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Postnatal maturation of mammalian cardiomyocytes proceeds rapidly after birth, with most of the myocytes exiting cell cycle, becoming binucleated, and adopting oxidative phosphorylation as the primary metabolic route. The triggers and transcriptional programs regulating cardiomyocyte maturation have not been fully understood yet. We performed single cell RNA-Seq in postnatal rat hearts in order to identify the important factors for this process.Single cell RNA-Seq profiling was performed of postnatal day 1 and day 7 rat hearts, and we found that members of the AP-1 transcription factors showed a transient upregulation in the maturing cardiomyocytes, suggesting their functional involvement in the process. Activating members of the AP-1 family by palmitate or adrenergic stimulation inhibited cardiomyocyte cytokinesis and promoted cardiomyocyte maturation. In contrast, knocking down AP-1 members Atf3 and Jun promoted cardiomyocyte cytokinesis, reduced polyploidy and inhibited maturation. Mechanistically, RNA-Seq results and rescue experiments indicated that AP-1 members activate the expression of fatty acid metabolic genes to promote cardiomyocyte maturation. Finally, intraperitoneal injection of AP-1 inhibitor T-5224 in neonatal mice inhibits cardiomyocyte maturation in vivo.Our results are the first evidence implicating AP-1 transcription factors in postnatal cardiomyocyte maturation both in vitro and in vivo, which expand our understanding of the molecular mechanism of cardiomyocyte maturation, and may lead to novel therapies to treat congenital heart diseases.Postnatal cardiomyocyte maturation is a crucial process of cardiac development that determines fitness of the adult heart, and can be affected by multiple congenital heart diseases which lead to adult heart conditions. Our finding that AP-1 transcription factors transiently activated by multiple cues such as fatty acid and adrenergic signal promote cardiomyocyte maturation provided novel targets for therapeutic intervention, which may be applied during the narrow time window of postnatal cardiomyocyte maturation to treat congenital heart diseases and limit their impact on the adult heart.

The role of adaptive immunity in myocardial recovery post myocardial infarction (MI), particularly the immune response by B lymphocytes, remains elusive. Bone marrow immune microenvironment in response to MI is remotely regulated by the hypothalamic pituitary adrenal (HPA) axis. We utilized the cardioprotective actions of SGLT2 inhibitor to identify and characterize bone marrow B cell subsets that respond to myocardial injury. Initially, we preformed ligation of left anterior descendant (LAD) coronary artery in male C57BL/6J mice to monitor the dynamic changes of immune cells across tissues. Mechanistic insights from mouse models demonstrated arrest of bone marrow B cell maturation and function 24 h post MI. A secondary MI model (twice MIs) in mice was established for the first time to evaluate the dosage-dependent cardioprotection of empagliflozin (EMPA). Single-cell RNA-Seq further demonstrated that EMPA restored bone marrow naïve B cell (B220

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular mortality in patients with diabetes mellitus but the protective mechanism remains elusive. Here we demonstrated that the SGLT2 inhibitor, Empagliflozin (EMPA), suppresses cardiomyocytes autosis (autophagic cell death) to confer cardioprotective effects. Using myocardial infarction (MI) mouse models with and without diabetes mellitus, EMPA treatment significantly reduced infarct size, and myocardial fibrosis, thereby leading to improved cardiac function and survival. In the context of ischemia and nutritional glucose deprivation where autosis is already highly stimulated, EMPA directly inhibits the activity of the Na+/H+ exchanger 1 (NHE1) in the cardiomyocytes to regulate excessive autophagy. Knockdown of NHE1 significantly rescued glucose deprivation-induced autosis. In contrast, overexpression of NHE1 aggravated the cardiomyocytes death in response to starvation, which was effectively rescued by EMPA treatment. Furthermore, in vitro and in vivo analysis of NHE1 and Beclin 1 knockout mice validated that EMPA’s cardioprotective effects are at least in part through downregulation of autophagic flux. These findings provide new insights for drug development, specifically targeting NHE1 and autosis for ventricular remodeling and heart failure after MI in both diabetic and non-diabetic patients.

Graphical abstract, Mitochondria, as the energy factory of cells, participate in metabolism processes and play a critical role in the maintenance of human life activities. Mitochondria belong to semi-automatic organelles, which have their own genome different from nuclear genome. Mitochondrial DNA (mtDNA) mutations can cause a series of diseases and threaten human health. However, an effective approach to edit mitochondrial DNA, though long-desired, is lacking. In recent years, gene editing technologies, represented by restriction endonucleases (RE) technology, zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, CRISPR system and pAgo-based system have been comprehensively explored, but the application of these technologies in mitochondrial gene editing is still to be explored and optimized. The present study highlights the progress and limitations of current mitochondrial gene editing technologies and approaches, and provides insights for development of novel strategies for future attempts.

