Mono-(2-ethylhexyl)-phthalate potentiates methylglyoxal-induced blood-brain barrier damage via mitochondria-derived oxidative stress and bioenergetic perturbation.

Phthalates in contaminated foods and personal care products are one of the most frequently exposed chemicals with a public health concern. Phthalate exposure is related to cardiovascular diseases, including diabetic vascular complications and cerebrovascular diseases, yet the mechanism is still unclear. The blood-brain barrier (BBB) integrity disruption is strongly associated with cardiovascular and neurological disease exacerbation. We investigated BBB damage by di-(2-ethylhexyl) phthalate (DEHP) or its metabolite mono-(2-ethylhexyl) phthalate (MEHP) using brain endothelial cells and rat models. BBB damage by the subthreshold level of MEHP, but not a DEHP, significantly increased by the presence of methylglyoxal (MG), a reactive dicarbonyl compound whose levels increase in the blood in hyperglycemic conditions in diabetic patients. Significant potentiation in apoptosis and autophagy activation, mitochondria-derived reactive oxygen species (ROS) production, and mitochondrial metabolic disturbance were observed in brain ECs by co-exposure to MG and MEHP. N-acetyl cysteine (NAC) restored autophagy activation as well as tight junction protein impairment induced by co-exposure to MG and MEHP. Intraperitoneal administration of MG and MEHP significantly altered mitochondrial membrane potential and tight junction integrity in rat brain endothelium. This study may provide novel insights into enhancing phthalate toxicity in susceptible populations, such as diabetic patients.

Is Nuclear Factor Erythroid 2-Related Factor 2 a Target for the Intervention of Cytokine Storms?

The term "cytokine storm" describes an acute pathophysiologic state of the immune system characterized by a burst of cytokine release, systemic inflammatory response, and multiple organ failure, which are crucial determinants of many disease outcomes. In light of the complexity of cytokine storms, specific strategies are needed to prevent and alleviate their occurrence and deterioration. Nuclear factor erythroid 2-related factor 2 (NRF2) is a CNC-basic region-leucine zipper protein that serves as a master transcription factor in maintaining cellular redox homeostasis by orchestrating the expression of many antioxidant and phase II detoxification enzymes. Given that inflammatory response is intertwined with oxidative stress, it is reasonable to assume that NRF2 activation limits inflammation and thus cytokine storms. As NRF2 can mitigate inflammation at many levels, it has emerged as a potential target to prevent and treat cytokine storms. In this review, we summarized the cytokine storms caused by different etiologies and the rationale of interventions, focusing mainly on NRF2 as a potential therapeutic target.

Amine-modified nanoplastics promote the procoagulant activation of isolated human red blood cells and thrombus formation in rats.

BACKGROUND: Microplastics (MPs) and nanoplastics (NPs) formed from decomposed plastic are increasing environmental threats. Although MPs and NPs exposed through various routes enter the systemic circulation, the potential toxicity of those is largely unknown. We investigated whether polystyrene NPs (PS-NPs) promote the coagulation activity of red blood cells (RBCs). RESULTS: We tested several types of PS-NPs using human RBCs and found that amine-modified 100 nm PS-NPs were the most potent. We measured the uptake of PS-NPs using flow cytometry and confocal microscopy. Electron microscopy revealed morphological changes of RBCs by PS-NPs. PS-NPs induced the externalization of phosphatidylserine, generation of microvesicles in RBCs, and perturbations in the intracellular microenvironment. PS-NPs increased the activity of scramblases responsible for phospholipid translocation in RBCs. PS-NPs modulated the functional interaction to adjacent tissues and coagulation cascade, enhancing RBC adhesion and thrombin generation. Our observations in human RBCs were consistent with those in isolated rat RBCs, showing no inter-species differences. In rat venous thrombosis models, the intravenous administration of PS-NPs enhanced thrombus formation. CONCLUSION: Amine-modified PS-NPs induce the prothrombotic activation of RBCs causing thrombus formation. We believe that our study will contribute to understanding the potential toxicity of amine-modified polystyrene particles in blood cells and cardiovascular systems.

