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Neutrophils are the most abundant circulating white blood cells and one of the major cell types of the innate immune system. Neutrophil extracellular traps (NETs) are a result of the extracellular release of nuclear chromatin from the ruptured nuclear envelope and plasma membrane. The externalized chromatin is an ancient defense weapon for animals to entrap and kill microorganisms in the extracellular milieu, thus protecting animals ranging from lower invertebrates to higher vertebrates. Although the externalized chromatin has the advantage of acting as anti-infective to protect against infections, extracellular chromatin might be problematic in higher vertebrate animals as they have an adaptive immune system that can trigger further immune or autoimmune responses. NETs and their associated nuclear and/or cytoplasmic components may induce sterile inflammation, immune, and autoimmune responses, leading to various human diseases. Though important in human pathophysiology, the cellular and molecular mechanisms of NET formation (also called NETosis) are not well understood. Given that nuclear chromatin forms the backbone of NETs, the nucleus is the root of the nuclear DNA extracellular traps. Thus, nuclear chromatin decondensation, along with the rupture of nuclear envelope and plasma membrane, is required for nuclear chromatin extracellular release and NET formation. So far, most of the literature focuses on certain signaling pathways, which are involved in NET formation but without explanation of cellular events and morphological changes described above. Here, we have summarized emerging evidence and discuss new mechanistic understanding, with our perspectives, in NET formation in neutrophils.

A facile “plug-and-go” strategy for the fabrication of Janus membranes (JMs) is proposed that uses different combinational substrates with opposite wettability, and some suitable substrates are tightly integrated through a certain pressure. The excellent compactness and controllability of the optimal JMs using foam Ni and superhydrophilic Cu mesh are demonstrated in multifunctional applications, including unidirectional liquid transportation in air–water systems, on-demand immiscible oil–water separation, droplet collection in liquid–liquid systems, and on–off switch control. The dual function of the resulting JMs as water and oil diodes is attributed to the interpenetrating structure and similar thicknesses of the formed hydrophobic and hydrophilic layers, which minimize the penetration distance of the Janus membrane in both directions. This strategy provides a common route for the fabrication of novel composite materials with greater diversity and composability regarding membrane pore size and thickness.

Design and fabrication of Janus interface materials as diodes for both liquids and gases are a great challenge in the field of materials science. Here, a Janus titanium mesh with superhydrophobicity and high oleophobicity was fabricated using economical and environmentally friendly electrochemical etching and wet-chemistry methods. Switchable, spontaneous, and unidirectional transport ability for both water and oil fluids was realized in the smart mesh by ultraviolet (UV) irradiation. Unidirectional fluid transport only occurred for fluids with specific surface tension values, and the fluid selectivity was dependent on the UV irradiation time. Underwater bubble unidirectional permeation through the mesh was also achieved using a mesh irradiated for approximately 7 h. More importantly, the mechanism is proposed and elucidated in detail, and the proposed mechanism can more reasonably explain the antigravity unidirectional transport of liquid droplets.

In idiopathic (primary) membranous glomerulopathy (MGN), there is a phenomenon of subepithelial deposits (stages 1 and 2) transitioned to intramembranous deposits, with lucent resolving features (stages 3 and 4). This phenomenon has not been described in other types of immune complex mediated glomerulonephritis with either subendothelial or mesangial deposits. The goal of this study was to evaluate what unique immunostaining pattern could occur in primary MGNs with intramembranous resolving features. PLA2R and IgG4 immunostains were performed in 50 primary MGNs, and 39 secondary MGNs after the clinical history was reviewed. Primary MGNs with resolving features were further evaluated in detail. A total of 84% (42/50) of primary MGN cases had diffuse positive immunostaining for IgG4 in the glomeruli, and most of them were also positive for PLA2R staining. Eight of the remaining primary MGN cases (8/50) with positive PLA2R but negative IgG4 staining in the glomeruli had diffuse resolving features as observed by electron microscopy. All secondary MGNs were stained negatively for both IgG4 and PLA2R except for one case with positive IgG4 staining but negative staining for PLA2R. Our data indicate that IgG4 staining on paraffin tissue is a very reliable screening tool to confirm the presence of primary MGN. Primary MGN with PLA2R+/IgG4- stains were seen in those with intramembranous resolving features. This finding is consistent with the known weak-binding capacity of IgG4 to the glomerular basement membranes. The transitional phenomenon from PLA2R+/IgG4+ subepithelial deposits to PLA2R+/IgG4- intramembranous resolving deposits in primary MGN implies that there may be a continuous metabolic activity from podocyte to glomerular basement membrane.

Systemic vasculitis is diverse group of autoimmune disorders which are characterized by inflammation of blood vessel walls with deep aching and burning pain. Their underlying etiology and pathophysiology still remain poorly understood. Extracellular vesicles (EVs), including exosomes, microvesicles (MVs), and apoptotic bodies, are membrane vesicular structures that are released either during cell activation, or when cells undergo programmed cell death, including apoptosis, necroptosis, and pyroptosis. Although EVs were thought as cell dusts, but now they have been found to be potently active since they harbor bioactive molecules, such as proteins, lipids, nucleic acids, or multi-molecular complexes. EVs can serve as novel mediators for cell-to-cell communications by delivery bioactive molecules from their parental cells to the recipient cells. Earlier studies mainly focused on MVs budding from membrane surface. Recent studies demonstrated that EVs may also carry molecules from cytoplasm or even from nucleus of their parental cells, and these EVs may carry autoantigens and are important in vasculitis. EVs may play important roles in vasculitis through their potential pathogenic involvements in inflammation, autoimmune responses, procoagulation, endothelial dysfunction/damage, angiogenesis, and intimal hyperplasia. EVs have also been used as specific biomarkers for diagnostic use or disease severity monitoring. In this review, we have focused on the aspects of EV biology most relevant to the pathogenesis of vasculitis, discussed their perspective insights, and summarized the exist literature on EV relevant studies in vasculitis, therefore provides an integration of current knowledge regarding the novel role of EVs in systemic vasculitis.

