 positive staining. Conversely, myocytes with less extensive vacuole formation showed RIP1 and NF-κB positive staining, though not positivity for other cell death markers. CONCLUSIONS Autophagy was extensively detected in end-stage heart failure and its progression, resulted in secondary cell death, with occurrence of oncosis and necroptosis exceeding that of apoptosis. Conversely, activation of the RIP1/NF-κB pathway was associated with cell survival.

Spasmogenic Effects of the Proteasome Inhibitor Carfilzomib on Coronary Resistance, Vascular Tone and Reactivity.

BACKGROUND: Carfilzomib (CFZ) is a new proteasome inhibitor used for the treatment of multiple myeloma. Besides heart failure, angina and myocardial ischemia occurred following administration of CFZ, which is not contraindicated in patients with recent myocardial infarction/unstable angina excluded from the safety trials. AIM OF STUDY: To test the effects of CFZ (10-9 to 10-7mol/L) on vascular tone and reactivity in the isolated rabbit heart and aorta. METHODS AND RESULTS: CFZ administered by bolus injection to the isolated heart increased coronary perfusion pressure (CPP) at all tested concentrations and mildly raised left ventricular pressure and heart rate, only at the highest concentration. Addition of CFZ directly into the organ bath increased the basal tone of isolated aortic strips with contraction plateau reached after 10min. This spasmogenic effect doubled following ablation of the endothelium. Pretreatment with CFZ amplified the vasospastic action exerted by KCl, noradrenaline (NA) and angiotensin II (A) on aortic strips, and impaired vasodilation following administration of nitroglycerin (NTG) and nifedipine (NFP) on the contraction plateau induced by KCl, NA and A. Aortic strips pretreated with CFZ exhibited impaired relaxation, as compared to untreated strips, following administration of acetylcholine (Ach), an endothelium-dependent vasodilating agent, on the plateau of NA contraction (p<0.05). CONCLUSIONS: CFZ increased CPP, resting vasoconstricting tone and the spasmogenic effect of different agents. Preincubation with CFZ decreased the anti-spasmogenic activity of NTG and NFP, as well as reduced by over 50% the vasodilating effect of Ach, suggesting that CFZ can impair vasodilation via an endothelium dependent mechanism. Further studies are warranted to establish its clinical safety in patients with known CAD and prior history of coronary spasm.

Induced peroxidase and cytoprotective enzyme expressions support adaptation of HUVECs to sustain subsequent H2O2 exposure.

H2O2 mediates autocrine and paracrine signaling in the vasculature and can propagate endothelial dysfunction. However, it is not clear how endothelial cells withstand H2O2 exposure and promote H2O2-induced vascular remodeling. To understand the innate ability of endothelial cells for sustaining excess H2O2 exposure, we investigated the genotypic and functional regulation of redox systems in primary HUVECs following an H2O2 treatment. Primary HUVECs were exposed to transient H2O2 exposure and consistent H2O2 exposure. Following H2O2 treatments for 24, 48 and 72 h, we measured O2(-) production, mitochondrial membrane polarization (MMP), and gene expressions of pro-oxidative enzymes, peroxidase enzymes, and cytoprotective intermediates. Our results showed that the 24 h H2O2 exposure significantly increased O2(-) levels, hyperpolarized MMP, and downregulated CAT, GPX1, TXNRD1, NFE2L2, ASK1, and ATF2 gene expression in HUVECs. At 72 h, HUVECs in both treatment conditions were shown to adapt to reduce O2(-) levels and normalize MMP. An upregulation of GPX1, TXNRD1, and HMOX1 gene expression and a recovery of NFE2L2 and PRDX1 gene expression to control levels were observed in both consistent and transient treatments at 48 and 72 h. The response of endothelial cells to excess levels of H2O2 involves a complex interaction amongst O2(-) levels, mitochondrial membrane polarization and anti- and pro-oxidant gene regulation. As a part of this response, HUVECs induce cytoprotective mechanisms including the expression of peroxidase and antioxidant enzymes along with the downregulation of pro-apoptotic genes. This adaptation assists HUVECs to withstand subsequent exposures to H2O2.