Acute myocardial infarction (AMI) induces blood leukocytosis, which correlates inversely with patient survival. The molecular mechanisms leading to leukocytosis in the infarcted heart, remain poorly understood. Using an AMI mouse model, we identified gasdermin D (GSDMD) in activated leukocytes early in AMI. We demonstrated that GSDMD is required for enhanced early mobilization of neutrophils to the infarcted heart. Loss of GSDMD resulted in attenuated IL-1β release from neutrophils and subsequent decreased neutrophils and monocytes in the infarcted heart. Knockout of GSDMD in mice significantly reduced infarct size, improved cardiac function, and increased survival post AMI. Through a series of bone marrow transplantation studies and leukocytes depletion experiments, we further clarified that excessive bone marrow derived and GSDMD-dependent early neutrophil production and mobilization (24 hours post AMI), contributed to the detrimental immunopathology after AMI. Pharmacological inhibition of GSDMD also conferred cardioprotection post AMI, through reduction of scar size and enhancement of heart function. Our study provides new mechanistic insights into molecular regulation of neutrophil generation and mobilization after AMI, and supports GSDMD as a new target for improved ventricular remodeling and reduced heart failure after AMI.

Patients with AMI and hyperglycemia upon hospital admission exhibited poorer prognosis compared with those without hyperglycemia. It is unknown whether SGLT2 inhibitors can also improve nondiabetic myocardial infarction (MI) with acute hyperglycemia and the underlying mechanisms. Here we demonstrated that hyperglycemia patients were more likely to have worse cardiac function levels, such as with Killip III/IV during hospitalization. Glucose injection-induced nondiabetic MI accompanied by acute hyperglycemia in WT mice, manifested lower survival compared with control. A significant increase in both survival and LV function was observed when treated with empagliflozin (EMPA). In addition, EMPA attenuated fibrosis and autophagy of border cardiac tissue in mice with MI accompanied by acute hyperglycemia. Applying Beclin1

Host genes critical for viral infection are effective antiviral drug targets with tremendous potential due to their universal characteristics against different subtypes of viruses and minimization of drug resistance. Accordingly, we execute a genome-wide CRISPR-Cas9 screen with multiple rounds of survival selection. Enriched in this screen are several genes critical for host sialic acid biosynthesis and transportation, including the cytohesin 2 (CYTH2), tetratricopeptide repeat protein 24 (TTC24), and N-acetylneuraminate synthase (NANS), which we confirm are responsible for efficient influenza viral infection. Moreover, we reveal that CYTH2 is required for the early stage of influenza virus infection by mediating endosomal trafficking. Furthermore, CYTH2 antagonist SecinH3 blunts influenza virus infection in vivo. In summary, these data suggest that CYTH2 is an attractive target for developing host-directed antiviral drugs and therapeutics against influenza virus infection.

Impaired wound healing and ulcer complications are a leading cause of death in diabetic patients. In this study, we report the design and synthesis of a cyclometalated iridium(III) metal complex 1a as a stabilizer of hypoxia-inducible factor-1α (HIF-1α). In vitro biophysical and cellular analyses demonstrate that this compound binds to Von Hippel-Lindau (VHL) and inhibits the VHL–HIF-1α interaction. Furthermore, the compound accumulates HIF-1α levels in cellulo and activates HIF-1α mediated gene expression, including VEGF, GLUT1, and EPO. In in vivo mouse models, the compound significantly accelerates wound closure in both normal and diabetic mice, with a greater effect being observed in the diabetic group. We also demonstrate that HIF-1α driven genes related to wound healing (i.e. HSP-90, VEGFR-1, SDF-1, SCF, and Tie-2) are increased in the wound tissue of 1a-treated diabetic mice (including, db/db, HFD/STZ and STZ models). Our study demonstrates a small molecule stabilizer of HIF-1α as a promising therapeutic agent for wound healing, and, more importantly, validates the feasibility of treating diabetic wounds by blocking the VHL and HIF-1α interaction., Impaired wound healing is a serious complication in diabetic patients, and is associated with reduced HIF1α stability. Here, the authors design a small molecule that stabilizes HIF1α by blocking its interaction with VHL and show that it promotes wound healing in mouse models of diabetes.