Systemic vasculitis is a heterogeneous group of multisystem autoimmune disorders characterized by inflammation of blood vessels. Although many progresses in diagnosis and immunotherapies have been achieved over the past decades, there are still many unanswered questions about vasculitis from pathological understanding to more advanced therapeutics. Extracellular vesicles (EVs) are double-layer phospholipid membrane vesicles harboring various cargoes. EVs can be classified into exosomes, microvesicles (MVs), and apoptotic bodies depending on their size and origin of cellular compartment. EVs can be released by almost all cell types and may be involved in physical and pathological processes including, inflammation and autoimmune responses. In systemic vasculitis, EVs may have pathogenic involvement in inflammation, autoimmune responses, thrombosis, endothelium injury, angiogenesis and intimal hyperplasia. EV-associated redox reaction may also be involved in vasculitis pathogenesis by inducing inflammation, endothelial injury and thrombosis. Additionally, EVs may serve as specific biomarkers for diagnosis or monitoring of disease activity and therapeutic efficacy, i.e. AAV-associated renal involvement. In this review, we have discussed the recent advances of EVs, especially their roles in pathogenesis and clinical involvements in vasculitis.

The kidney is a complicated and important internal organ receiving approximately 20% of the cardiac output and mediates numerous pathophysiologic actions. These include selectively filtering macromolecules of the blood, exquisite reclaimation of electrolyctes, urine concentration via an elegant osmotic mechanisms, and excretion of an acid load. In addition, the renal tubules carry out secretory functions and produce hormones and cytokines. The kidney receives innervation and hormonal regulation. Therefore, dysfunction of the kidney leads to retention of metabolic waste products, and/or significant proteinuria and hematuria. In the past several decades, the role of extracellular vesicles (EV) in intercellular communications, and the uptake of extracellular vesicles by recipient cells through phagocytosis and endocytosis have been elucidated. The new knowledge on EVs expands over the classical mechanisms of cellular interaction, and may change our way of thinking of renal pathophysiology in the subcellular scale. Based on some ultrastructural discoveries in the kidney, this review will focus on the role of extracellular vesicles in intercellular communications, their internalization by recipient cells, and their relationship to renal pathology.

The nuclear lamina is essential for the structural integration of the nuclear envelope. Nuclear envelope rupture and chromatin externalization is a hallmark of the formation of neutrophil extracellular traps (NETs). NET release was described as a cellular lysis process; however, this notion has been questioned recently. Here, we report that during NET formation, nuclear lamin B is not fragmented by destructive proteolysis, but rather disassembled into intact full-length molecules. Furthermore, we demonstrate that nuclear translocation of PKCα, which serves as the kinase to induce lamin B phosphorylation and disassembly, results in nuclear envelope rupture. Decreasing lamin B phosphorylation by PKCα inhibition, genetic deletion, or by mutating the PKCα consensus sites on lamin B attenuates extracellular trap formation. In addition, strengthening the nuclear envelope by lamin B overexpression attenuates NET release in vivo and reduces levels of NET-associated inflammatory cytokines in UVB-irradiated skin of lamin B transgenic mice. Our findings advance the mechanistic understanding of NET formation by showing that PKCα-mediated lamin B phosphorylation drives nuclear envelope rupture for chromatin release in neutrophils.

Hepatocellular injury is the pathological hallmark of hepatitis and a crucial driver for the progression of liver diseases, while the treatment options are commonly restricted. Interleukin-22 (IL-22) has attracted special attention as a potent survival factor for hepatocytes that both prevents and repairs the injury of hepatocytes through activation of STAT3 signaling pathway. We hypothesized that the ability to generate potent expression of IL-22 locally for the treatment of severe hepatocellular injury in hepatitis was a promising strategy to enhance efficacy and overcome off-target effects. Accordingly, we developed a polypeptide penetratin-based hybrid nanoparticle system (PDPIA) carrying IL-22 gene by a self-assembly process. This nanocomplex modified with penetratin featured direct translocation across the cellular or endosomal membrane but mild zeta-potential to facilitate the high cellular internalization and endosomal escape of the gene cargos as well as scarcely Kupffer cells uptake. More importantly, PDPIA afforded preferential liver accumulation and predominant hepatocytes internalization following systemic administration, which showed pharmacologically suitable organ and sub-organ-selective properties. Subsequent studies confirmed a considerable protective role of PDPIA in a model of severe hepatitis induced by concanavalin A, evidenced by reduced hepatocellular injury and evaded immune response. The locally expressed IL-22 by PDPIA activated STAT3/Erk signal transduction, and thus promoted hepatocyte regeneration, inhibited reactive oxygen species (ROS) accumulation as well as prevented the dysfunction of mitochondrial. In addition, this system did not manifest side effects or systemic toxicity in mice. Collectively, the high versatility of PDPIA rendered its promising applications might be an effective agent to treat various hepatic disorders.

Microvesicles (MVs) have been shown to be involved in pathophysiology of ischemic heart diseases. However, the underlying mechanisms are still unclear. Here we investigated the effects of MVs derived from ischemic preconditioning (IPC-MVs) on myocardial ischemic/reperfusion (I/R) injury in rats. Myocardial IPC model was elicited by three cycles of ischemia and reperfusion of the left anterior descending (LAD) coronary artery. IPC-MVs from the peripheral blood of the above animal model were isolated by ultracentrifugation and characterized by flow cytometry and transmission electron microscopy. IPC-MVs were administered intravenously (7 mg/kg) at 5 min before reperfusion procedure in I/R injury model which was induced by 30-min ischemia and 120-min reperfusion of LAD in rats. We found that total IPC-MVs and different phenotypes, including platelet-derived MVs (PMVs), endothelial cell-derived MVs (EMVs), leucocyte-derived MVs and erythrocyte-derived MVs (RMVs) were all isolated which were identified membrane vesicles (

// Yao Wang 1 , Su Wei 1 , Yi-Lu Wang 1 , Miao Liu 1 , Man Shang 1 , Qi Zhang 4 , Yan-Na Wu 1 , Ming-Lin Liu 2, 3 , Jun-Qiu Song 1 and Yan-Xia Liu 1 1 Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China 2 Section of Endocrinology, Department of Medicine, Temple University School of Medicine, Philadelphia, PA19140, USA 3 Section of Endocrinology, Department of Medicine, Temple University School of Medicine, Philadelphia, PA 19140, USA 4 Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Correspondence to: Yan-Xia Liu, email: liu_yanxia126@126.com Jun-Qiu Song, email: song_junqiu@126.com Keywords: microvesicles, ischemia/reperfusion, apoptosis, Bcl-2/Bax, GRP78 Received: October 17, 2016 Accepted: March 28, 2017 Published: April 26, 2017 ABSTRACT Objective: To investigate the effects of circulating microvesicles derived from myocardial ischemia (I-MVs) on apoptosis in myocardial ischemia/reperfusion (I/R) injury in rats. Methods: I-MVs from rats undergoing myocardial left anterior descending (LAD) coronary artery ligation were isolated by ultracentrifugation from circulating blood and characterized by flow cytometry. I-MVs were administered intravenously (4.8 mg/kg) at 5 min before reperfusion procedure in I/R injury model which was induced by 30-min of ischemia and 120-min of reperfusion of LAD in rats. Results: Treatment with I-MVssignificantly reduced the size of myocardial infarction, the activities of serum CK-MB and LDH, and the number of apoptotic cardiomyocytes. The activities of caspase 3, caspase 9 and caspase 12 in myocardium were also decreased significantly with I-MVs treatment. Moreover, the expression of Bax was decreased but Bcl-2 was increased. The expression of glucose regulated protein 78 (GRP78), sarco/endoplasmic reticulum Ca 2+ -ATPase 2 (SERCA2) and phosphorylated phospholamban (p-PLB) were increased after being treated with I-MVs. Conclusion: I-MVs could protect hearts from I/R injury in rats through SERCA2 and p-PLB of calcium regulatory proteins to alleviate intrinsic myocardial apoptosis including mitochondrial and endoplasmic reticulum pathways.