Hyperglycemia induces differential change in oxidative stress at gene expression and functional levels in HUVEC and HMVEC.

BACKGROUND: Endothelial dysfunction precedes pathogenesis of vascular complications in diabetes. In recent years, the mechanisms of endothelial dysfunction were investigated to outline strategies for its treatment. However, the therapies for dysfunctional endothelium resulted in multiple clinical trial failures and remain elusive. There is a need for defining hyperglycemia-induced endothelial dysfunction with both generic and specific dysfunctional changes in endothelial cells (EC) using a systems approach. In this study, we investigated hyperglycemia-induced endothelial dysfunction in HUVEC and HMVEC. We investigated hyperglycemia-induced functional changes (superoxide (O2⁻), and hydrogen peroxide (H2O2) production and mitochondrial membrane polarization) and gene expression fingerprints of related enzymes (nitric oxide synthase, NAD(P)H oxidase, and reactive oxygen species (ROS) neutralizing enzymes) in both ECs. METHOD: Gene expression of NOS2, NOS3, NOX4, CYBA, UCP1, CAT, TXNRD1, TXNRD2, GPX1, NOX1, SOD1, SOD2, PRDX1, 18s, and RPLP0 were measured using real-time PCR. O2⁻ production was measured with dihydroethidium (DHE) fluorescence measurement. H2O2 production was measured using Amplex Red assay. Mitochondrial membrane polarization was measured using JC-10 based fluorescence measurement. RESULTS: We showed that the O2⁻ levels increased similarly in both ECs with hyperglycemia. However, these endothelial cells showed significantly different underlying gene expression profile, H2O2 production and mitochondrial membrane polarization. In HUVEC, hyperglycemia increased H2O2 production, and hyperpolarized mitochondrial membrane. ROS neutralizing enzymes SOD2 and CAT gene expression were downregulated. In contrast, there was an upregulation of nitric oxide synthase and NAD(P)H oxidase and a depolarization of mitochondrial membrane in HMVEC. In addition, ROS neutralizing enzymes SOD1, GPX1, TXNRD1 and TXNRD2 gene expression were significantly upregulated in high glucose treated HMVEC. CONCLUSION: Our findings highlighted a unique framework for hyperglycemia-induced endothelial dysfunction. We showed that multiple pathways are differentially affected in these endothelial cells in hyperglycemia. High occurrences of gene expression changes in HMVEC in this study supports the hypothesis that microvasculature precedes macrovasculature in epigenetic regulation forming vascular metabolic memory. Identifying genomic phenotype and corresponding functional changes in hyperglycemic endothelial dysfunction will provide a suitable systems biology approach for understanding underlying mechanisms and possible effective therapeutic intervention.

Length-dependent effect of single-walled carbon nanotube exposure in a dynamic cell growth environment of human alveolar epithelial cells.

Despite the great use of nanomaterials for engineering and medical applications, nanomaterials may have adverse consequences by accidental exposure, because of their nanoscale size, composition and shape. Like many nanomaterials, carbon nanotubes (CNTs) have been used for many proven applications, but the size of the CNTs makes them more readily become airborne and can therefore create the risk of being inhaled by a worker. In this study, we evaluated single-walled CNT (SWCNT)-induced effects on cellular responses such as cell proliferation, inflammatory response and oxidative stress in dynamic cell growth condition. A dynamic cell growth environment was established to mimic the dynamic changes in the amount of circumferential and longitudinal expansion and contraction occurred during normal breathing movement in the lung. Two different length (short: outer diameter (OD) 1-2 nm, length 0.5-2 µm; long: OD 1-2 nm, length 5-30 µm) of SWCNTs were used at different exposure concentrations (5, 10 and 20 µg/ml) during the different exposure duration (24, 48 and 72 h). Dynamic environment facilitated altered interaction between SWCNTs and A549 monolayer. Cellular responses in dynamic condition were significantly different from those in static condition. Moreover, cellular responses were dependent on the length of SWCNTs both in static and dynamic cell growth conditions.