Cardiovascular disease is the leading cause of death worldwide. In spite of the mature managements of myocardial infarction (MI), post-MI reperfusion (I/R) injury results in high morbidity and mortality. Cardiomyocyte Ca2+ overload is a major factor of I/R injury, initiating a cascade of events contributing to cardiomyocyte death and myocardial dysfunction. Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a critical role in cardiomyocyte death response to I/R injury, whose activation is a key feature of myocardial I/R in causing intracellular mitochondrial swelling, endoplasmic reticulum (ER) Ca2+ leakage, abnormal myofilament contraction, and other adverse reactions. CaMKII is a multifunctional serine/threonine protein kinase, and CaMKIIδ, the dominant subtype in heart, has been widely studied in the activation, location, and related pathways of cardiomyocytes death, which has been considered as a potential targets for pharmacological inhibition. In this review, we summarize a brief overview of CaMKII with various posttranslational modifications and its properties in myocardial I/R injury. We focus on the molecular mechanism of CaMKII involved in regulation of cell death induced by myocardial I/R including necroptosis and pyroptosis of cardiomyocyte. Finally, we highlight that targeting CaMKII modifications and cell death involved pathways may provide new insights to understand the conversion of cardiomyocyte fate in the setting of myocardial I/R injury.

Morbidity and mortality from acute myocardial infarction (AMI) remains substantial although interventional coronary reperfusion strategies are widely use and successful. MI remains the most common cause of heart failure (HF) worldwide. Here we demonstrated that Panax Notoginseng saponins (PNS), the extract of Panax notoginseng, exerts cardioprotective effect in AMI and the underlying mechanism refers to inducing cardiomyocyte autophagy, antiplatelet aggregation, enhancing endothelial migration and angiogenesis. PNS was initially tested to rescue the myocardial infarct size and cardiac function in left anterior descending (LAD) ligation-operated mice to mimic AMI. RNA-seq to profile transcriptome changes in the heart by treatment with PNS were then conducted. PNS and its main constituents Rg1 and Rd directly inhibited platelet aggregation of healthy subjects with VerifyNow Aspirin and P2Y12 assays but less affecting on coagulation compared with dual-antiplatelet (DAPT). In addition, wound healing scratch assay and heart staining demonstrated that PNS and its main constituents Rg1 and R1 significant enhanced the migration of endothelial cells and angiogenesis in response to MI injury. Interestingly, PNS rather than its constituents enhanced glucose deprivation (GD)-induced autophagy through phosphorylation of AMPK Thr172 and CaMKII Thr287 in cardiomyocytes. These findings provide new insights for drug development from natural products like PNS against ischemia heart diseases and HF post MI.

Aims To investigate the characteristics and clinical significance of myocardial injury in patients with severe coronavirus disease 2019 (COVID-19). Methods and results We enrolled 671 eligible hospitalized patients with severe COVID-19 from 1 January to 23 February 2020, with a median age of 63 years. Clinical, laboratory, and treatment data were collected and compared between patients who died and survivors. Risk factors of death and myocardial injury were analysed using multivariable regression models. A total of 62 patients (9.2%) died, who more often had myocardial injury (75.8% vs. 9.7%; P < 0.001) than survivors. The area under the receiver operating characteristic curve of initial cardiac troponin I (cTnI) for predicting in-hospital mortality was 0.92 [95% confidence interval (CI), 0.87–0.96; sensitivity, 0.86; specificity, 0.86; P < 0.001]. The single cut-off point and high level of cTnI predicted risk of in-hospital death, hazard ratio (HR) was 4.56 (95% CI, 1.28–16.28; P = 0.019) and 1.25 (95% CI, 1.07–1.46; P = 0.004), respectively. In multivariable logistic regression, senior age, comorbidities (e.g. hypertension, coronary heart disease, chronic renal failure, and chronic obstructive pulmonary disease), and high level of C-reactive protein were predictors of myocardial injury. Conclusion The risk of in-hospital death among patients with severe COVID-19 can be predicted by markers of myocardial injury, and was significantly associated with senior age, inflammatory response, and cardiovascular comorbidities.

Visual Abstract, Highlights • Optimal treatment for patients with diabetes and myocardial infarction remains a challenge. • Hyperglycemia- and hyperinsulinemia-induced miR-24 reduction and O-GlcNAcylation in the diabetic heart contributes to poor survival in diabetic myocardial I/R and increased infarct size post-I/R. • Overexpression of miR-24 in murine hearts significantly reduces myocardial infarct size. • miR-24 targets multiple key proteins including O-GlcNac transferase, ATG4A (a key protein in autophagy), and BIM (a pro-apoptosis protein) to protect the myocardium from I/R injury. • miR-24 is a promising therapeutic candidate for diabetic I/R injury., Summary Management for patients with diabetes experiencing myocardial infarction remains a challenge. Here the authors show that hyperglycemia- and hyperinsulinemia-induced microRNA-24 (miR-24) reduction and O-GlcNAcylation in the diabetic heart contribute to poor survival and increased infarct size in diabetic myocardial ischemia/reperfusion (I/R). In a mouse model of myocardial I/R, pharmacological or genetic overexpression of miR-24 in hearts significantly reduced myocardial infarct size. Experimental validation revealed that miR-24 targets multiple key proteins, including O-GlcNac transferase, ATG4A, and BIM, to coordinately protect the myocardium from I/R injury. These results establish miR-24 as a promising therapeutic candidate for diabetic I/R injury.