Neutrophil extracellular trap formation (NETosis) is a dynamic process featuring nuclear chromatin extrusion and extracellular trap formation (1) in which ruptures of nuclear envelope and plasma membrane are prerequisite events. Thiam et al. (1) analyze several important cellular events, including cytoskeleton organization, during NETosis using time-lapse microscopy. They conclude that NETosis begins with rapid disassembly of actin cytoskeleton, based on the microscopy analysis of a few hundred neutrophils (1). However, other studies have found that pharmacologic (2, 3) or genetic (4) inhibition of actin assembly decreases NET formation, indicating the role of functional actin cytoskeleton in NETosis induction. To systematically explore the dynamic changes of actin, our time course study with flow cytometry analysis … [↵][1]1Email: lium1{at}pennmedicine.upenn.edu. [1]: #xref-corresp-1-1

Extracellular vesicles (EVs) including exosomes, microvesicles (MVs), and apoptotic bodies, are small membrane vesicular structures that are released during cell activation, senescence, or programmed cell death, including apoptosis, necroptosis, and pyroptosis. EVs serve as novel mediators for long-distance cell-to-cell communications and can transfer various bioactive molecules, such as encapsulated cytokines and genetic information from their parental cells to distant target cells. In the context of obesity, adipocyte-derived EVs are implicated in metabolic homeostasis serving as novel adipokines. In particular, EVs released from brown adipose tissue or adipose-derived stem cells may help control the remolding of white adipose tissue towards browning and maintaining metabolic homeostasis. Interestingly, EVs may even serve as mediators for the transmission of metabolic dysfunction across generations. Also, EVs have been recognized as novel modulators in various metabolic disorders, including insulin resistance, diabetes mellitus, and non-alcoholic fatty liver disease. In this review, we summarize the latest progress from basic and translational studies regarding the novel effects of EVs on metabolic diseases. We also discuss EV-mediated cross-talk between adipose tissue and other organs/tissues that are relevant to obesity and metabolic diseases, as well as the relevant mechanisms, providing insight into the development of new therapeutic strategies in obesity and metabolic diseases.

Human insulin-like growth factor-1 (IGF-1) plays important roles in development and regeneration of skeletal muscles and bones but requires daily injections or surgical implantation. Current clinical IGF-1 lacks e-peptide and is glycosylated, reducing functional efficacy. In this study, codon-optimized Pro-IGF-1 with e-peptide (fused to GM1 receptor binding protein CTB or cell penetrating peptide PTD) was expressed in lettuce chloroplasts to facilitate oral delivery. Pro-IGF-1 was expressed at high levels in the absence of the antibiotic resistance gene in lettuce chloroplasts and was maintained in subsequent generations. In lyophilized plant cells, Pro-IGF-1 maintained folding, assembly, stability and functionality up to 31 months, when stored at ambient temperature. CTB-Pro-IGF-1 stimulated proliferation of human oral keratinocytes, gingiva-derived mesenchymal stromal cells and mouse osteoblasts in a dose-dependent manner and promoted osteoblast differentiation through upregulation of ALP, OSX and RUNX2 genes. Mice orally gavaged with the lyophilized plant cells significantly increased IGF-1 levels in sera, skeletal muscles and was stable for several hours. When bioencapsulated CTB-Pro-IGF-1 was gavaged to femoral fractured diabetic mice, bone regeneration was significantly promoted with increase in bone volume, density and area. This novel delivery system should increase affordability and patient compliance, especially for treatment of musculoskeletal diseases.

Excessive release of interleukin-1β (IL-1β) is well-known to provoke cascades of inflammatory responses thus contributing to the pathogenesis of alcohol-induced steatohepatitis (ASH), but the cellular mechanism that regulates IL-1β release during ASH remains unclear. Herein, we identified that gasdermin D (GSDMD) membrane pore is critical in mediating IL-1β hypersecretion from chronic ethanol or acetaldehyde-stimulated macrophages. Deletion of GSDMD reduced IL-1β release and ameliorated alcoholic steatohepatitis in vivo. These findings uncovered a novel mechanism regarding the IL-1β release in ASH, and also indicated the therapeutic potential of IL-1β blockade. Interleukin-1 receptor antagonist (IL-1Ra) is protective to ASH by blocking IL-1β, but it has a short biological half-life (4–6 h) and lower liver concentrations. Thus, we constructed a therapeutic plasmid pVAX1-IL-1Ra-ApoAI (pVAX1-IA) encoding IL-1Ra anchored to the liver-targeting protein apolipoprotein A-I (ApoAI), and developed hepatocyte-specific nanobiologics (Glipo-pVAX1-IA) by galactose functionalization for local and prolonged expression of IL-1Ra in liver. Data presented here showed that Glipo-pVAX1-IA facilitated efficient uptake of gene cargos by hepatocytes. The biodistribution studies confirmed a predominant hepatocytes internalization, but a minimal kupffer cells uptake of Glipo-pVAX1-IA following intravenous injection. The locally secreted IL-1Ra attenuated alcohol-induced steatohepatisis and infiltration of inflammatory cells. Together, our results unraveled the critical role of GSDMD membrane pore in IL-1β hypersecretion and highlighted the hepatocyte-specific Glipo-pVAX1-IA nanobiologics as a promising therapeutic strategy for ASH.

