First-in-Class Humanized Antibody against Alternatively Spliced Tissue Factor Augments Anti-Metastatic Efficacy of Chemotherapy in a Preclinical Model of Pancreatic Ductal Adenocarcinoma.

Alternatively spliced tissue factor (asTF) promotes the progression of pancreatic ductal adenocarcinoma (PDAC) by activating ß1-integrins on PDAC cell surfaces. hRabMab1, a first-in-class humanized inhibitory anti-asTF antibody we recently developed, can suppress PDAC primary tumor growth as a single agent. Whether hRabMab1 has the potential to suppress metastases in PDAC is unknown. Following in vivo screening of three asTF-proficient human PDAC cell lines, we chose to make use of KRAS G12V-mutant human PDAC cell line PaCa-44, which yields aggressive primary orthotopic tumors with spontaneous spread to PDAC-relevant anatomical sites, along with concomitant severe leukocytosis. The experimental design featured orthotopic tumors formed by luciferase labeled PaCa-44 cells; administration of hRabMab1 alone or in combination with gemcitabine/paclitaxel (gem/PTX); and the assessment of the treatment outcomes on the primary tumor tissue as well as systemic spread. When administered alone, hRabMab1 exhibited poor penetration of tumor tissue; however, hRabMab1 was abundant in tumor tissue when co-administered with gem/PTX, which resulted in a significant decrease in tumor cell proliferation; leukocyte infiltration; and neovascularization. Gem/PTX alone reduced primary tumor volume, but not metastatic spread; only the combination of hRabMab1 and gem/PTX significantly reduced metastatic spread. RNA-seq analysis of primary tumors showed that the addition of hRabMab1 to gem/PTX enhanced the downregulation of tubulin binding and microtubule motor activity. In the liver, hRabMab1 reduced liver metastasis as a single agent. Only the combination of hRabMab1 and gem/PTX eliminated tumor cell-induced leukocytosis. We here demonstrate for the first time that hRabMab1 may help suppress metastasis in PDAC. hRabMab1's ability to improve the efficacy of chemotherapy is significant and warrants further investigation.

Molecular Insights into the MLCK Activation by CaM.

Calmodulin (CaM) is a universal regulatory protein that modulates numerous cellular processes by using calcium (Ca2+) as the signal. In smooth muscle cells (SMC), one major target of CaM is myosin light chain kinase (MLCK), a kinase that phosphorylates the myosin regulatory light chain and thereby regulates cell contraction. In the absence of CaM, MLCK remains inhibited by its autoinhibitory domain (AID). While it is well established that CaM activates MLCK, the molecular interactions between these two proteins remain elusive due to the lack of structural data. In this work, we constructed a molecular model of mammalian CaM (mCaM) in complex with MLCK leveraging AlphaFold, published biochemical data, and protein-protein docking. The model, along with a strategic set of CaM mutants including a inhibitory variant soybean CaM isoform 4 (sCaM-4), was subject to molecular dynamics (MD) simulations. Using principal component analysis (PCA), we mapped out the transition path for the removal of the AID from the MLCK kinase domain to provide molecular basis of MLCK activation. Additionally, we established MLCK conformations that correspond to the active and inactive states of the kinase. We showed that mCaM and sCaM-4 cause MLCK to undergo the transition to the active and inactive states, respectively. Using two structural metrics, we computed the probabilities of MLCK activation by different CaM variants, which were in good agreement with the experimental data. Distributions along these metrics revealed that different inhibitory CaM variants impair MLCK activation through unique mechanisms. We finally identified molecular contacts that contribute to the MLCK activation by CaM. Overall, we report a de novo molecular model of CaM-MLCK that provides insights into the molecular mechanism of MLCK activation by CaM. The mechanism requires effective removal of the AID while preserving an active configuration of the kinase domain. This mechanism may be shared by other MLCK isoforms and potentially other structurally similar kinases with CaM-mediated regulatory domains.

Potential utility of a multi-component coagulation factor panel to calculate MELD scores and assess the risk of portal vein thrombosis in chronic liver disease.