MARTX toxin of Vibrio vulnificus induces RBC phosphatidylserine exposure that can contribute to thrombosis.

V. vulnificus-infected patients suffer from hemolytic anemia and circulatory lesions, often accompanied by venous thrombosis. However, the pathophysiological mechanism of venous thrombosis associated with V. vulnificus infection remains largely unknown. Herein, V. vulnificus infection at the sub-hemolytic level induced shape change of human red blood cells (RBCs) accompanied by phosphatidylserine exposure, and microvesicle generation, leading to the procoagulant activation of RBCs and ultimately, acquisition of prothrombotic activity. Of note, V. vulnificus exposed to RBCs substantially upregulated the rtxA gene encoding multifunctional autoprocessing repeats-in-toxin (MARTX) toxin. Mutant studies showed that V. vulnificus-induced RBC procoagulant activity was due to the pore forming region of the MARTX toxin causing intracellular Ca2+ influx in RBCs. In a rat venous thrombosis model triggered by tissue factor and stasis, the V. vulnificus wild type increased thrombosis while the ΔrtxA mutant failed to increase thrombosis, confirming that V. vulnificus induces thrombosis through the procoagulant activation of RBCs via the mediation of the MARTX toxin.

Pb-Induced Eryptosis May Provoke Thrombosis Prior to Hemolysis.

Lead (Pb) is a common metal, which can be toxic to the human body via the pollution of water or food, and can cause anemia and other diseases. However, what happens before hemolysis and anemia caused by Pb poisoning is unclear. Here, we demonstrated Pb can cause procoagulant activity of erythroid cells leading to thrombosis before hemolysis. In freshly isolated human erythroid cells, we observed that Pb resulted in hemolysis in both concentration- and time-dependent manners, but that no lysis occurred in Pb-exposed erythroid cells (≤20 µM for 1 h). Pb treatment did not cause shape changes at up to 0.5 h incubation but at 1 h incubation echinocyte and echino-spherocyte shape changes were observed, indicating that Pb can exaggerate a concentration- and time-dependent trend of shape changes in erythroid cells. After Pb treatment, ROS-independent eryptosis was shown with no increase of reactive oxygen species (ROS), but with an increase of [Ca2+]i and caspase 3 activity. With a thrombosis mouse model, we observed increased thrombus by Pb treatment (0 or 25 mg/kg). In brief, prior to hemolysis, we demonstrated Pb can cause ROS-independent but [Ca2+]i-dependent eryptosis, which might provoke thrombosis.

A Comprehensive Understanding of the Genomic Bone Tumor Landscape: A Multicenter Prospective Study.

Complexity and heterogeneity increases the difficulty of diagnosis and treatment of bone tumors. We aimed to identify the mutational characterization and potential biomarkers of bone tumors. In this study, a total of 357 bone tumor patients were recruited and the next generation sequencing (NGS)-based YuanSu450 panel, that includes both DNA and RNA sequencing, was performed for genomic alteration identification. The most common mutated genes in bone tumors included TP53, NCOR1, VEGFA, RB1, CCND3, CDKN2A, GID4, CCNE1, TERT, and MAP2K4. The amplification of genes such as NCOR1, VEGFA, and CCND3 mainly occurred in osteosarcoma. Germline mutation analysis reveal a high frequency of HRD related mutations (46.4%, 13/28) in this cohort. With the assistance of RNA sequencing, 16.8% (19/113) gene fusions were independently detected in 20% (16/79) of patients. Nearly 34.2% of patients harbored actionable targeted mutations, of which the most common mutation is CDKN2A deletion. The different mutational characterizations between juvenile patients and adult patients indicated the potential effect of age in bone tumor treatment. According to the genomic alterations, the diagnosis of 26 (7.28%) bone tumors were corrected. The most easily misdiagnosed bone tumor included malignant giant cell tumors of bone (2.8%, 10/357) and fibrous dysplasia of bone (1.7%, 6/357). Meanwhile, we found that the mutations of MUC16 may be a potential biomarker for the diagnosis of mesenchymal chondrosarcomas. Our results indicated that RNA sequencing effectively complements DNA sequencing and increased the detection rate of gene fusions, supporting that NGS technology can effectively assist the diagnosis of bone tumors.