Argonaute proteins are present and conserved in all domains of life. Recently characterized prokaryotic Argonaute proteins (pAgos) participates in host defense by DNA interference. Here, we report that the Natronobacterium gregoryi Argonaute (NgAgo) enhances gene insertions or deletions in Pasteurella multocida and Escherichia coli at efficiencies of 80–100%. Additionally, the effects are in a homologous arms-dependent but guide DNA- and potential enzyme activity-independent manner. Interestingly, such effects were also observed in other pAgos fragments including Thermus thermophilus Argonaute (TtAgo), Aquifex aeolicus Argonaute (AaAgo) and Pyrococcus furiosus Argonaute (PfAgo). The underlying mechanism of the NgAgo system is a positive selection process mainly through its PIWI-like domain interacting with recombinase A (recA) to enhance recA-mediated DNA strand exchange. Our study reveals a novel system for enhancing homologous sequence-guided gene editing in bacteria.

Diabetes mellitus (DM) is a growing international concern. Considerable mortality and morbidity associated with diabetes mellitus arise predominantly from thrombotic cardiovascular events. Oxidative stress‐mediated mitochondrial damage contributes significantly to enhanced thrombosis in DM. A basal autophagy process has recently been described as playing an important role in normal platelet activation. We now report a substantial mitophagy induction (above basal autophagy levels) in diabetic platelets, suggesting alternative roles for autophagy in platelet pathology. Using a combination of molecular, biochemical, and imaging studies on human DM platelets, we report that platelet mitophagy induction serves as a platelet protective mechanism that responds to oxidative stress through JNK activation. By removing damaged mitochondria (mitophagy), phosphorylated p53 is reduced, preventing progression to apoptosis, and preserving platelet function. The absence of mitophagy in DM platelets results in failure to protect against oxidative stress, leading to increased thrombosis. Surprisingly, this removal of damaged mitochondria does not require contributions from transcription, as platelets lack a nucleus. The considerable energy and resources expended in “prepackaging” the complex mitophagy machinery in a short‐lived normal platelet support a critical role, in anticipation of exposure to oxidative stress.

Purpose of review Von Willebrand factor (VWF) is a large multidomain, multimeric glycoprotein that plays an essential role in regulating the balance between blood clotting and bleeding. Aberrant VWF regulation can lead to a spectrum of diseases extending from bleeding disorders [Von Willebrand disease (VWD)] to aberrant thrombotic thrombocytopenic purpura (TTP). Understanding the biology of VWF expression and secretion is essential for developing novel targeted therapies for VWF-related hemostasis disorders. Recent findings A number of recent elegant in-vitro and in-vivo studies will be highlighted, including the discovery of intronic splicing in the VWF gene, microRNA-regulated VWF gene expression, and syntaxin binding protein and autophagy mediated VWF secretion. Compared with the already established critical role of VWF in VWD and TTP pathophysiology, additional clinical studies have clarified and reinforced the association of elevated plasma levels of VWF with an increased risk of stroke, myocardial infarction, venous thrombosis, and diabetic thrombotic complications. Moreover, experimental mouse models of ischemic stroke and myocardial infarction have further supported VWF as a potential therapeutic target. Summary VWF biosynthesis, maturation, and secretion is a complex process, which mandates tight regulation. Significant progress has been made in our understandings of VWF expression and secretion and its association with thrombotic diseases, contributing to the development of novel targeting VWF drugs for prevention and treatment of deficient and enhanced hemostasis.

Mycobacterium bovis (M. bovis), the most common pathogens of tuberculosis (TB), is virulent to human and cattle, and transmission between cattle and humans warrants reconsideration concerning food safety and public health. Recently, efforts have begun to analyze cellular proteomic responses induced by Mycobacterium tuberculosis (M. tb). However, the underlying mechanisms by which virulent M. bovis affects human hosts are not fully understood. For the present study, we utilized a global and comparative labeling strategy of isobaric tag for relative and absolute quantitation (iTRAQ) to assess proteomic changes in the human monocyte cell line (THP-1) using a vaccine strain and two virulent strains H37Rv and M. bovis. We measured 2,032 proteins, of which 61 were significantly differentially regulated. Ingenuity Pathway Analysis was employed to investigate the canonical pathways and functional networks involved in the infection. Several pathways, most notably the phagosome maturation pathway and TNF signaling pathway, were differentially affected by virulent strain treatment, including the key proteins CCL20 and ICAM1. Our qRT-PCR results were in accordance with those obtained from iTRAQ. The key enzyme MTHFD2, which is mainly involved in metabolism pathways, as well as LAMTOR2 might be effective upon M. bovis infection. String analysis also suggested that the vacuolar protein VPS26A interacted with TBC1D9B uniquely induced by M. bovis. In this study, we have first demonstrated the application of iTRAQ to compare human protein alterations induced by virulent M. bovis infections, thus providing a conceptual understanding of mycobacteria pathogenesis within the host as well as insight into preventing and controlling TB in human and animal hosts' transmission.

SummaryLeukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal haemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout (KO), shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bβ3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using two different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice.Supplementary Material to this article is available online at www.thrombosis-online.com.

von Willebrand factor (VWF) is a large adhesive glycoprotein with established functions in hemostasis. It serves as a carrier for factor VIII and acts as a vascular damage sensor by attracting platelets to sites of vessel injury. VWF size is important for this latter function, with larger multimers being more hemostatically active. Functional imbalance in multimer size can variously cause microvascular thrombosis or bleeding. The regulation of VWF multimeric size and platelet-tethering function is carried out by ADAMTS13, a plasma metalloprotease that is constitutively active. Unusually, protease activity of ADAMTS13 is controlled not by natural inhibitors but by conformational changes in its substrate, which are induced when VWF is subject to elevated rheologic shear forces. This transforms VWF from a globular to an elongated protein. This conformational transformation unfolds the VWF A2 domain and reveals cryptic exosites as well as the scissile bond. To enable VWF proteolysis, ADAMTS13 makes multiple interactions that bring the protease to the substrate and position it to engage with the cleavage site as this becomes exposed by shear. This article reviews recent literature on the interaction between these 2 multidomain proteins and provides a summary model to explain proteolytic regulation of VWF by ADAMTS13.

The platelet-tethering function of von Willebrand factor (VWF) is proteolytically regulated by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which cleaves the Tyr1605-Met1606 (P1-P1′) bond in the VWF A2 domain. To date, most of the functional interactions between ADAMTS13 and VWF that have been characterized involve VWF residues that are C terminal to the scissile bond. We now demonstrate that the substrate P3 position in VWF, Leu1603, is a critical determinant of VWF proteolysis. When VWF Leu1603 was substituted with Ser, Ala, Asn, or Lys in a short VWF substrate, VWF115, proteolysis was either greatly reduced or ablated (up to 400-fold reduction in k cat /K m ). As Leu1603 must interact with residues proximate to the Zn 2+ ion coordinated in the active center of ADAMTS13, we sought the corresponding S3 interacting residues. Substitution of 10 candidate residues in the metalloprotease domain of ADAMTS13 identified two spatially separated clusters centered on Leu198 or Val195 (acting with Leu232 and Leu274, or with Leu151, respectively), as possible subsites interacting with VWF. These experimental findings using the short VWF115 substrate were replicated using full-length VWF. It is hypothesized that VWF Leu1603 interacts with ADAMTS13 Leu198/Leu232/Leu274 and that Val195/Leu151 may form part of a S1 subsite. The recognition of VWF Leu1603 by ADAMTS13, in conjunction with previously reported remote exosites C terminal of the cleavage site, suggests a mechanism whereby the VWF P1-P1′ scissile bond is brought into position over the active site for cleavage. Together with recently characterized remote exosite interactions, these findings provide a general framework for understanding the ADAMTS family substrate interactions.

Objective The possibility of the preventive and therapeutic effects of Chinese traditional patent medicine for myocardial infarction has been reported in the literature, but there have been few studies. This paper presents three tendencies for the secondary prevention of myocardial infarction and presents a systematic evaluation of current research evidence related to the use of Chinese traditional patent medicine in secondary prevention of myocardial infarction, with a goal of making objective recommendations for patient rehabilitation. Method Literature was retrieved from traditional Chinese medicine websites, MEDLINE, CNKI, VIP, CINAHL, The Cochrane Library, EMBASE, HealthSTAR, and Academic ASAP to locate research-based scientific evidence related to the use of Chinese traditional patent medicine for myocardial infarction. Results Some Chinese traditional patent medicine therapies alone or in combination with routine rehabilitation therapies might be useful in patients with myocardial infarction, but we found insufficient evidence to support the use of most Chinese traditional patent medicine therapies in the secondary prevention of myocardial infarction. Conclusion Difficulty in accessing information regarding traditional Chinese medicine has implications for health education in both myocardial infarction and patient safety. Some Chinese traditional patent medicine therapies hold promise for patients in the secondary prevention of myocardial infarction but further research is essential in all areas of traditional Chinese medicine to confirm its efficacy.