It’s not clear how nuclear envelope (NE) is ruptured for chromatin externalization during NETosis. The membrane rupture during neutrophil NET release was described as a membrane lysis process, this notion, however, has been questioned. Here, we found that lamin B, the structural NE component, was involved in NETosis. Unexpectedly, lamin B was not fragmented by destructive proteolysis, but rather disassembled into its intact full-length molecule, in NETotic cells with ruptured NE. In the mechanistic study, our experiments demonstrated that cytosolic PKCα translocated to the nucleus, where it serves as a NETotic lamin kinase to induce lamin B phosphorylation, following by lamina disassembly and NE rupture. To determine causality, we found that decreasing lamin B phosphorylation, by PKCα inhibition or genetic deletion, or mutation at the PKCα consensus phosphorylation sites of lamin B, attenuated extracellular trap formation. Importantly, strengthening NE by lamin B overexpression attenuated neutrophil NETosisin vivoand alleviated exhibition of NET-associated inflammatory cytokines in UVB irradiated skin of lamin B transgenic mice. These findings advance our understanding of NETosis process and elucidate a cellular mechanism that PKCα-mediated lamin B phosphorylation drives nuclear envelope rupture for NET release in neutrophils.Graphical Abstract

High mobility group box 1 (HMGB1) is a nuclear protein that can be released from activated or dead cells. Extracellular HMGB1 can serve as a "danger signal" and novel cytokine that mediates sterile inflammation. In addition to its soluble form, extracellular HMGB1 can also be carried by membrane microvesicles. However, the cellular mechanisms responsible for nuclear HMGB1 translocation to the plasma membrane and release onto membrane microvesicles have not been investigated. Tobacco smoking is a major cause of sterile inflammation in many diseases. Smoking also increases blood levels of HMGB1. In this study, we found that exposure of macrophages to tobacco smoke extract (TSE) stimulated HMGB1 expression, redistribution, and release into the extracellular milieu both as a soluble molecule and, surprisingly, as a microvesicle-associated form (TSE-MV). Inhibition of chromosome region maintenance-1 (CRM1), a nuclear exporter, attenuated TSE-induced HMGB1 redistribution from the nucleus to the cytoplasm, and then its release on TSE-MVs. Our study demonstrates a novel mechanism for the translocation of nuclear HMGB1 to the plasma membrane, and then its release in a microvesicle-associated form. J. Cell. Physiol. 231: 2319-2326, 2016. © 2016 Wiley Periodicals, Inc.

Lupus nephritis (LN) is a major cause for overall morbidity and mortality in patients with systemic lupus erythematosus (SLE), while its pathogenic mechanisms are still not well understood. Extracellular vesicles (EVs) are membrane vesicles that are released from almost all cell types. EVs can be subdivided into exosomes, microvesicles, and apoptotic bodies. Latest studies have shown that EVs can be released during several cellular events, including cell activation, autophagy, and several types of programed cell death, i.e. apoptosis, necroptosis, pyroptosis, and NETosis. Emerging evidence demonstrates that EVs harbor different bioactive molecules, including nucleic acids, proteins, lipids, cytokines, immune complexes (ICs), complements, and other molecules, some of which may contribute to pathogenesis of autoimmune diseases. EVs can serve as novel information shuttle to mediate local autocrine or paracrine signals to nearby cells, and distant endocrine signals to cells located far away. In LN, EVs may have pathogenic effects by transportation of autoantigens or complements, promotion of IC deposition or complement activation, and stimulation of inflammatory responses, renal tissue injury, or microthrombus formation. Additionally, EVs released from kidney cells may serve as specific biomarkers for diagnosis or monitoring of disease activity and therapeutic efficacy. In this review, we will summarize the latest progress about EV generation from basic research, their potential pathologic effects on LN, and their clinical implications. The cutting-edge knowledge about EV research provides insights into novel therapeutic strategy, new tools for diagnosis or prognosis, and evaluation approaches for treatment effectiveness in LN.

Objective— This study aims to determine whether and how the enriched metabolites of endothelial extracellular vesicles (eEVs) are critical for cigarette smoke-induced direct injury of endothelial cells and the development of pulmonary hypertension, rarely explored in contrast to long-investigated mechanisms secondary to chronic hypoxemia. Approach and Results— Metabonomic screen of eEVs from cigarette-smoking human subjects reveals prominent elevation of spermine—a polyamine metabolite with potent agonist activity for the extracellular CaSR (calcium-sensing receptor). CaSR inhibition with the negative allosteric modulator Calhex231 or CaSR knockdown attenuates cigarette smoke-induced pulmonary hypertension in rats without emphysematous changes in lungs or chronic hypoxemia. Cigarette smoke exposure increases the generation of spermine-positive eEVs and their spermine content. Immunocytochemical staining and immunogold electron microscopy recognize the spermine enrichment not only within the cytosol but also on the outer surface of eEV membrane. The repression of spermine synthesis, the inhibitory analog of spermine, N 1 -dansyl-spermine, Calhex231, or CaSR knockdown profoundly suppresses eEV exposure-mobilized cytosolic calcium signaling, pulmonary artery constriction, and smooth muscle cell proliferation. Confocal imaging of immunohistochemical staining demonstrates the migration of spermine-positive eEVs from endothelium into smooth muscle cells in pulmonary arteries of cigarette smoke-exposed rats. The repression of spermine synthesis or CaSR knockout results in attenuated development of pulmonary hypertension induced by an intravascular administration of eEVs. Conclusions— Cigarette smoke enhances eEV generation with spermine enrichment at their outer surface and cytosol, which activates CaSR and subsequently causes smooth muscle cell constriction and proliferation, therefore, directly leading to the development of pulmonary hypertension.