BACKGROUND: Current quantitative approaches to assess chronic liver disease (CLD) severity have limitations. Further, portal vein thrombosis (PVT) pre-liver transplant (LT) is a major contributor to morbidity in CLD; the means of detecting and/or predicting PVT are limited. We sought to explore whether plasma coagulation factor activity levels can serve as a substitute for prothrombin time/international normalized ratio (PT/INR) in the Model for End-stage Liver Disease (MELD), and/or help assess the risk of PVT. METHODS: Plasma activity levels of Factor V (FV), Factor VIII (FVIII), Protein C (PC), and Protein S (PS) and the concentrations of D-dimer, sP-selectin, and asTF were assessed in two cohorts of CLD patients (ambulatory, n = 42; LT, n = 43). RESULTS: FV and PC activity levels strongly correlated with MELD scores, which enabled the development of a novel scoring system based on multiple linear regressions of the correlations of FV and PC activity with MELD-Na that substitutes PT/INR. Six-month and 1-year follow-up revealed that our novel approach was non-inferior to MELD-Na at predicting mortality. A significant inverse correlation between FVIII activity levels and PVT was found in the LT cohort (p = 0.010); FV and PS activity levels were in-trend (p = 0.069, p = 0.064). We developed a logistic regression-based compensation score to identify patients at risk of PVT. CONCLUSIONS: We demonstrate that FV and PC activity levels may be used to replace PT/INR in MELD scoring. We also show the potential of using the combination of FV, FVIII, and PS activity levels to assess the risk of PVT in CLD.

STIM1 ablation impairs exercise-induced physiological cardiac hypertrophy and dysregulates autophagy in mouse hearts.

Cardiac stromal interaction molecule 1 (STIM1), a key mediator of store-operated Ca2+ entry (SOCE), is a known determinant of cardiomyocyte pathological growth in hypertrophic cardiomyopathy. We examined the role of STIM1 and SOCE in response to exercise-dependent physiological hypertrophy. Wild-type (WT) mice subjected to exercise training (WT-Ex) showed a significant increase in exercise capacity and heart weight compared with sedentary (WT-Sed) mice. Moreover, myocytes from WT-Ex hearts displayed an increase in length, but not width, compared with WT-Sed myocytes. Conversely, exercised cardiac-specific STIM1 knock-out mice (cSTIM1KO-Ex), although displaying significant increase in heart weight and cardiac dilation, evidenced no changes in myocyte size and displayed a decreased exercise capacity, impaired cardiac function, and premature death compared with sedentary cardiac-specific STIM1 knock-out mice (cSTIM1KO-Sed). Confocal Ca2+ imaging demonstrated enhanced SOCE in WT-Ex myocytes compared with WT-Sed myocytes with no measurable SOCE detected in cSTIM1KO myocytes. Exercise training induced a significant increase in cardiac phospho-Akt Ser473 in WT mice but not in cSTIM1KO mice. No differences were observed in phosphorylation of mammalian target of rapamycin (mTOR) and glycogen synthase kinase (GSK) in exercised versus sedentary cSTIM1KO mice hearts. cSTIM1KO-Sed mice showed increased basal MAPK phosphorylation compared with WT-Sed that was not altered by exercise training. Finally, histological analysis revealed exercise resulted in increased autophagy in cSTIM1KO but not in WT myocytes. Taken together, our results suggest that adaptive cardiac hypertrophy in response to exercise training involves STIM1-mediated SOCE. Our results demonstrate that STIM1 is involved in and essential for the myocyte longitudinal growth and mTOR activation in response to endurance exercise training.NEW & NOTEWORTHY Store-operated Ca2+ entry (SOCE) has been implicated in pathological cardiac hypertrophy; however, its role in physiological hypertrophy is unknown. Here we report that SOCE is also essential for physiological cardiac hypertrophy and functional adaptations in response to endurance exercise. These adaptations were associated with activation of AKT/mTOR pathway and curtailed cardiac autophagy and degeneration. Thus, SOCE is a common mechanism and an important bifurcation point for signaling paths involved in physiological and pathological hypertrophy.

NaV1.6 dysregulation within myocardial T-tubules by D96V calmodulin enhances proarrhythmic sodium and calcium mishandling.