Construction of survival-related co-expression modules and identification of potential prognostic biomarkers of osteosarcoma using WGCNA.

Background: Osteosarcoma (OS) is a primary malignant bone tumor. Patients with different immune characteristics respond differently to chemotherapy and have a lower chance of survival. The potential pathogenesis and therapeutic targets of OS must be investigated further. Methods: OS expression profile data and clinical information were downloaded from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) and the Gene Expression Omnibus (GEO) databases. The immune-related gene set was obtained from the ImmPort database, and the immune-related gene expression profiles were used for non-negative matrix factorization (NMF) cluster analysis of patients in the 2 databases to find the best clustering number. In the TARGET database, OS patients were classified into low-risk and high-risk groups based on the differences in their survival rates. Weighted correlation network analysis (WGCNA) was applied to the low-risk and high-risk groups to determine the module with the lowest conservatism in order to differentiate the prognosis of the 2 groups. Results: A total of 500 key genes associated with poor prognosis were identified. Gene Ontology (GO) enrichment analysis revealed that the biological processes of these genes were primarily focused on the regulation of small guanosine triphosphatase (GTPase) mediated signal transduction, collagen-containing extracellular matrix, and Rho GTPase binding. A random survival forest identified EPHB3, TEAD1, and KRR1P1 as key genes. Their expression level was linked to overall survival. We discovered that the core genes were associated to immune cell infiltration. Simultaneously, paired survival analysis of two genes revealed differences in survival. We also reverse-predicted the main genes and developed their competitive endogenous RNA (ceRNA) network. Finally, utilizing the CellMiner database, we observed that the genes TEAD1 and EPHB3 were connected to drug sensitivity. Conclusions: In this study, we identified the modules and key genes related to the poor prognosis of OS patients by using WGCNA, and verified their impact on the prognosis of OS patients and their role in the immune microenvironment of OS. In addition, targeted gene related antitumor drugs were screened out. The discoveries may lead to novel molecular targets and treatment methods for OS patients. Keywords: Osteosarcoma (OS); weighted gene co-expression network analysis (WGCNA); gene.

CNC-bZIP protein NFE2L1 regulates osteoclast differentiation in antioxidant-dependent and independent manners.

Fine-tuning of osteoclast differentiation (OD) and bone remodeling is crucial for bone homeostasis. Dissecting the mechanisms regulating osteoclastogenesis is fundamental to understanding the pathogenesis of various bone disorders including osteoporosis and arthritis. Nuclear factor erythroid 2-related factor 1 (NFE2L1, also known as NRF1), which belongs to the CNC-bZIP family of transcription factors, orchestrates a variety of physiological processes and stress responses. While Nfe2l1 gene may be transcribed into multiple alternatively spliced isoforms, the biological function of the different isoforms of NFE2L1 in bone metabolism, osteoclastogenesis in particular, has not been reported. Here we demonstrate that knockout of all isoforms of Nfe2l1 transcripts specifically in the myeloid lineage in mice [Nfe2l1(M)-KO] results in increased activity of osteoclasts, decreased bone mass and worsening of osteoporosis induced by ovariectomy and aging. In comparison, LysM-Cre-mediated Nfe2l1 deletion has no significant effect on the osteoblast and osteocytes. Mechanistic investigations using bone marrow cells and RAW 264.7 cells revealed that deficiency of Nfe2l1 leads to accelerated and elevated OD, which is attributed, at least in part, to enhanced accumulation of ROS in the early stage of OD and expression of nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1α (Nfatc1/α). In addition, NFE2L1 regulates the transcription of multiple antioxidant genes and Nfatc1/α and OD in an isoform-specific manner. While long isoforms of NFE2L1 function as accelerators of induction of Nfatc1/α and antioxidant genes and OD, the short isoform NFE2L1-453 serves as a brake that keeps the long isoforms' accelerator effects in check. These findings provide a novel insight into the regulatory roles of NFE2L1 in osteoclastogenesis and highlight that NFE2L1 is essential in regulating bone remodeling and thus may be a valuable therapeutic target for bone disorders.