Multi-walled carbon nanotube-induced inflammatory response and oxidative stress in a dynamic cell growth environment.

BACKGROUND: Rapid increase in multi-walled carbon nanotube (MWCNT) production for their industrial and biomedical applications has led to concerns over the effects of MWCNTs on human health and the environment. Both animal and in vitro studies have provided important findings about MWCNT-induced effects on the lung cells or tissues. In vitro studies have provided a considerable amount of fundamental information on MWCNT-induced effects on the specific lung cells. However, the cell culture systems used in those studies were limited by the absence of dynamic nature of lung tissues. We hypothesized that MWCNT-induced cellular responses such as proliferation, inflammation, and oxidative stress under dynamic cell growth environment may differ from those under static cell growth environment. RESULTS: In this study, we used a dynamic cell growth condition to mimic mechanically dynamic environment of the lung and characterized interleukin 8 (IL-8), reactive oxygen species (ROS), glutathione (GSH), and cell proliferation for three days following exposure of MWCNTs at different concentrations (5, 10, and 20 µg/ml) to A549 cell monolayer under both static and dynamic cell growth conditions. Our results demonstrated the distinct differences in the levels of inflammatory response and oxidative stress between static and dynamic cell growth conditions. CONCLUSIONS: In conclusion, the dynamic cell growth system used in this study provided important changes in cellular responses that were not found in the static cell growth system and were similar to animal studies. The dynamic cell growth system can be considered as a viable alternative to in vivo test system in combination with existing in vitro static cell growth systems to evaluate the effect of MWCNTs on cellular responses in the respiratory system.

High-frequency ultrasound for intraoperative margin assessments in breast conservation surgery: a feasibility study.

BACKGROUND: In addition to breast imaging, ultrasound offers the potential for characterizing and distinguishing between benign and malignant breast tissues due to their different microstructures and material properties. The aim of this study was to determine if high-frequency ultrasound (20-80 MHz) can provide pathology sensitive measurements for the ex vivo detection of cancer in margins during breast conservation surgery. METHODS: Ultrasonic tests were performed on resected margins and other tissues obtained from 17 patients, resulting in 34 specimens that were classified into 15 pathology categories. Pulse-echo and through-transmission measurements were acquired from a total of 57 sites on the specimens using two single-element 50-MHz transducers. Ultrasonic attenuation and sound speed were obtained from time-domain waveforms. The waveforms were further processed with fast Fourier transforms to provide ultrasonic spectra and cepstra. The ultrasonic measurements and pathology types were analyzed for correlations. The specimens were additionally re-classified into five pathology types to determine specificity and sensitivity values. RESULTS: The density of peaks in the ultrasonic spectra, a measure of spectral structure, showed significantly higher values for carcinomas and precancerous pathologies such as atypical ductal hyperplasia than for normal tissue. The slopes of the cepstra for non-malignant pathologies displayed significantly greater values that differentiated them from the normal and malignant tissues. The attenuation coefficients were sensitive to fat necrosis, fibroadenoma, and invasive lobular carcinoma. Specificities and sensitivities for differentiating pathologies from normal tissue were 100% and 86% for lobular carcinomas, 100% and 74% for ductal carcinomas, 80% and 82% for benign pathologies, and 80% and 100% for fat necrosis and adenomas. Specificities and sensitivities were also determined for differentiating each pathology type from the other four using a multivariate analysis. The results yielded specificities and sensitivities of 85% and 86% for lobular carcinomas, 85% and 74% for ductal carcinomas, 100% and 61% for benign pathologies, 84% and 100% for fat necrosis and adenomas, and 98% and 80% for normal tissue. CONCLUSIONS: Results from high-frequency ultrasonic measurements of human breast tissue specimens indicate that characteristics in the ultrasonic attenuation, spectra, and cepstra can be used to differentiate between normal, benign, and malignant breast pathologies.