Calmodulin (CaM) plays critical roles in cardiomyocytes, regulating Na+ (NaV) and L-type Ca2+ channels (LTCCs). LTCC dysregulation by mutant CaMs has been implicated in action potential duration (APD) prolongation and arrhythmogenic long QT (LQT) syndrome. Intriguingly, D96V-CaM prolongs APD more than other LQT-associated CaMs despite inducing comparable levels of LTCC dysfunction, suggesting dysregulation of other depolarizing channels. Here, we provide evidence implicating NaV dysregulation within transverse (T) tubules in D96V-CaM-associated arrhythmias. D96V-CaM induced a proarrhythmic late Na+ current (INa) by impairing inactivation of NaV1.6, but not the predominant cardiac NaV isoform NaV1.5. We investigated arrhythmia mechanisms using mice with cardiac-specific expression of D96V-CaM (cD96V). Super-resolution microscopy revealed close proximity of NaV1.6 and RyR2 within T-tubules. NaV1.6 density within these regions increased in cD96V relative to WT mice. Consistent with NaV1.6 dysregulation by D96V-CaM in these regions, we observed increased late NaV activity in T-tubules. The resulting late INa promoted aberrant Ca2+ release and prolonged APD in myocytes, leading to LQT and ventricular tachycardia in vivo. Cardiac-specific NaV1.6 KO protected cD96V mice from increased T-tubular late NaV activity and its arrhythmogenic consequences. In summary, we demonstrate that D96V-CaM promoted arrhythmias by dysregulating LTCCs and NaV1.6 within T-tubules and thereby facilitating aberrant Ca2+ release.

Effect of pre-orthodontic trainer treatment on the masticatory and perioral muscles in patients with class II subdivision 1 malocclusion evaluated by surface EMG.

INTRODUCTION: Myofunctional appliances have been shown to have a positive effect on the muscles in the facial area. Surface electromyography (sEMG) is one of the reliable methods used to investigate these effects.

Type of correlation between bite force and EMG activity of the temporalis and masseter muscles during maximal and submaximal clenching.

INTRODUCTION: Maximal bite force is an important determinant of masticatory function and is essential for the estimation of dental status. Bite force is usually measured by gnathodynamometry.

Integrin regulation by tissue factor promotes cancer stemness and metastatic dissemination in breast cancer.

Tissue Factor (TF) is the initiator of blood coagulation but also functions as a signal transduction receptor. TF expression in breast cancer is associated with higher tumor grade, metastasis and poor survival. The role of TF signaling on the early phases of metastasis has never been addressed. Here, we show an association between TF expression and metastasis as well as cancer stemness in 574 breast cancer patients. In preclinical models, blockade of TF signaling inhibited metastasis tenfold independent of primary tumor growth. TF blockade caused a reduction in epithelial-to-mesenchymal-transition, cancer stemness and expression of the pro-metastatic markers Slug and SOX9 in several breast cancer cell lines and in ex vivo cultured tumor cells. Mechanistically, TF forms a complex with ß1-integrin leading to inactivation of ß1-integrin. Inhibition of TF signaling induces a shift in TF-binding from α3ß1-integrin to α6ß4 and dictates FAK recruitment, leading to reduced epithelial-to-mesenchymal-transition and tumor cell differentiation. In conclusion, TF signaling inhibition leads to reduced pro-metastatic transcriptional programs, and a subsequent integrin ß1 and ß4-dependent reduction in metastasic dissemination.

SARS-CoV-2, platelets, and endothelium: coexistence in space and time, or a pernicious ménage à trois?

As we enter year 3 of SARS-CoV-2 pandemic, long-term consequences of COVID-19 have become a major public health issue worldwide; however, the molecular and cellular underpinnings of 'long COVID' remain very poorly understood. A paradigm has recently emerged that thrombo-inflammatory consequences of SARS-CoV-2's impact on endothelial cells and platelets likely play a significant role in the development of chronic symptomatology associated with COVID-19. In this brief overview, we discuss the recent findings pertaining to the detection of SARS-CoV-2 virions in vascular cell subtypes, the contribution of the coagulation system to the development of 'long COVID', and the potential role of stem/progenitor cells in the viral and thrombotic dissemination in this disorder.

Ero1α-Dependent ERp44 Dissociation From RyR2 Contributes to Cardiac Arrhythmia.