Panax ginseng C.A. Meyer is a well-known medicinal herb native to China and Korea, and has been used as a herbal remedy in eastern Asia for thousands of years. However, there is different evidence of ginseng efficacy between traditional Chinese medicine (TCM), modern pharmacological experiments and clinical trials. In TCM, ginseng is a highly valued herb and has been applied to a variety of pathological conditions and illnesses such as hypodynamia, anorexia, shortness of breath, palpitation, insomnia, impotence, hemorrhage and diabetes. Modern pharmacological experiments have proved that ginseng possesses multiple constituents (ginsenosides, polysaccharides, peptides, polyacetylenic alcohols, etc.) and actions (central nervous system effects, neuroprotective effect, immunomodulation, anticancer, etc.), ginsenosides as the active ingredients, especially, having antioxidant, antiinflammatory, antiapoptotic and immunostimulant properties. Recently, ginseng has been studied in a number of randomized controlled trials investigating its effect mainly on physical and psychomotor performance, cognitive function, immunomodulation, diabetes mellitus, cardiovascular risk factors, quality of life, as well as adverse effects. Equivocal results have been demonstrated for many of these indications. Because of the poor quality of most clinical trials on ginseng, reliable clinical data in humans are still lacking. Therefore, a broader understanding of medical knowledge and reasoning on ginseng is necessary.

Platelets play a key role in thrombosis and haemostasis, which can be either beneficial or deleterious depending on the circumstances. Multiple factors, such as genetic polymorphisms, pathological state and lifestyle, are thought to be associated with platelet hyperreactivity. Platelet activation occurs through the complex process of transmembrane signalling, with a cascade of biochemical interactions leading to platelet activation. Transmembrane signalling involves many different molecules with different enzymatic activity and/or function. Based on the signalling pathways involved in platelet activation, there are four possible targets of antiplatelet drugs: (i) inhibition of agonist generation; (ii) receptor inhibition; (iii) G-protein inhibition; and (iv) inhibition of enzymatic cascades. However, both established and novel antiplatelet drugs have their own advantages and disadvantages. Because of the problems associated with the use of current antiplatelet drugs, such as resistance, optimal dosage and safety, future strategies for the development of new antiplatelet drugs and new treatment regimens may include consideration of the following: (i) a shift from single targets within the signalling cascade to multiple targets; (ii) a shift from therapy with a single drug to combination therapy; and (iii) investigating drugs in current clinical use for novel antiplatelet properties.

Case recruitment of large-scale clinical trials should be strictly checked in quality and quantity for it is the key to clinical trial. This study discusses the main difficulties and countermeasures in the case recruitment of large sample, multi-center clinical trials according to the national research project "Myocardial Infarction Secondary Prevention Study in Traditional Chinese Medicine".