Effect of exposure conditions on SWCNT-induced inflammatory response in human alveolar epithelial cells.

There has been an increasing interest in the development and applications of carbon nanotubes (CNTs) due to their huge potential in industrial and medical applications, but the toxicological properties of these materials have not been well characterized, especially the effects of nanoparticle exposure under different conditions on cellular responses. Nano-structured particles are potentially hazardous when they deposit in the respiratory system. In this study, we characterized the effects of single walled CNT (SWCNT) exposure on interleukin-8 (IL-8) expression in human alveolar epithelial cells (A549) under various exposure conditions. We measured the level of IL-8 expression in the presence and absence of serum following exposure of SWCNTs. The results demonstrated that IL-8 expression was enhanced in the presence of serum. When A549 cells were exposed to low concentrations of SWCNTs, IL-8 expression kept increasing, even after removal of SWCNTs from the media. In addition, SWCNT exposure under dynamic cell growth condition induced changes in IL-8 expression.

Effects of diesel particulate matters on inflammatory responses in static and dynamic culture of human alveolar epithelial cells.

Diesel particulate matter (DPM) possesses the potential to induce acute and chronic health issues upon occupational and daily exposure. Many recent studies have focused on understanding molecular mechanisms to depict DPM's side effects inside the lung using static in vitro cell culture models. These studies have provided abundant fundamental information on DPM's adverse effects on cellular responses, but these systems were limited by the absence of dynamic nature to access relevant cellular responses and functionality. We hypothesized that the exposure of DPM under dynamic environment may affect the levels of cellular inflammation and reactive oxygen species, which may be different from those under static environments. In this study, we used the dynamic cell growth condition to mimic mechanically dynamic environment similar to the normal breathing in vivo. We also used high (20, 10, and 5 ppm) and low (3, 1, 0.1, and 0.01 ppm) ranges of DPM exposure to mimic different levels of exposure, respectively. Following 24-, 48-, and 72-h exposure of DPM, Interleukin-8 (IL-8), C-reactive protein (CRP), reactive oxygen species (ROS), and total amount of protein were analyzed. Our results demonstrated the distinct differences in the profiles of inflammatory mediators (IL-8, CRP, and ROS) between the static and dynamic cell growth conditions.

Ultrasonic differentiation of normal versus malignant breast epithelial cells in monolayer cultures.

Normal and malignant mammary epithelial cells were studied using laboratory measurements, wavelet analysis, and numerical simulations of monolayer cell cultures to determine whether microscopic breast cancer can be detected in vitro with high-frequency ultrasound. Pulse-echo waveforms were acquired by immersing a broadband, unfocused 50-MHz transducer in the growth media of cell culture well plates and collecting the first reflection from the well bottoms. The simulations included a multilayer pulse-reflection model and a model of two-dimensional arrays of spherical cells and nuclei. The results show that normal and malignant cells produce time-domain signals and spectral features that are significantly different.

Shear-regulated uptake of nanoparticles by endothelial cells and development of endothelial-targeting nanoparticles.

The purpose of this research project was to develop nanoparticles with improved targeting, adhesion, and cellular uptake to activated or inflamed endothelial cells (ECs) under physiological flow conditions. Our hypothesis is that by mimicking platelet binding to activated ECs through the interaction between platelet glycoprotein Ibalpha (GP Ibalpha) and P-selectin on activated endothelial cells, GP Ibalpha-conjugated nanoparticles could exhibit increased targeting and higher cellular uptake in injured or activated endothelial cells under physiological flow conditions. To test this hypothesis, fluorescent-carboxylated polystyrene nanoparticles were selected for the study as a model particle because of its narrow size distribution as a "proof-of-concept." Using confocal microscopy, fluorescent measurements, and protein assays, cellular uptake properties were characterized for these polystyrene nanoparticles. The study also found that conjugation of 100-nm polystyrene nanoparticles with glycocalicin (the extracellular segment of GP Ibalpha) significantly increased the particle adhesion on P-selectin-coated surfaces and cellular uptake of nanoparticles by activated endothelial cells under physiological flow conditions. The results demonstrate that these novel endothelial-targeting nanoparticles could be the first step toward developing a targeted and sustained drug delivery system that can improve shear-regulated particle adhesion and cellular uptake.