BACKGROUND: Oxidative stress in cardiac disease promotes proarrhythmic disturbances in Ca2+ homeostasis, impairing luminal Ca2+ regulation of the sarcoplasmic reticulum (SR) Ca2+ release channel, the RyR2 (ryanodine receptor), and increasing channel activity. However, exact mechanisms underlying redox-mediated increase of RyR2 function in cardiac disease remain elusive. We tested whether the oxidoreductase family of proteins that dynamically regulate the oxidative environment within the SR are involved in this process. METHODS: A rat model of hypertrophy induced by thoracic aortic banding (TAB) was used for ex vivo whole heart optical mapping and for Ca2+ and reactive oxygen species imaging in isolated ventricular myocytes (VMs). RESULTS: The SR-targeted reactive oxygen species biosensor ERroGFP showed increased intra-SR oxidation in TAB VMs that was associated with increased expression of Ero1α (endoplasmic reticulum oxidoreductase 1 alpha). Pharmacological (EN460) or genetic Ero1α inhibition normalized SR redox state, increased Ca2+ transient amplitude and SR Ca2+ content, and reduced proarrhythmic spontaneous Ca2+ waves in TAB VMs under ß-adrenergic stimulation (isoproterenol). Ero1α overexpression in Sham VMs had opposite effects. Ero1α inhibition attenuated Ca2+-dependent ventricular tachyarrhythmias in TAB hearts challenged with isoproterenol. Experiments in TAB VMs and human embryonic kidney 293 cells expressing human RyR2 revealed that an Ero1α-mediated increase in SR Ca2+-channel activity involves dissociation of intraluminal protein ERp44 (endoplasmic reticulum protein 44) from the RyR2 complex. Site-directed mutagenesis and molecular dynamics simulations demonstrated a novel redox-sensitive association of ERp44 with RyR2 mediated by intraluminal cysteine 4806. ERp44-RyR2 association in TAB VMs was restored by Ero1α inhibition, but not by reducing agent dithiothreitol, as hypo-oxidation precludes formation of covalent bond between RyR2 and ERp44. CONCLUSIONS: A novel axis of intraluminal interaction between RyR2, ERp44, and Ero1α has been identified. Ero1α inhibition exhibits promising therapeutic potential by stabilizing RyR2-ERp44 complex, thereby reducing spontaneous Ca2+ release and Ca2+-dependent tachyarrhythmias in hypertrophic hearts, without causing hypo-oxidative stress in the SR.

Obesity and the Bidirectional Risk of Cancer and Cardiovascular Diseases in African Americans: Disparity vs. Ancestry.

Cardiovascular disease (CVD) and cancer often occur in the same individuals, in part due to the shared risk factors such as obesity. Obesity promotes adipose inflammation, which is pathogenically linked to both cardiovascular disease and cancer. Compared with Caucasians, the prevalence of obesity is significantly higher in African Americans (AA), who exhibit more pronounced inflammation and, in turn, suffer from a higher burden of CVD and cancer-related mortality. The mechanisms that underlie this association among obesity, inflammation, and the bidirectional risk of CVD and cancer, particularly in AA, remain to be determined. Socio-economic disparities such as lack of access to healthy and affordable food may promote obesity and exacerbate hypertension and other CVD risk factors in AA. In turn, the resulting pro-inflammatory milieu contributes to the higher burden of CVD and cancer in AA. Additionally, biological factors that regulate systemic inflammation may be contributory. Mutations in atypical chemokine receptor 1 (ACKR1), otherwise known as the Duffy antigen receptor for chemokines (DARC), confer protection against malaria. Many AAs carry a mutation in the gene encoding this receptor, resulting in loss of its expression. ACKR1 functions as a decoy chemokine receptor, thus dampening chemokine receptor activation and inflammation. Published and preliminary data in humans and mice genetically deficient in ACKR1 suggest that this common gene mutation may contribute to ethnic susceptibility to obesity-related disease, CVD, and cancer. In this narrative review, we present the evidence regarding obesity-related disparities in the bidirectional risk of CVD and cancer and also discuss the potential association of gene polymorphisms in AAs with emphasis on ACKR1.

Distributed synthesis of sarcolemmal and sarcoplasmic reticulum membrane proteins in cardiac myocytes.