Diabetes are common diseases threatening health worldwide. Over the past several years it has become clear that alterations in the expression of microRNA (miRNA) genes contribute to the pathogenesis of diabetes [1, 2]. miRNAs are small (~22 nt) regulatory RNA molecules that functionally modulate the activity of specific mRNA targets involving in a wide range of physiologic and pathologic processes [3]. miRNA expression profiling of diabetes has identified signatures associated with diagnosis, progression, prognosis and response to treatment [1]. Here, we focus on one specific miRNA: miR-24, which is involved in both diabetes and diabetic complications. miR-24 is highly conserved in various species and is clustered with miR-23 and miR-27 on human chromosome 9 (miR-24-1) and 19 (miR-24-2). Both miR-24-1 and miR-24-2 generate the same mature product: miR-24. The levels of miR-24 in plasma of diabetes were significantly lower than that in healthy controls [4]. Notably, plasma levels of miR-24 inversely correlated with HbA1c [4]. miR-24 was highly expressed in the lungs, heart, pancreases, and kidneys but poorly expressed in the liver and muscle. Consistent with human observations, miR-24 levels were decreased 2-3 fold in STZ-induced mice and db/db diabetic mice lungs, and 5-fold in db/db mice plasma [4]. In vitro cultured cell line analyses indicate miR-24 is top highly expressed in endothelial cells and beta cells (MIN6 cells) [4, 5]. The expression of miR-24 was decreased response to high glucose culture condition in endothelial cells [4] but was reported upregulated by glucose and palmitate in beta cells [6]. miR-24 was identified targeting multiple 3′UTR sites and genes, directly regulating von Willebrand Factor (VWF) expression and related enzymes or proteins involved in the process of VWF synthesis, maturation and secretion [4]. These lend insight into miR-24-based coordinate regulation of VWF and provide a mechanism for the high plasma VWF levels and thrombotic complications observed with Type 2 diabetes. Thrombotic cardiovascular diseases is the leading cause of mortality among diabetic patients. Of particular interest is miR-24. In the heart, miR-24 has been reported to target BCL2L11 (BIM) and junctophilin-2 in cardiomyocyte, and targeting transcription factor GATA2, p21-activated kinase PAK4 and eNOS in endothelial cells [7]. In the vascular system, miR-24 was found to target chitinase 3-like 1 to limit vascular inflammation and matrix metalloproteinase-14 in macrophage to retard atherosclerotic plaque progression [7]. However, all of these findings are based on in vitro cultured cells and animal studies without an association with cardiovascular risk in human studies. Several studies have reported contrasting results in mouse myocardial infarction models. Some groups suggested that inhibition of miR-24 had beneficial therapeutic effects on myocardial infarction, whereas others supported improved recovery after myocardial infarction with miR-24 mimic therapy [7]. In line with these later studies, we identified decreased miR-24 in plasma and the heart under diabetic conditions [4]. Theoretically, miR-24 replacement therapy for diabetes should improve the heart functions and prevent cardiovascular complications. Our recent studies established the causality of diabetes and miR-24, and presented a novel mechanism for miR-24 down-regulation through hyperglycemia-induced activation of c-Myc [4]. Whether insulin resistance and hyperinsulinemia also regulate the expression or stability of miR-24 in diabetes remains unclear. Our unpublished data suggests that intensively insulin treatment for diabetic patients may contribute to diabetic thrombosis through miR-24. On the other hand, miRNAs (including miR-24) inactivation in β-cells of adult mice results in a striking diabetic phenotype [5]. These suggest that diabetic impaired miR-24 expression may feedback aggravate the diabetic conditions. In contrast, another recent study implicated miR-24 in the suppression of two maturity-onset diabetes of young (MODY) genes (HNF1α and NeuroD) [6]. miR-24 was found an increase in islets in the HFD-fed mice and db/db mice. But our unpublished results suggest that both aging and diabetes severity influence the miR-24 expression and downstream regulatory networks that fine-tune insulin expression and secretion in beta cells. That also partially explains the inconsistent cellular phenotype: knockdown of miR-24 did not affect the insulin expression in MIN6 cells but downregulated insulin in primary cultured islets [5]. In summary, miR-24 has multiple targets in beta cells, cardiomyocytes, endothelial cells, and macrophages, all of which are involved in diabetic complications. In turn, miR-24 itself is feedback regulated by glucolipotoxicity and impaired insulin pathways. For miR-24-based therapeutics, large-animal studies are required but miR-24 has not been studied in large-animal models to date. Based on our recent findings that decreased miR-24 in diabetes increases the risk of diabetic thrombotic and vascular complications, miR-24 replacement therapy seems to be promising for diabetic patients. However, such miR-24 therapeutics also involve challenges as observations of miR-24 upregulation in cancers. The delivery dose and time should be well controlled. Another challenge is to achieve overexpression and cell-specific delivery of miR-24, which has divergent effects in endothelial cells versus cardiac cells. Nevertheless, the use of miR-24 mimics or inhibitors to control the diabetic pathophysiological processes offers new therapeutic opportunities.

An elevated level of von Willebrand factor (VWF) in diabetic patients is associated with increased risk of thrombotic cardiovascular events. The underlying mechanism of how VWF expression is upregulated in diabetes mellitus is poorly understood. We now report that hyperglycemia-induced repression of microRNA-24 (miR-24) increases VWF expression and secretion in diabetes mellitus. In diabetic patients and diabetic mouse models (streptozotocin/high-fat diet-induced and db/db mice), miR-24 is reduced in both tissues and plasma. Knockdown of miR-24 in mice leads to increased VWF mRNA and protein levels and enhanced platelet tethering (spontaneous thrombosis). miR-24 tightly controls VWF levels through pleiotropic effects, including direct binding to the 3′ untranslated region of VWF and targeting FURIN and the histamine H1 receptor, known regulators of VWF processing and secretion in endothelial cells. We present a novel mechanism for miR-24 downregulation through hyperglycemia-induced activation of aldose reductase, reactive oxygen species, and c-Myc. These findings support a critical role for hyperglycemic repression of miR-24 in VWF-induced pathology. miR-24 represents a novel therapeutic target to prevent adverse thrombotic events in patients with diabetes mellitus.

Platelets play a key role in thrombosis and haemostasis, which can be either beneficial or deleterious depending on the circumstance. Antiplatelet therapy is the 'cornerstone' in the prevention and treatment of thrombotic diseases. Platelet activation is a complex process known as transmembrane signaling which then serves to activate the platelet via a cascade of biochemical interactions. Currently available strategies of antiplatelet therapy are generally based on the signaling pathway of platelet activation, which possess the characters that mono-agent, mono-target, and mainly irreversible inhibition. Therefore, both established and novel antiplatelet agents have their own pros and cons and such problems as resistance, drug-drug interaction, discontinuation and monitoring, etc. have been appeared. Due to the problems existing in current antiplatelet agents, future new strategies for antiplatelet targets, agent-developing and treatment might probably include three aspects: targeting the factors associated with platelet hyperactivity, developing novel antiplatelet agents with multiple targets, mild and reversible properties from natural products, and keeping healthy lifestyle and emphasizing prevention.