Microvesicles (MVs, also known as microparticles) are small vesicles that originate from plasma membrane of almost all eukaryotic cells during apoptosis or activation. MVs can serve as extracellular vehicles to transport bioactive molecules from their parental cells to recipient target cells, thereby serving as novel mediators for intercellular communication. Importantly, more and more evidence indicates that MVs could play important roles in early pathogenesis and subsequent progression of cardiovascular and metabolic diseases. Elevated plasma concentrations of MVs, originating from red blood cells, leukocytes, platelets, or other organs and tissues, have been reported in various cardiometabolic diseases. Circulating MVs could serve as potential biomarkers for disease diagnosis or therapeutic monitoring. In this review, we summarized recently-published studies in the field and discussed the role of MVs in the pathogenesis of cardiometabolic diseases. The emerging values of MVs that serve as biomarker for non-invasive diagnosis and prognosis, as well as their roles as novel therapeutic targets in cardiometabolic diseases, were also described. Keywords: Microvesicle, Microparticle, Exosome, Cardiometabolic disease, Biomarker

Microvesicles (MVs), also known as microparticles, are small membrane vesicles released from different cell types under different conditions. MVs have been detected in the circulation and in organs/tissues in various diseases, including diabetes. Patients with different types of diabetes and complications have different cellular MV patterns. Studies have shown that MVs may mediate vascular thrombosis, vascular inflammation, angiogenesis, and other pathological processes of the disease through their procoagulant, pro-inflammatory, pro-angiogenic, proteolytic, and other properties. Therefore, MVs contribute to the development of diabetic macrovascular and microvascular complications. In addition, clinical studies have indicated that changes in MV number and composition may reflect the pathophysiological conditions of disease, and therefore, may serve as potential biomarkers for diagnostic and prognostic use. Understanding MVs' involvement in the pathophysiological conditions may provide insight into disease mechanisms and would also be helpful for the development of novel therapeutic strategies in the future. Here, we review the latest publications from our group and other groups and focus on the involvement of MVs in diabetic complications.

We have read with great interest the article by Kato and colleagues regarding the ‘Apoptosis-derived membrane vesicles drive the cGAS–STING pathway and enhance type I IFN production in systemic lupus erythematosus’.1 However, we are concerned that the authors studied exclusively blood serum, that is, all blood samples from their patients with lupus and healthy controls were coagulated ex vivo before analysis. At least three major changes can occur to the population of membrane microvesicles (MVs) during coagulation in a test tube. First, a subset of circulating MVs participate in, and become consumed by, clotting. For example, studies from our group and others indicated that over half of the total membrane MVs in blood plasma …

Endoplasmic reticulum (ER) stress is increased in obesity and is postulated to be a major contributor to many obesity-related pathologies. Little is known about what causes ER stress in obese people. Here, we show that insulin upregulated the unfolded protein response (UPR), an adaptive reaction to ER stress, in vitro in 3T3-L1 adipocytes and in vivo, in subcutaneous (sc) adipose tissue of nondiabetic subjects, where it increased the UPR dose dependently over the entire physiologic insulin range (from ∼35 to ∼1,450 pmol/L). The insulin-induced UPR was not due to increased glucose uptake/metabolism and oxidative stress. It was associated, however, with increased protein synthesis, with accumulation of ubiquitination associated proteins, and with multiple posttranslational protein modifications (acetylations, methylations, nitrosylations, succinylation, and ubiquitinations), some of which are potential causes for ER stress. These results reveal a new physiologic role of insulin and provide a putative mechanism for the development of ER stress in obesity. They may also have clinical and therapeutic implications, e.g., in diabetic patients treated with high doses of insulin.

The objectives of safety evacuation in the subway were directly related to the safety of Personal life. How to effectively ease the evacuation of the public crowded and prevent casualties had become a focus in the domain of current domestic and international public safety, as well as an important component of MRT design. Once emergency happened, the capability of emergency evacuation is the important factor to reduce the loss and ensure the life safety. Based on the analysis of characteristics of subway station emergency established on the basis of evaluation index system using fuzzy network analysis to subway station emergency evacuation capacity in comprehensive evaluation Based on the analysis of subway station emergency characteristics to established evaluation index system and used fuzzy network analysis to evaluate the subway station emergency evacuation capacity in comprehensive.

Diabetic retinopathy (DR) is a common complication of diabetes and has been recognized as the leading cause of blindness in adults. Several interrelated molecular pathways are involved in the development of DR. Microvesicles (MVs) are cell membrane vesicles, which carry many biologic molecules, such as mRNAs, microRNAs, transcription factors, membrane lipids, membrane receptors, and other proteins. They may be involved in intercellular communication that can promote inflammation, angiogenesis, and coagulation. Recent studies have indicated that changes in the number and composition of MVs may reflect the pathologic conditions of DR. At present, MVs are well recognized as being involved in the pathophysiological conditions of tumors and cardio-metabolic diseases. However, the roles of MVs in DR have yet to be investigated. In this review, we provide an overview of DR-induced microvascular injury that is caused by MVs derived from endothelial and circulating cells, and discuss the possible mechanisms by which MVs can lead to endothelial dysfunction, coagulation and inflammation. In addition, the protective effects of preconditioned MVs and stem cell-derived MVs are also described . Understanding the involvement of MVs in the pathophysiological conditions of DR may provide insight into the disease mechanisms and may suggest novel therapeutic strategies for DR in the future.

Background Vascular endothelial dysfunction is the closely related determinant of ischemic heart disease (IHD). Endothelial dysfunction and ischemia/reperfusion injury (IRI) have been associated with an increase in microvesicles (MVs) in vivo. However, the potential contribution of endothelial microvesicles (EMVs) to myocardial damage is unclear. Here we aimed to investigate the role of EMVs derived from hypoxia/reoxygenation (H/R) -treated human umbilical vein endothelial cells (HUVECs) on cultured H9c2 cardiomyocytes. Results H/R injury model was established to induce HUVECs to release H/R-EMVs. The H/R-EMVs from HUVECs were isolated from the conditioned culture medium and characterized. H9c2 cardiomyocytes were then incubated with 10, 30, 60 μg/mL H/R-EMVs for 6 h. We found that H9c2 cells treated by H/R-EMVs exhibited reduced cell viability, increased cell apoptosis and reactive oxygen species (ROS) production. Moreover mechanism studies demonstrated that H/R-EMVs could induce the phosphorylation of p38 and JNK1/2 in H9c2 cells in a dose-dependent manner. In addition, H/R-EMVs contained significantly higher level of ROS than EMVs generated from untreated HUVECs, which might be a direct source to trigger a cascade of myocardial damage. Conclusion We showed that EMVs released during H/R injury are pro-apoptotic, pro-oxidative and directly pathogenic to cardiomyocytes in vitro. EMVs carry ROS and they may impair myocardium by promoting apoptosis and oxidative stress. These findings provide new insights into the pathogenesis of IRI.

To establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles (MVs) from myocardial ischemic preconditioning (IPC) treated rats (IPC-MVs), and to investigate the effects of IPC-MVs on ischemia/reperfusion (I/R) injury in rats.Myocardial IPC was elicited by three.cycles of 5-min ischemia and 5-min reperfusion of the left anterior descending (LAD) coronary artery. Platelet-free plasma (PFP) was isolated through two steps of centrifugation at room temperature from the peripheral blood, and IPC-MVs were isolated by ultracentrifugation from PFR PFP was incubated with anti-CD61, anti-CD144, anti-CD45 and anti-Erythroid Cells, and added 1, 2 µm latex beads to calibrate and absolutely count by flow cytometry. For functional research, I/R injury was induced by 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg were infused via the femoral vein in myocardial I/R injured rats. Mean arterial blood pressure (MAP), heart rate (HR) and ST-segment of electro-cardiogram (ECG) were monitored throughout the experiment. Changes of myocardial morphology were observed after hematoxylin-eosin (HE) staining. The activity of plasma lactate dehydrogenase (LDH) was tested by Microplate Reader. Myocardial infarct size was measured by TTC staining.Total IPC-MVs and different phenotypes, including platelet-derived MVs (PMVs), endothelial cell-derived MVs (EMVs), leucocyte-derived MVs (LMVs) and erythrocyte-derived MVs (RMVs) were all isolated which were identified membrane vesicles (1 Vm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats (P0.05, respectively). In addition, at the end of 120-min reperfusion in I/R injured rats, IPC-MVs markedly increased HR (P0.01), decreased ST-segment and LDH activity (P0.05, P0.01). The damage of myocardium was obviously alleviated and myocardial infarct size was significantly lowered after IPC-MVs treatment (P0.01).The method of flow cytometry was successfully established to detect the phenotypes and concentration alteration of IPC-MVs, including PMVs, EMVs, LMVs and RMVs. Furthermore, circulating IPC-MVs protected myocardium against I/R injury in rats.

Objective— Cholesterol enrichment occurs in vivo when phagocytes ingest retained and aggregated lipoproteins, damaged or senescent cells, and related debris. We previously reported that enrichment of human monocyte/macrophages with unesterified cholesterol (UC) triggers the release of highly procoagulant microvesicles ([MVs], also called microparticles) through induction of apoptosis. We determined whether UC-induced MVs (UCMVs) might transmit endogenous danger signals and, if so, what molecular processes might be responsible for their production, recognition, and detoxification. Methods and Results— Injection of UCMVs into rats provoked extensive leukocyte rolling and adherence to postcapillary venules in vivo. Likewise, exposure of mouse aortic explants or cultured human endothelial cells to UCMVs augmented the adhesion of human monocytes by several fold and increased endothelial cell intercellular adhesion molecule-1 via nuclear factor-κB activation. To explore molecular mechanisms, we found that UC enrichment of human monocytes, in the absence of other stimuli, induced mitochondrial complex II–dependent accumulation of superoxide and peroxides. A subset of these moieties was exported on UCMVs and mediated endothelial activation. Strikingly, aortic explants from mice lacking lectin–like oxidized low–density lipoprotein receptor-1, a pattern-recognition receptor, were essentially unable to respond to UCMVs, whereas simultaneously treated explants from wild-type mice responded robustly by increasing monocyte recruitment. Moreover, high-density lipoprotein and its associated enzyme paraoxonase-1 exerted unexpected roles in the detoxification of UCMVs. Conclusion— Overall, our study implicates MVs from cholesterol–loaded human cells as novel carriers of danger signals. By promoting maladaptive immunologic and thrombotic responses, these particles may contribute to atherothrombosis and other conditions in vivo.

High mobility group box 1 (HMGB1) is a nuclear protein that can be released from activated or dead cells. Extracellular HMGB1 can serve as a "danger signal" and novel cytokine that mediates sterile inflammation. In addition to its soluble form, extracellular HMGB1 can also be carried by membrane microvesicles. However, the cellular mechanisms responsible for nuclear HMGB1 translocation to the plasma membrane and release onto membrane microvesicles have not been investigated. Tobacco smoking is a major cause of sterile inflammation in many diseases. Smoking also increases blood levels of HMGB1. In this study, we found that exposure of macrophages to tobacco smoke extract (TSE) stimulated HMGB1 expression, redistribution, and release into the extracellular milieu both as a soluble molecule and, surprisingly, as a microvesicle-associated form (TSE-MV). Inhibition of chromosome region maintenance-1 (CRM1), a nuclear exporter, attenuated TSE-induced HMGB1 redistribution from the nucleus to the cytoplasm, and then its release on TSE-MVs. Our study demonstrates a novel mechanism for the translocation of nuclear HMGB1 to the plasma membrane, and then its release in a microvesicle-associated form. J. Cell. Physiol. 231: 2319-2326, 2016. © 2016 Wiley Periodicals, Inc.

During apoptosis or activation, cells can release a subcellular structure, called a membrane microvesicle (also known as microparticle) into the extracellular environment. Microvesicles bud-off as a portion of cell membrane with its associated proteins and lipids surrounding a cytosolic core that contains intracellular proteins, lipids, and nucleic acids (DNA, RNA, siRNA, microRNA, lncRNA). Biologically active molecules on the microvesicle surface and encapsulated within can act on recipient cells as a novel mode of intercellular communication. Apoptosis has long been known to be involved in the development of diseases of autoimmunity. Abnormally persistent microvesicles, particularly apoptotic microvesicles, can accelerate autoimmune responses locally in specific organs and tissues as well as systemically. In this review, we focus on studies implicating microvesicles in the pathogenesis of autoimmune diseases and their complications.

The effect of B2O3 addition on the sintering, microstructure and the microwave dielectric properties of LiNb0.6Ti0.5O3 ceramics have been investigated. It is found that low-level doping of B2O3 (≤2 wt.%) can significantly improve the densification and dielectric properties of LiNb0.6Ti0.5O3 ceramics. Due to the liquid phase effect of B2O3 addition, LiNb0.6Ti0.5O3 ceramics could be sintered to a theoretical density higher than 95% even at 880 °C. No secondary phase was observed for the B2O3-doped ceramics. There is no obvious degradation in dielectric properties for the ceramics with B2O3 additions. In the case of 1 wt.% B2O3 addition, the ceramics sintered at 880 °C show good microwave dielectric properties of ɛr = 70, Q × f = 5400 GHz, τf = −6.39 ppm/°C. It represents that the ceramics could be promising for multilayer low-temperature co-fired ceramics (LTCC) applications.