It is widely assumed that synthesis of membrane proteins, particularly in the heart, follows the classical secretory pathway with mRNA translation occurring in perinuclear regions followed by protein trafficking to sites of deployment. However, this view is based on studies conducted in less-specialized cells, and has not been experimentally addressed in cardiac myocytes. Therefore, we undertook direct experimental investigation of protein synthesis in cardiac tissue and isolated myocytes using single-molecule visualization techniques and a novel proximity-ligated in situ hybridization approach for visualizing ribosome-associated mRNA molecules for a specific protein species, indicative of translation sites. We identify here, for the first time, that the molecular machinery for membrane protein synthesis occurs throughout the cardiac myocyte, and enables distributed synthesis of membrane proteins within sub-cellular niches where the synthesized protein functions using local mRNA pools trafficked, in part, by microtubules. We also observed cell-wide distribution of membrane protein mRNA in myocardial tissue from both non-failing and hypertrophied (failing) human hearts, demonstrating an evolutionarily conserved distributed mechanism from mouse to human. Our results identify previously unanticipated aspects of local control of cardiac myocyte biology and highlight local protein synthesis in cardiac myocytes as an important potential determinant of the heart's biology in health and disease.

Acute Detubulation of Ventricular Myocytes Amplifies the Inhibitory Effect of Cholinergic Agonist on Intracellular Ca2+ Transients.

Muscarinic receptors expressed in cardiac myocytes play a critical role in the regulation of heart function by the parasympathetic nervous system. How the structural organization of cardiac myocytes affects the regulation of Ca2+ handling by muscarinic receptors is not well-defined. Using confocal Ca2+ imaging, patch-clamp techniques, and immunocytochemistry, the relationship between t-tubule density and cholinergic regulation of intracellular Ca2+ in normal murine ventricular myocytes and myocytes with acute disruption of the t-tubule system caused by formamide treatment was studied. The inhibitory effect of muscarinic receptor agonist carbachol (CCh, 10 µM) on the amplitude of Ca2+ transients, evoked by field-stimulation in the presence of 100 nM isoproterenol (Iso), a ß-adrenergic agonist, was directly proportional to the level of myocyte detubulation. The timing of the maximal rate of fluorescence increase of fluo-4, a Ca2+-sensitive dye, was used to classify image pixels into the regions functionally coupled or uncoupled to the sarcolemmal Ca2+ influx (ICa). CCh decreased the fraction of coupled regions and suppressed Ca2+ propagation from sarcolemma inside the cell. Formamide treatment reduced ICa density and decreased sarcoplasmic reticulum (SR) Ca2+ content. CCh did not change SR Ca2+ content in Iso-stimulated control and formamide-treated myocytes. CCh inhibited peak ICa recorded in the presence of Iso by ∼20% in both the control and detubulated myocytes. Reducing ICa amplitude up to 40% by changing the voltage step levels from 0 to -25 mV decreased Ca2+ transients in formamide-treated but not in control myocytes in the presence of Iso. CCh inhibited CaMKII activity, whereas CaMKII inhibition with KN93 mimicked the effect of CCh on Ca2+ transients in formamide-treated myocytes. It was concluded that the downregulation of t-tubules coupled with the diminished efficiency of excitation-contraction coupling, increases the sensitivity of Ca2+ release and propagation to muscarinic receptor-mediated inhibition of both ICa and CaMKII activity.

Functional Characteristics and Regulated Expression of Alternatively Spliced Tissue Factor: An Update.

In human and mouse, alternative splicing of tissue factor's primary transcript yields two mRNA species: one features all six TF exons and encodes full-length tissue factor (flTF), and the other lacks exon 5 and encodes alternatively spliced tissue factor (asTF). flTF, which is oftentimes referred to as "TF", is an integral membrane glycoprotein due to the presence of an alpha-helical domain in its C-terminus, while asTF is soluble due to the frameshift resulting from the joining of exon 4 directly to exon 6. In this review, we focus on asTF-the more recently discovered isoform of TF that appears to significantly contribute to the pathobiology of several solid malignancies. There is currently a consensus in the field that asTF, while dispensable to normal hemostasis, can activate a subset of integrins on benign and malignant cells and promote outside-in signaling eliciting angiogenesis; cancer cell proliferation, migration, and invasion; and monocyte recruitment. We provide a general overview of the pioneering, as well as more recent, asTF research; discuss the current concepts of how asTF contributes to cancer progression; and open a conversation about the emerging utility of asTF as a biomarker and a therapeutic target.