Titanium dioxide nanoparticles enhance thrombosis through triggering the phosphatidylserine exposure and procoagulant activation of red blood cells.

BACKGROUND: Expanding biomedical application of anatase titanium dioxide (TiO2) nanoparticles (NPs) is raising the public concern on its potential health hazards. Here, we demonstrated that TiO2 NPs can increase phosphatidylserine (PS) exposure and procoagulant activity of red blood cells (RBCs), which may contribute to thrombosis. RESULTS: We conducted in vitro studies using RBCs freshly isolated from healthy male volunteers. TiO2 NPs exposure (≦ 25 µg/mL) induced PS exposure and microvesicles (MV) generation accompanied by morphological changes of RBCs. While ROS generation was not observed following the exposure to TiO2 NPs, intracellular calcium increased and caspase-3 was activated, which up-regulated scramblase activity, leading to PS exposure. RBCs exposed to TiO2 NPs could increase procoagulant activity as measured by accelerated thrombin generation, and enhancement of RBC-endothelial cells adhesion and RBC-RBC aggregation. Confirming the procoagulant activation of RBC in vitro, exposure to TiO2 NPs (2 mg/kg intravenously injection) in rats increased thrombus formation in the venous thrombosis model. CONCLUSION: Collectively, these results suggest that anatase TiO2 NPs may harbor prothrombotic risks by promoting the procoagulant activity of RBCs, which needs attention for its biomedical application.

The roles of NFE2L1 in adipocytes: Structural and mechanistic insight from cell and mouse models.

Adipocytes play pivotal roles in maintaining energy homeostasis by storing lipids in adipose tissue (AT), regulating the flux of lipids between AT and the circulation in response to the body's energy requirements and secreting a variety of hormones, cytokines and other factors. Proper AT development and function ensure overall metabolic health. Nuclear factor erythroid 2-related factor 1 (NFE2L1, also known as NRF1) belongs to the CNC-bZIP family and plays critical roles in regulating a wide range of essential cellular functions and varies stress responses in many cells and tissues. Human and rodent Nfe2l1 genes can be transcribed into multiple splice variants resulting in various protein isoforms, which may be further modified by a variety of post-translational mechanisms. While the long isoforms of NFE2L1 have been established as master regulators of cellular adaptive antioxidant response and proteasome homeostasis, the exact tissue distribution and physiological function of NFE2L1 isoforms, the short isoforms in particular, are still under intense investigation. With regard to key roles of NFE2L1 in adipocytes, emerging data indicates that deficiency of Nfe2l1 results in aberrant adipogenesis and impaired AT functioning. Intriguingly, a single nucleotide polymorphism (SNP) of the human NFE2L1 gene is associated with obesity. In this review, we summarize the most significant findings regarding the specific roles of the multiple isoforms of NFE2L1 in AT formation and function. We highlight that NFE2L1 plays a fundamental regulatory role in the expression of multiple genes that are crucial to AT metabolism and function and thus could be an important target to improve disease states involving aberrant adipose plasticity and lipid homeostasis.

Comprehensive metabolomic profiling of osteosarcoma based on UHPLC-HRMS.

INTRODUCTION: Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. An increasing number of studies have demonstrated that tumor proliferation and metastasis are closely related to complex metabolic reprogramming. However, there are limited data to provide a comprehensive metabolic picture of osteosarcoma. OBJECTIVES: Our study aims to identify aberrant metabolic pathways and seek potential adjuvant biomarkers for osteosarcoma. METHODS: Serum samples were collected from 65 osteosarcoma patients and 30 healthy controls. Nontargeted metabolomic profiling was performed by liquid chromatography-mass spectrometry (LC-MS) based on univariate and multivariate statistical analyses. RESULTS: The OPLS-DA model analysis identified clear separations among groups. We identified a set of differential metabolites such as higher serum levels of adenosine-5-monophosphate, inosine-5-monophosphate and guanosine monophosphate in primary OS patients compared to healthy controls, and higher serum levels of 5-aminopentanamide, 13(S)-HpOTrE (FA 18:3 + 2O) and methionine sulfoxide in lung metastatic OS patients compared to primary OS patients, revealing aberrant metabolic features during the proliferation and metastasis of osteosarcoma. We found a group of metabolites especially lactic acid and glutamic acid, with AUC values of 0.97 and 0.98, which could serve as potential adjuvant diagnostic biomarkers for primary osteosarcoma, and a panel of 2 metabolites, 5-aminopentanamide and 13(S)-HpOTrE (FA 18:3 + 2O), with an AUC value of 0.92, that had good monitoring ability for lung metastases. CONCLUSIONS: Our study provides new insight into the aberrant metabolic features of osteosarcoma. The potential biomarkers identified here may have translational significance.