To investigate the effects of microvesicles (MVs) derived from hypoxia/reoxygenation (H/R)-treated human umbilical vein endothelial cells (HUVECs) on endothelium-dependent relaxation of rat thoracic aortic rings.H/R injury model was established to induce HUVECs to release H/R-EMVs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized using 1 μm latex beads and anti-PE-CD144 by flow cytometry. Thoracic aortic rings of rats were incubated with 2.5, 5, 10, 20 μg/ml H/R-EMVs derived from H/R-treated HUVECs for 4 hours, and their endothelium-dependent relaxation in response to acetylcholine (ACh) or endothelium-independent relaxation in response to sodium nitroprusside (SNP) was recorded in vitro. The nitric oxide (NO) production of ACh-treated thoracic aortic rings of rats was measured using Griess reagent. The expression of endothelial NO synthase (eNOS) and phosphorylated eNOS (p-eNOS, Ser-1177) in the thoracic aortic rings of rats was detected by Western blotting. Furthermore, the levels of SOD and MDA in H/R-EMVs-treated thoracic aortic rings of rats were measured using SOD and MDA kit.H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The membrane vesicles (1 μm) induced by H/R were CD144 positive. ACh-induced relaxation and NO production of rat thoracic aortic rings were impaired by H/R-EMVs treatment in a concentration-dependent manner (P0.05, P0.01). The expression of total eNOS (t-eNOS) was not affected by H/R-EMVs. However, the expression of p-eNOS decreased after treated with H/R-EMVs. The activity of SOD decreased and the level of MDA increased in H/R-EMVs treated rat thoracic aortic rings (P0.01).ACh induced endothelium-dependent relaxation of thoracic aortic rings of rats was impaired by H/R-EMVs in a concentration-dependent manner. The mechanisms included a decrease in NO production, p-eNOS expression and an increase in oxidative stress.

Background Cigarette smoking is associated with an increased risk of type 2 diabetes mellitus (T2DM). Smokers exhibit low circulating levels of total adiponectin (ADPN) and high-molecular-weight (HMW) ADPN multimers. Blood concentrations of HMW ADPN multimers closely correlate with insulin sensitivity for handling glucose. How tobacco smoke exposure lowers blood levels of ADPN, however, has not been investigated. In the current study, we examined the effects of tobacco smoke exposure in vitro and in vivo on the intracellular and extracellular distribution of ADPN and its HMW multimers, as well as potential mechanisms. Findings We found that exposure of cultured adipocytes to tobacco smoke extract (TSE) suppressed total ADPN secretion, and TSE administration to mice lowered their plasma ADPN concentrations. Surprisingly, TSE caused intracellular accumulation of HMW ADPN in cultured adipocytes and in the adipose tissue of wild-type mice, while preferentially decreasing HMW ADPN in culture medium and in plasma. Importantly, we found that TSE up-regulated the ADPN retention chaperone ERp44, which colocalized with ADPN in the endoplasmic reticulum. In addition, TSE down-regulated DsbA-L, a factor for ADPN secretion. Conclusions Tobacco smoke exposure traps HMW ADPN intracellularly, thereby blocking its secretion. Our results provide a novel mechanism for hypoadiponectinemia, and may help to explain the increased risk of T2DM in smokers.

The basis for accelerated atherosclerosis in diabetes is unclear. Diabetes is associated with loss of heparan sulfate (HS) from the liver, which may impede lipoprotein clearance and thereby worsen atherosclerosis. To study hepatic HS loss in diabetes, we examined regulation of HS N-deacetylase/N-sulfotransferase-1 (NDST), a key enzyme in hepatic HS biosynthesis. Hepatic NDST mRNA, protein, and enzymatic activity were suppressed by >50% 2 weeks after induction of type 1 diabetes in rats. Treatment of diabetic rats with enalapril, an ACE inhibitor, had no effect on hyperglycemia or hepatic NDST mRNA levels, yet increased hepatic NDST protein and enzymatic activity. Similar results were obtained in diabetic animals treated with losartan, which blocks the type 1 receptor for angiotensin II (AngII). Consistent with these findings, diabetic livers exhibited increased ACE expression, and addition of AngII to cultured hepatoma cells reduced NDST activity and protein. We conclude that diabetes substantially suppresses hepatic NDST mRNA, protein, and enzymatic activity. AngII contributes to suppression of NDST protein and enzymatic activity, whereas mRNA suppression occurs independently. Suppression of hepatic NDST may contribute to diabetic dyslipidemia, and stimulation of NDST activity by AngII inhibitors may provide cardiovascular protection.

Objective— Oxidized low-density lipoprotein (Ox-LDL)is implicated in the pathogenesis of atherosclerosis. Circulating oxidation-specific epitopes on plasma Ox-LDL has been linked with coronary artery disease, but its determinants and its association with early development of atherosclerosis in familial combined hyperlipidemia (FCHL) has not been very well studied. This study aimed to investigate the determinants of the circulating Ox-LDL and the association between Ox-LDL and carotid intima-media thickness (IMT) in asymptomatic members of FCHL families. Methods and Results— Ox-LDL, susceptibility of LDL to oxidation in vitro, plasma 8-isoprostane and antioxidants, lipids and lipoproteins, LDL particle size, and carotid IMT were measured in 150 asymptomatic FCHL family members. Affected FCHL family members had reduced LDL particle size and lag time for LDL oxidation, increased plasma levels of Ox-LDL, increased plasma urate and α-tocopherol, and a trend for the increase of 8-isoprostane as compared with nonaffected FCHL. Ox-LDL was independently associated with serum LDL cholesterol, apoB, and 8-isoprostane in multivariate analysis but only univariately correlated with LDL particle size and lag time for LDL oxidation. In addition, Ox-LDL was significantly associated with carotid mean IMT independently of other clinical and biochemical variables in a multivariate model. Conclusion— Serum LDL cholesterol, apoB levels, and 8-isoprostane were the most important determinants of Ox-LDL. Ox-LDL is independently associated with carotid IMT in asymptomatic FCHL family members and can be used as a marker of early atherosclerosis in FCHL.