A First-In-Class, Humanized Antibody Targeting Alternatively Spliced Tissue Factor: Preclinical Evaluation in an Orthotopic Model of Pancreatic Ductal Adenocarcinoma.

In 2021, pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer deaths in the United States. This is largely due to a lack of symptoms and limited treatment options, which extend survival by only a few weeks. There is thus an urgent need to develop new therapies effective against PDAC. Previously, we have shown that the growth of PDAC cells is suppressed when they are co-implanted with RabMab1, a rabbit monoclonal antibody specific for human alternatively spliced tissue factor (asTF). Here, we report on humanization of RabMab1, evaluation of its binding characteristics, and assessment of its in vivo properties. hRabMab1 binds asTF with a KD in the picomolar range; suppresses the migration of high-grade Pt45.P1 cells in Boyden chamber assays; has a long half-life in circulation (~ 5 weeks); and significantly slows the growth of pre-formed orthotopic Pt45.P1 tumors in athymic nude mice when administered intravenously. Immunohistochemical analysis of tumor tissue demonstrates the suppression of i) PDAC cell proliferation, ii) macrophage infiltration, and iii) neovascularization, whereas RNAseq analysis of tumor tissue reveals the suppression of pathways that promote cell division and focal adhesion. This is the first proof-of-concept study whereby a novel biologic targeting asTF has been investigated as a systemically administered single agent, with encouraging results. Given that hRabMab1 has a favorable PK profile and is able to suppress the growth of human PDAC cells in vivo, it comprises a promising candidate for further clinical development.

Enhanced Efficacy of Combination of Gemcitabine and Phosphatidylserine-Targeted Nanovesicles against Pancreatic Cancer.

Phosphatidylserine (PS) is often externalized in viable pancreatic cancer cells and is therapeutically targetable using PS-selective drugs. One of the first-line treatments for advanced pancreatic cancer disease, gemcitabine (GEM), provides only marginal benefit to patients. We therefore investigated the therapeutic benefits of combining GEM and the PS-targeting drug, saposin C-dioleoylphosphatidylserine (SapC-DOPS), for treating pancreatic ductal adenocarcinoma (PDAC). Using cell-cycle analyses and a cell surface PS-based sorting method in vitro, we observed an increase in surface PS as cells progress through the cell cycle from G1 to G2/M. We also observed that GEM treatment preferentially targets G1 phase cells that have low surface PS, resulting in an increased median surface PS level of PDAC cells. Inversely, SapC-DOPS preferentially targets high surface PS cells that are predominantly in the G2/M phase. Finally, combination therapy in subcutaneous and orthotopic PDAC tumors in vivo with SapC-DOPS and GEM or Abraxane (Abr)/GEM (one of the current standards of care) significantly inhibits tumor growth and increases survival compared with individual treatments. Our studies confirm a surface PS and cell cycle-based enhancement of cancer cytotoxicity following SapC-DOPS treatment in combination with GEM or Abr/GEM. Thus, PDAC patients treated with Abr/GEM may benefit from concurrent administration of SapC-DOPS.

Alternatively spliced tissue factor levels are elevated in the plasma of patients with chronic liver diseases.

OBJECTIVES: In patients with chronic liver diseases, hypercoagulability can contribute to the progression of fibrosis and complications of cirrhosis. Tissue factor (TF) is a transmembrane glycoprotein that initiates the extrinsic pathway of blood coagulation. Recent investigations have established that TF is elevated in patients with pancreatic cancer, blood disorders, diabetes, and cardiovascular disease. Alternatively spliced tissue factor (asTF), a secreted form of TF, induces angiogenesis and exhibits low-level procoagulant activity. The aim of this study was to investigate whether the circulating levels of asTF are elevated in the plasma of patients with liver disease. MATERIALS AND METHODS: In a single-center study, we retrospectively analyzed asTF plasma levels in healthy participants and patients having stage F0-F3 liver fibrosis, liver cirrhosis, as well as hepatocellular carcinoma (HCC). AsTF plasma levels were measured using a sandwich enzyme-linked immunosorbent assay. Values were expressed as median with interquartile range (IQR). RESULTS: The lowest median plasma asTF concentration (94 pg/ml, IQR: 33-275) was found in the healthy control group. The patients with low-grade liver fibrosis (F0-F1 group) displayed the highest median asTF concentration (404 pg/ml, IQR: 277-789). Significant differences between the asTF levels in the plasma of healthy participants and those in patients with grade F0-F1 fibrosis (P<0.001), patients with grade F2-F3 fibrosis (P=0.019), patients with cirrhosis (P=0.004), and patients with HCC (P<0.001) were found using a Wilcoxon rank-sum test. Treatment-naive patients with HCC had significantly higher asTF levels (P=0.018) than those receiving treatment. AsTF levels were found to increase with worsening Child-Pugh scores and heightened liver disease activity. CONCLUSION: AsTF levels are elevated in patients with chronic liver diseases, which increase with worsening Child-Pugh scores and decrease following HCC therapy.