Comparison of local control and survival outcomes between surgical and non-surgical local therapy on pelvic Ewing's sarcoma patients: a meta-analysis.

BACKGROUND: The efficacy of surgical therapy to nonsurgical therapy is still a controversial topic in pelvic Ewing's sarcoma (ES) management. We perform a systemic review and meta-analysis to compare the effect of local control (LC) and survival outcomes between surgical and nonsurgical local therapy on pelvic ES patients with systemic chemotherapy. METHODS: Published retrospective studies searched from PubMed, Embase, Cochrane and Web of Science databases that investigated the effects of surgical and nonsurgical local therapy on the LC and survival outcomes of patients with pelvic ES treated with chemotherapy were included in our study. Our primary outcome was the LC rate and progression-free survival (PFS) rate. The effect of confounders of extend of disease, surgical margin and chemotherapy respond on PFS was analyzed in subgroups. RESULTS: Ten studies with 782 pelvic ES patients were included in our analysis. Surgical patients showed higher LC and PFS rate comparing to nonsurgical patients [LC: risk ratio (RR) 0.72, 95% CI: 0.52-1.00, P=0.05, I2=0%; PFS: RR 0.72, 95% CI: 0.61-0.86, P=0.000, I2=15%]. Localized patients showed higher PFS with surgical therapy than nonsurgical patients (RR 0.67, 95% CI: 0.51-0.88, P=0.003).Patients with adequate resection and good chemotherapy respond improved PFS comparing to nonsurgical patients (adequate resection vs. nonsurgical: RR 0.59, 95% CI: 0.46-0.76, P<0.001, I2=0%; good respond vs. nonsurgical: RR 0.56, 95% CI: 0.41-0.77, P<0.001, I2=21%). But patients with inadequate resection and poor chemotherapeutic respond shows no statistical different PFS comparing to nonsurgical patients (inadequate resection vs. nonsurgical: RR 1.11, 95% CI: 0.87-1.41, P=0.41, I2=0%; poor respond vs. nonsurgical: RR 1.17, 95% CI: 0.90-1.52, P=0.25, I2=0%). CONCLUSIONS: Surgical therapy is primarily recommended in localized, resectable, good chemotherapeutic respond pelvic ES. Inadequate resection and poor chemotherapeutic respond are negative prognostic factors in surgical patients and their surviving are not improved comparing with nonsurgical patients. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020149224.

Regional lymph node involvement is associated with poorer survivorship in patients with upper extremity osteosarcoma than with lower extremity osteosarcoma: a SEER analysis.

BACKGROUND: Although the upper limb is the second most common site of osteosarcoma, investigations into clinical manifestation differences between upper and lower limb patients are still sporadic. We retrospectively investigated the characteristics of these patients to gain a better understanding of the differences between upper and lower limb osteosarcoma patients. METHODS: This retrospective study involved patients diagnosed with extremity osteosarcoma between 1997 and 2016 collected from the Surveillance, Epidemiology, and End Results (SEER) database. Patient characteristics were analyzed with t-tests, rank sum tests and chi-square tests. Log-rank tests were applied to evaluate univariate significance, and Cox hazards models were performed in multivariate analysis. A binary logistics regression model was used to screen the risk factors related to lymph node involvement. RESULTS: In total, 1,882 patients, 1,588 (84.4%) with lower limb lesions and 294 (15.6%) with upper limb lesions were enrolled in our study. The patients with upper limb osteosarcoma exhibited poorer 5-year overall survival (OS) than patients with lower limb osteosarcoma (54.8% vs. 63.2%, P=0.02). The upper limb patients had more lymph node involvement (5.6% vs. 2.7%, P=0.03), which was found to be an independent prognostic factor (P=0.000). Tumors located in the upper limbs and the presence of distal metastasis were risk factors related to lymph node involvement in the extremity (P<0.05). The upper limb patients were tended to suffer greater risk of being affected by both metastasis and lymph node involvement (15.7% vs. 9.4%, P=0.18). CONCLUSIONS: Upper limb osteosarcoma patients are characterized by more lymph node involvement than lower limb patients, leading to poorer OS. In addition, upper limb patients are at greater risk for both lymph node involvement and distal metastasis. Our results suggest that upper limb patients should be screened more thoroughly for regional lymph node involvement.