Thromb Haemost 2008; 100: 729–730 Platelet activation plays a central role in haemostasis and thrombosis. Antiplatelet therapy is considered to be the ‘cornerstone’in the prevention and treatment of thrombotic diseases such as coronary artery disease (CAD), stroke and peripheral arterial disease (PAD). While dual antiplatelet therapy with aspirin and clopidogrel is currently the standard therapy after coronary stent implantation to prevent a life-threatening stent thrombosis, the concept of antiplatelet drug ‘resistance’ or non-responsiveness has also received increasing attention over recent years, and at least aspirin resistance, clopidogrel resistance and glycoprotein IIb/IIIa (GpIIb-IIIa) inhibitors resistance have been widely reported (1, 2). Therefore, to illuminate the underlying mechanisms of antiplatelet drug resistance such clinical issues are of great importance. In this issue of Thrombosis and Haemostasis, Beres et al. (3) identify a potential novel mechanism of clopidogrel resistance, high platelet α-2 adrenergic receptor. The extent of the platelet aggregation response in vitro to adenosine diphosphate (ADP) and the inhibition of vasodilatorstimulated phosphoprotein (VASP) phosphorylation have been used to define clopidogrel resistance in the large majority of studies that have been published so far.The incidence of clopidogrel resistance varied between 5% and 85.8% (4–6), while aspirin resistance was 5.5% ~ 61% (7). The potential mechanisms involved in the variable response to clopidogrel have been fully summarized by De Miguel et al. (8). However, many mechanisms mentioned are still controversial, e.g. polymorphisms of P2Y12 do not seem to play additional roles in modulating the individual response (9, 10). Only ‘metabolic activity of the hepatic cytochrome P450’ and ‘clopidogrel bioavailability’ have been well established. Currently, Beres et al. (3) have studied the influence of platelet α-2 adrenoreceptor on increased residual platelet ractivity. Interestingly, their results demonstrate that α-2 adrenergic receptor contributes to high on-treatment platelet reactivity and consequently, to clopidogrel resistance through preserving P2Y12 receptor function. It is known that human platelets exhibit both adrenergic and dopaminergic receptors that are influenced by different catecholamines. α-2 adrenergic receptors prevail on the platelet membrane; through their stimulation, catecholamines potentiate the effects of other agonists (e.g. ADP) and, at higher concentrations, initiate platelet responses, including aggregation, secretion and arachidonate pathway activation (11). Especially, epinephrine amplifies the response elicited by a number of agonists for platelet aggregation (12). Using five different agonists (ADP, epinephrine, collagen, collagen-related peptide, and ristocetin) and a broad range of agonist concentrations,Yee et al. (13) found that epinephrine was especially reliable and efficient in detecting platelet hyperreactivity, and excellent reproducibility persisted for up to three years. Moreover, platelet hyperreactivity is not limited to epinephrine-mediated aggregation, but can generally be observed with multiple forms of platelet stimulation, and is associated with each major phase of platelet function: From adhesion (increased agglutination in response to low-dose ristocetin) to activation (increased P-selectin expression after granule release) to aggregation (increased aggregation in response to multiple different stimuli) (14).These findings suggest an underlying mechanism that occurs relatively early in the progression of platelet activity (i.e. in proximity to epinephrine’s interaction with the α2A-adrenergic receptor) and that is shared by multiple pathways affecting distinct aspects of platelet function (14). Through studying 121 stable angina patients on standard dual antiplatelet therapy (75 mg clopidogrel and 100 mg acetylsalicylic acid), Beres et al. (3) also found similar results that patients with high adrenergic activity have significantly increased baseline ADPand collagen-induced platelet aggregation. In addition, another study demonstrated that platelet α-2 adrenergic receptor plays a significant role in thrombus stabilization (15). Therefore, epinephrine-platelet receptor signal pathway may play a central role in the platelet hyperreactivity relevant for thrombotic disease. Many platelet agonists function through G-protein-coupled receptors (16). Epinephrine activates the α-2 adrenergic receptor that couples to Gz in platelets, while ADP activates P2Y12 receptor that couples to Gi and P2Y1 to Gq, respectively (1). Although different in downstream events of the signal transduction pathways, platelet response to both epinephrine and ADP are independent of adenylyl cyclase inhibition which is shared by α-2A adre