A single high-fat meal provokes pathological erythrocyte remodeling and increases myeloperoxidase levels: implications for acute coronary syndrome.

High-fat meal (HFM) consumption can produce acute lipemia and trigger myocardial infarction in patients with atherosclerosis, but the mechanisms are poorly understood. Erythrocytes (red blood cells, RBCs) intimately interact with inflammatory cells and blood vessels and play a complex role in regulating vascular function. Chronic high-fat feeding in mice induces pathological RBC remodeling, suggesting a novel link between HFM, RBCs, and vascular dysfunction. However, whether acute HFM can induce RBC remodeling in humans is unknown. Ten healthy individuals were subjected to biochemical testing and assessment of endothelial-dependent flow-mediated dilation (FMD) before and after a single HFM or iso-caloric meal (ICM). Following the HFM, triglyceride, cholesterol, and free fatty acid levels were all significantly increased, in conjunction with impaired post-prandial FMD. Additionally, peripheral blood smears demonstrated microcytes, remodeled RBCs, and fatty monocytes. Increased intracellular ROS and nitration of protein band 3 was detected in RBCs following the HFM. The HFM elevated plasma and RBC-bound myeloperoxidase (MPO), which was associated with impaired FMD and oxidation of HDL. Monocytic cells exposed to lipid in vitro released MPO, while porcine coronary arteries exposed to fatty acids ex vivo took up MPO. We demonstrate in humans that a single HFM induces pathological RBC remodeling and concurrently elevates MPO, which can potentially enter the blood vessel wall to trigger oxidative stress and destabilize vulnerable plaques. These novel findings may have implications for the short-term risk of HFM consumption and alimentary lipemia in patients with atherosclerosis.

Deletion of the Duffy antigen receptor for chemokines (DARC) promotes insulin resistance and adipose tissue inflammation during high fat feeding.

OBJECTIVE: Inflammation in adipose tissues in obesity promotes insulin resistance and metabolic disease. The Duffy antigen receptor for chemokines (DARC) is a promiscuous non-signaling receptor expressed on erythrocytes and other cell types that modulates tissue inflammation by binding chemokines such as monocyte chemoattractant protein-1 (MCP-1) and by acting as a chemokine reservoir. DARC allelic variants are common in humans, but the role of DARC in modulating obesity-related metabolic disease is unknown. METHODS: We examined body weight gain, tissue adiposity, metabolic parameters and inflammatory marker expression in wild-type and DARC knockout mice fed a chow diet (CD) and high fat diet (HFD). RESULTS: Compared to wild-type mice, HFD-fed DARC knockout mice developed glucose intolerance and insulin resistance independent of increases in body weight or adiposity. Interestingly, insulin sensitivity was also diminished in lean male DARC knockout mice fed a chow diet. Insulin production was not reduced by DARC gene deletion, and plasma leptin levels were similar in HFD fed wild-type and DARC knockout mice. MCP-1 levels in plasma rose significantly in the HFD fed wild-type mice, but not in the DARC knockout mice. Conversely, adipose tissue MCP-1 levels were higher, and more macrophage crown-like structures were detected, in the HFD fed DARC knockout mice as compared with the wild-type mice, consistent with augmented adipose tissue inflammation that is not accurately reflected by plasma levels of DARC-bound MCP-1 in these mice. CONCLUSIONS: These findings suggest that DARC regulates metabolic function and adipose tissue inflammation, which may impact obesity-related disease in ethnic populations with high frequencies of DARC allelic variants.