Multimodal Risk-Adapted Treatment in Surgical Patients With Synovial Sarcoma: A Preoperative Nomogram-Guided Adjuvant Treatment Strategy.

Background: Synovial sarcoma is characterized by heterogeneous clinical manifestations, making it difficult to evaluate individual patients' prognoses and design personal treatment schemes. We established an effective preoperative nomogram to predict cancer-specific survival (CSS) and present a risk-adapted adjuvant treatment strategy in surgical patients with synovial sarcoma. Methods: This retrospective study included patients from the Surveillance, Epidemiology, and End Results (SEER) database who were diagnosed with synovial sarcoma between 1996 and 2015. The patients were randomly divided into training and validation groups. The predictors were selected using univariate and multivariate Cox hazards models. The nomogram performance was verified for its discriminatory ability and calibration. We further stratified the patients into different risk groups according to the nomogram scores and compared the efficacy of chemotherapy, radiotherapy, and combination of radiotherapy and chemotherapy. Results: There were 915 patients enrolled in our study, with 874 patients either alive or dead due to synovial sarcoma. We established a nomogram to predict 5-year CSS based on independent factors, including sex, age, grade, tumor size, location, and extent (all p < 0.05). Our model showed a consistently good discriminatory ability and calibration for predicting 5-year CSS in both the training (c-index = 0.78, 95% CI 0.75-0.81) and validation (c-index = 0.73, 95% CI 0.68-0.78). Based on their nomogram scores, we divided patients into 5 groups. Compared to patients without adjuvant treatment, nomogram I patients with adjuvant treatment had no improvements in 5-year CSS (100.0% vs. 100.0%), nomogram II patients had higher 5-year CSS with radiotherapy or chemotherapy (92.9% vs. 72.2%, p = 0.015), nomogram III patients had higher 5-year CSS with combination of chemotherapy and radiotherapy (70.1% vs. 47.2%, p = 0.004), nomogram IV patients had higher 5-year CSS with radiotherapy (41.3% vs. 15.6%, p = 0.015), and nomogram V patients had no improvements in 5-year CSS rates with adjuvant treatment (28.9% vs. 16.9%, p = 0.18). Conclusion: The nomogram showed a satisfactory discriminatory ability and calibration for predicting 5-year CSS in synovial sarcoma patients. Based on this nomogram, we stratified synovial sarcoma patients according to risk levels, which enabled us to provide a useful grouping scheme that can inform multimodal risk-adapted treatment in synovial sarcoma.

Antithrombotic effect of SP-8008, a benzoic acid derivative, through the selective inhibition of shear stress-induced platelet aggregation.