There is increasing evidence that oxidation of low-density lipoprotein (LDL) plays an important role in atherogenesis.To explore the LDL oxidizability and its determinants in familial combined hyperlipidemia (FCHL) patients with different phenotypes.The study included 59 FCHL family members with different lipid phenotypes, 39 non-affected relatives, and 30 spouses as healthy controls.The lag time for LDL oxidation was significantly shorter in FCHL patients with different lipid phenotypes as compared to healthy controls. There were no significant differences in the propagation rate and conjugated diene formation and alpha-tocopherol content in LDL between the FCHL groups and healthy controls. Plasma concentrations of alpha-tocopherol in all FCHL patients and uric acid in FCHL patients with IIB and IV phenotypes were significantly higher than in healthy controls. Plasma total peroxyl radical trapping capacity measured (TRAPmea) and TRAPcalc tended to be higher in affected FCHL groups, but the difference was significant only for IIB phenotype. The peak LDL particle size in the combined group of FCHL patients was significantly smaller than in healthy controls. The lag time for LDL oxidation correlated significantly with LDL size both in the group of FCHL family members (r = 0.477, P0.001) and in the healthy controls (r = 0.482, P0.01).LDL from FCHL patients irrespectively of lipid phenotypes is more susceptible to oxidation in vitro than LDL from healthy controls. This increased susceptibility of LDL to oxidation in vitro seems to be attributed to the abundance of small dense LDL particles and not to the defect of antioxidant capacity in FCHL

Microparticles are small membrane fragments shed primarily from blood and endothelial cells during either activation or apoptosis. There is mounting evidence suggesting that microparticles perform a large array of biological functions and contribute to various diseases. Of these disease processes, a significant link has been established between microparticles and venous thromboembolism. Advances in research on the role of microparticles in thrombosis have yielded crucial insights into possible mechanisms, diagnoses and therapeutic targets of venous thromboembolism. In this review, we discuss the definition and properties of microparticles and venous thromboembolism, provide a synopsis of the evidence detailing the contributions of microparticles to venous thromboembolism, and propose potential mechanisms, by which venous thromboembolism occurs. Moreover, we illustrate a possible role of microparticles in cancer-related venous thromboembolism.

To investigate the effects of endothelial microvesicles (EMVs) induced by calcium ionophore A23187 on H9c2 cardiomyocytes.Human umbilical vein endothelial cells (HUVECs) were treated with 10 micromol/L A23187 for 30 min. EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. EMVs were characterized using 1 and 2 microm latex beads and anti-PE-CD144 antibody by flow cytometry. For functional research, EMVs at different concentrations were cocultured with H9c2 cardiomyocytes for 6 h. Cell viability of H9c2 cells and the activity of LDH leaked from H9c2 cells were tested by colorimetry. Moreover, apoptosis of H9c2 cells was observed through Hoechst 33258 staining and tested by FITC-Annexin V/PI double staining.EMVs were induced by A23187 on HUVECs, and isolated by ultracentrifugation. We identified the membrane vesicles (1 microm) induced by A23187 were CD144 positive. In addition, the EMVs could significantly reduce the viability of H9c2 cells, and increase LDH leakage from H9c2 cells in a dose dependent manner (P0.05). Condensed nuclei could be observed with the increasing concentrations of EMVs through Hoechst 33258 staining. Furthermore, increased apoptosis rates of H9c2 cells could be assessed through FITC-Annexin V/PI double staining by flow cytometry.Microvesicles could be released from HUVECs after induced by A23187 through calcium influx, and these EMVs exerted a pro-apoptotic effect on H9c2 cells by induction of apoptosis.

OBJECTIVE: To search for determinants of endothelial dysfunction in type 2 diabetes. RESEARCH DESIGN AND METHODS: We performed a comprehensive analysis of cardiovascular risk markers and measured blood flow responses to endothelium-dependent (acetylcholine [ACh] and NG-monomethyl-L-arginine) and -independent (sodium nitroprusside [SNP]) vasoactive agents in 30 nonsmoking men with type 2 diabetes (age 51 +/- 1 years, BMI 27.8 +/- 0.4 kg/m2, HbA1c 7.4 +/- 0.3%) and 12 matched normal control men. RESULTS: ACh-induced vasodilation was 37% lower in type 2 diabetic (6.1 +/- 0.5) than in normal subjects (9.7 +/- 1.5 ml.dl-1.min-1, P < 0.01), while flows during SNP were similar (9.1 +/- 0.6 vs. 9.9 +/- 1.3 ml.dl-1.min-1, NS). The ratio of endothelium-dependent vs. -independent flow (ACh:SNP ratio) was 31% lower in type 2 diabetic (0.70 +/- 0.05) than in normal subjects (1.10 +/- 0.18, P < 0.01). Total (2.2 +/- 0.4 vs. 1.3 +/- 0.2 mmol/l, P < 0.05), VLDL, and intermediate-density lipoprotein triglycerides were significantly higher, and the mean LDL particle diameter was significantly smaller in type 2 diabetic than in normal subjects. The lag times for LDL oxidation by Cu2+ in vitro were similar in patients with type 2 diabetes (183 +/- 7) and in normal subjects (183 +/- 9 min, NS). Measured and calculated (sum of concentration of individual antioxidants in serum) total peroxyl radical-trapping capacities (TRAPs) were comparable between the groups. In the patients with type 2 diabetes, LDL size was significantly correlated with endothelium-dependent vasodilation (r = 0.43, P < 0.05), serum triglycerides (r = -0.75, P < 0.001), and the lag time for LDL oxidation in vitro (r = 0.38, P < 0.05). HbA1c was inversely correlated with the lag time for LDL oxidation in vitro (r = -0.41, P < 0.05) and TRAP. CONCLUSIONS: In summary, patients with type 2 diabetes exhibited impaired endothelium-dependent vasodilation in vivo, elevated serum triglycerides, decreased LDL size, and normal antioxidant capacity. Of these parameters, LDL size was significantly correlated with endothelial function.

Physical activity increases the production of oxygen free radicals, which may consume antioxidants and oxidize low-density lipoprotein (LDL). To determine whether this occurs during strenuous aerobic exercise, we studied 11 well-trained runners who participated in the Helsinki City Marathon. Blood samples were collected before, immediately after, and 4 days after the race to determine its effect on circulating antioxidants and LDL oxidizability in vitro. LDL oxidizability was increased as determined from a reduction in the lag time for formation of conjugated dienes both immediately after (180 ± 7 vs. 152 ± 4 min, P < 0.001) and 4 days after (155 ± 7 min, P < 0.001) the race. No significant changes in lipid-soluble antioxidants in LDL or in the peak LDL particle size were observed after the race. Total peroxyl radical trapping antioxidant capacity of plasma (TRAP) and uric acid concentrations were increased after the race, but, except for TRAP, these changes disappeared within 4 days. Plasma thiol concentrations were reduced after the race. No significant changes were observed in plasma ascorbic acid, α-tocopherol, β-carotene, and retinol concentrations after the marathon race. We conclude that strenuous aerobic exercise increases the susceptibility of LDL to oxidation in vitro for up to 4 days. Although the increase in the concentration of plasma TRAP reflects an increase of plasma antioxidant capacity, it seems insufficient to prevent the increased susceptibility of LDL to oxidation in vitro, which was still observed 4 days after the race.