Interplay Between Cytokine-Induced and Cyclic Equibiaxial Deformation-Induced Nitric Oxide Production and Metalloproteases Expression in Human Alveolar Epithelial Cells.

Ventilator-induced lung overdistension has been a growing concern in the management of mechanically ventilated patients. Mechanical ventilation triggers or enhances the net inflammatory and tissue remodeling activities. Although it has been shown that proinflammatory and tissue remodeling factors play important roles during airway remodeling, the interplay between them is not well understood. Thus, our objective was to study and characterize the molecular mechanism of cyclic equibiaxial deformation-induced airway inflammation and remodeling either in the presence or absence of a pre-existing inflammatory condition. This study was done using an in vitro dynamic model, which can simulate different mechanical ventilative conditions. Type II alveolar epithelial cell (A549) monolayers were exposed to the different levels of mechanical ventilative conditions using the Flexcell® Tension Plus™ 4000T system, which generated the different levels of cyclic equibiaxial deformation (5, 10, 15, and 20%) at 0.2 Hz deformation frequency. The production of nitric oxide (NO), the expression of metalloprotease-2 (MMP-2)/tissue inhibitor metalloprotease-2 (TIMP-2), and the activation of MMP-2 were measured under the different levels of cyclic equibiaxial deformation either in the presence or absence of TNF-α. Our study indicated that cyclic equibiaxial deformation-induced production of NO and MMP-2/TIMP-2. Higher levels of cyclic equibiaxial deformation increased the expression of the active form of MMP-2. In particular, in the presence of TNF-α, the more active form of MMP-2 was detected during both cyclic equibiaxial deformation and remodeling periods.

A combined strategy to reduce restenosis for vascular tissue engineering applications.

Biodegradable polymers including poly(l-lactic acid) (PLLA) have been used to develop cardiovascular prostheses such as vascular grafts and stents. However, implant-associated thrombosis, inflammation, and restenosis are still major obstacles for the utility of these devices. The lack of an endothelial cell (EC) lining (endothelialization) on the implants and the responses of the immune systems toward the implants have been associated with these complications. In our research strategy, we have combined the drug delivery principle with the strategies of tissue engineering, the controlled release of anti-inflammation drugs and enhanced endothelialization, to reduce the implant-associated adverse responses. We first integrated curcumin, an anti-inflammatory drug and anti-smooth muscle cell (SMC) proliferative drug, with PLLA. This curcumin-loaded PLLA material was then modified using adsorptive coating of adhesive proteins such as fibronectin, collagen-I, vitronectin, laminin, and matrigel to improve the endothelial cell (EC) adhesion and proliferation, and ECs were seeded on top of these modified surfaces. Our results showed steady drug release kinetics over the period of 50 days from curcumin-loaded PLLA materials. Additionally, integration of curcumin in PLLA increased the roughness of the scaffold at the nanometric scale using an atomic force microscopic analysis. Moreover, coating with fibronectin on curcumin-loaded PLLA surfaces gave the highest EC adhesion and proliferation compared to other adhesive proteins using PicoGreen DNA assays. The ability of our strategy to release the curcumin for producing anti-inflammation and anti-proliferation responses and to improve EC adhesion and growth after EC seeding suggests this strategy may reduce implant-associated adverse responses and be a better approach for vascular tissue engineering applications.

Pediatric uveitis.

Uveitis in children is an entity most pediatricians and ophthalmologists seldom encounter. It is prevalent in society, however, and the high incidence of sight-threatening complications in untreated children warrants a baseline knowl-edge of the diseases and disorders involved as well as a sense of when to refer to a specialist.