BACKGROUND AND PURPOSE: Bleeding is one of the most critical adverse effects of antithrombotic drugs, and many efforts have been made to discover novel antiplatelet agents without bleeding complications. Shear stress-induced platelet aggregation (SIPA), where the interaction of von Willebrand factor (vWF) and platelet glycoprotein (GP) Ib constitutes the initial step, is a promising target to overcome bleeding problems, as SIPA occurs only in pathological conditions. Here, we describe SP-8008, a novel modulator of vWF-GP Ib interactions and evaluated its antiplatelet/antithrombotic effects. EXPERIMENTAL APPROACH: Newly synthesized compounds were screened for antiplatelet effects in vitro, using human platelets exposed to high shear stress. Aggregation, intracellular calcium level, granule secretion, and integrin activation were assessed. Molecular modelling using virtual docking and flow cytometry were used to evaluate effects on vWF-GP Ib interactions. Antithrombotic effects in vivo were determined in rats, using arterial thrombosis and shear stress-specific thrombosis. Transection tail bleeding time was used to evaluate adverse effects. KEY RESULTS: SP-8008 was a potent inhibitor of SIPA, with IC50 of 1.44 ± 0.09 µM. SP-8008 effectively and broadly blocked shear stress-induced platelet activation events, without any significant toxicity. Importantly, SP-8008 was highly selective against SIPA, effectively interfering with vWF-GP Ib engagement. Most importantly, SP-8008 exerted significant antithrombotic effects in vivo in both shear stress-specific and arterial thrombosis, without prolonging bleeding time. CONCLUSIONS AND IMPLICATIONS: Our results demonstrated that SP-8008 can be a novel selective antiplatelet agent with improved safety profile.

Antithrombotic Effects of Paeoniflorin from Paeonia suffruticosa by Selective Inhibition on Shear Stress-Induced Platelet Aggregation.

Antiplatelet agents are important in the pharmacotherapeutic regime for many cardiovascular diseases, including thrombotic disorders. However, bleeding, the most serious adverse effect associated with current antiplatelet therapy, has led to many efforts to discover novel anti-platelet drugs without bleeding issues. Of note, shear stress-induced platelet aggregation (SIPA) is a promising target to overcome bleeding since SIPA happens only in pathological conditions. Accordingly, this study was carried out to discover antiplatelet agents selectively targeting SIPA. By screening various herbal extracts, Paeonia suffruticosa and its major bioactive constituent, paeoniflorin, were identified to have significant inhibitory effects against shear-induced aggregation in human platelets. The effects of paeoniflorin on intraplatelet calcium levels, platelet degranulation, and integrin activation in high shear stress conditions were evaluated by a range of in vitro experiments using human platelets. The inhibitory effect of paeoniflorin was determined to be highly selective against SIPA, through modulating von Willebrand Factor (vWF)-platelet glycoprotein Ib (GP Ib) interaction. The effects of paeoniflorin on platelet functions under high shear stress were confirmed in the ex vivo SIPA models in rats, showing the good accordance with the anti-SIPA effects on human platelets. Treatment with paeoniflorin significantly prevented arterial thrombosis in vivo from the dose of 10 mg/kg without prolonging bleeding time or blood clotting time in rats. Collectively, our results demonstrated that paeoniflorin can be a novel anti-platelet agent selectively targeting SIPA with an improved safety profile.

Dapsone Hydroxylamine, an Active Metabolite of Dapsone, Can Promote the Procoagulant Activity of Red Blood Cells and Thrombosis.

Dapsone hydroxylamine (DDS-NHOH), N-hydroxylated metabolite of a sulfonamide antibiotic, dapsone, is responsible for various adverse effects of dapsone that include methemoglobinemia, hemolytic anemia, and thrombosis. However, the mechanism underlying DDS-NHOH-induced thrombosis remains unclear. Here, we demonstrated that DDS-NHOH, but not dapsone, could increase prothrombotic risks through inducing the procoagulant activity of red blood cells (RBCs). In freshly isolated human RBCs in vitro, sub-hemolytic concentrations of DDS-NHOH (10-50 µM) increased phosphatidylserine (PS) exposure and augmented the formation of PS-bearing microvesicles (MV). Reactive oxygen species (ROS) generation and the subsequent dysregulation of enzymes maintaining membrane phospholipid asymmetry were found to induce the procoagulant activity of DDS-NHOH. Dapsone hydroxylamine also accelerated thrombin generation and enhanced RBC self-aggregation and adherence of RBCs to endothelial cells in vitro. Most importantly, both the single dose of 50 or 100 mg/kg (i.p.) DDS-NHOH and repeated doses of 10 mg/kg per day (i.p.) for 4 days increased thrombus formation in rats (six rats per dose) in vivo, substantiating a potential prothrombotic risk of DDS-NHOH. Collectively, these results demonstrated the central role of RBC procoagulant activity induced by DDS-NHOH in the thrombotic risk of dapsone.

Ginsenoside Rg3, a component of ginseng, induces pro-thrombotic activity of erythrocytes via hemolysis-associated phosphatidylserine exposure.

Ginseng and its active gradient, ginsenoside Rg3 (Rg3), are widely used for a variety of health benefits, but concerns over their misuses are increasing. Previously, it has been reported that Rg3 can cause hemolysis, but its health outcome remains unknown. Here, we demonstrated that Rg3 could promote the procoagulant activity of erythrocytes through the process of hemolysis, ultimately leading to increased thrombosis. In freshly isolated human erythrocytes, Rg3 caused pore formation and fragmentation of the erythrocyte membrane. Confocal microscopy observation and flow cytometric analysis revealed that remnant erythrocyte fragments after the exposure to Rg3 expressed phosphatidylserine (PS), which can promote blood coagulation through providing assembly sites for coagulation complexes. Rat in vivo experiments further confirmed that intravenous administration of Rg3 produced PS-bearing erythrocyte debris and increased thrombosis. Collectively, we demonstrated that Rg3 could induce the procoagulant activity of erythrocytes by generating PS-bearing erythrocyte debris through hemolysis, which might provoke thrombosis.

Effect of radiochemotherapy on the cognitive function and diffusion tensor and perfusion weighted imaging for high-grade gliomas: A prospective study.

This study aimed to explore the effects of radiochemotherapy on the neurocognitive function of patients with high-grade gliomas (HGG). The mini-mental state examination (MMSE), Montreal Cognitive Assessment (MoCA), event-related potential P300 (ERP-P300), and specific MRI parameters were compared, and the associations between specific MRI parameters and different doses of radiation were determined for before and up to 12 months after radiotherapy. There were no significant differences in MMSE, MoCA, or ERP-P300 before and after radiotherapy. Compared with pre-radiochemotherapy, fractional anisotropy (FA) in the contralateral hippocampus decreased at 6 and 9 months after radiotherapy. FA in the ipsilateral hippocampus before radiochemotherapy decreased compared with 6 months after radiotherapy. Compared to the end of radiotherapy, as well as 3- and 6-months post-radiotherapy, the regional cerebral blood volume (rCBV) in the genu of the corpus was significantly lower at 12 months post-radiotherapy. Some MRI parameters in different regions of the brain were negatively correlated with the mean and maximum dose. There was no significant effect of radiochemotherapy on the neurocognitive functioning of patients with HGGs found before radiochemotherapy until 12 months after radiotherapy. The radiation-induced FA decrease in the bilateral hippocampus preceded cognitive dysfunction, and DTI of the hippocampus may provide a useful biomarker for predicting radiation-induced neurocognitive impairment in patients with HGGs.

Co-Existence of Hypertensive and Anti-Hypertensive Constituents, Synephrine, and Nobiletin in Citrus unshiu Peel.

A single herb can contain multiple constituents with diverse bioactivities. We found that the extract of Citrus unshiu peel (CUP), induced abnormal vasoconstriction responses on the freshly isolated rat aortic rings in vitro. CUP stimulated the vasoconstriction alone, and it suppressed the phenylephrine-stimulated vasoconstriction. We studied the reasons behind this abnormal vasoconstriction pattern. Major constituents of CUP were determined and evaluated for their vaso-activities. Notably, synephrine, a contractile agonist, and nobiletin, newly identified to have anti-contractile activity co-existed in CUP. Synephrine and nobiletin competitively blocked or activated the same contractile targets resulting in contradicting and abnormal vasoconstriction responses. Accordingly, the vasoconstriction pattern varies significantly depending on the relative contents of synephrine and nobiletin in CUP. Interestingly, this response pattern could be observed with another plant extract, Acorus gramineus Sol. Collectively, we demonstrated that active ingredients with contradicting bioactivities could co-exist in a single plant extract, interact and produce abnormal response patterns in bioassay, which would give an important insight into the interpretation of unusual activity patterns induced by plant extracts.