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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 short release half-life of carbon monoxide (CO) is a major obstacle to the effective therapeutic use of carbon monoxide-releasing molecule-2 (CORM-2). The potential of CORM-2-entrapped ultradeformable liposomes (CORM-2-UDLs) to enhance the release half-life of CO and alleviate skin inflammation was investigated in the present study. CORM-2-UDLs were prepared by using soy phosphatidylcholine to form lipid bilayers and Tween 80 as an edge activator. The deformability of CORM-2-UDLs was measured and compared with that of conventional liposomes by passing formulations through a filter device at a constant pressure. The release profile of CO from CORM-2-UDLs was evaluated by myoglobin assay. In vitro and in vivo anti-inflammatory effects of CORM-2-UDLs were assessed in lipopolysaccharide-stimulated macrophages and TPA-induced ear edema model, respectively. The deformability of the optimized CORM-2-UDLs was 2.3 times higher than conventional liposomes. CORM-2-UDLs significantly prolonged the release half-life of CO from 30 s in a CORM-2 solution to 21.6 min. CORM-2-UDLs demonstrated in vitro anti-inflammatory activity by decreasing nitrite production and pro-inflammatory cytokine levels. Furthermore, CORM-2-UDLs successfully ameliorated skin inflammation by reducing ear edema, pathological scores, neutrophil accumulation, and inflammatory cytokines expression. The results demonstrate that CORM-2-UDLs could be used as promising therapeutics against acute skin inflammation., Graphical abstract The short release half-life of carbon monoxide is a major obstacle to the effective therapeutic use of carbon monoxide-releasing molecule-2 (CORM-2). Herein, CORM-2-entrapped ultradeformable liposomes (CORM-2-UDLs) were designed to prolong carbon monoxide release half-life and enhance skin permeation of CORM-2 for better anti-inflammatory effects in skin inflammation model.Image 1

Background The blood–brain barrier (BBB) maintains homeostasis of the brain environment by tightly regulating the entry of substances from systemic circulation. A breach in the BBB results in increased permeability to potentially toxic substances and is an important contributor to amplification of ischemic brain damage. The precise molecular pathways that result in impairment of BBB integrity remain to be elucidated. Autophagy is a degradation pathway that clears damaged or unnecessary proteins from cells. However, excessive autophagy can lead to cellular dysfunction and death under pathological conditions. Methods In this study, we investigated whether autophagy is involved in BBB disruption in ischemia, using in vitro cells and in vivo rat models. We used brain endothelial bEnd.3 cells and oxygen glucose deprivation (OGD) to simulate ischemia in culture, along with a rat ischemic stroke model to evaluate the role of autophagy in BBB disruption during cerebral ischemia. Results OGD 18 h induced cellular dysfunction, and increased permeability with degradation of occludin and activation of autophagy pathways in brain endothelial cells. Immunostaining revealed that occludin degradation is co-localized with ischemic autophagosomes. OGD-induced occludin degradation and permeability changes were significantly decreased by inhibition of autophagy using 3-methyladenine (3-MA). Enhanced autophagic activity and loss of occludin were also observed in brain capillaries isolated from rats with middle cerebral artery occlusion (MCAO). Intravenous administration of 3-MA inhibited these molecular changes in brain capillaries, and recovered the increased permeability as determined using Evans blue. Conclusions Our findings provide evidence that autophagy plays an important role in ischemia-induced occludin degradation and loss of BBB integrity.

Exenatide, a synthetic version of exendin-4, is a glucagon-like peptide-1 receptor agonist (GLP-1RA) used for treating diabetes, but its relatively short half-life is a major disadvantage. In this study, we attempted residue-specific mono-PEGylation to the middle of the amino acid backbone to extend its in vivo half-life. Exenatide was point-mutated from Lys to Cys at the 12th residue to yield a variant (K12C), and PEG-maleimide of varying molecular weights (MW) (5, 10, 20, 40 kD) was site-specifically conjugated to yield a mono-PEGylate with branched T-shape molecular structure. In another approach, we conjugated a bis-maleimide PEG (10 kD) to the middle of two K12Cs to yield an H-shape homodimer PEGylate In vitro bioactivity assays indicated that: (1) PEGylates conjugated with higher MW PEG lead to stronger receptor binding, (2) the branched form was superior to the linear configuration in the binding, and (3) both T-shape and H-shape mono-PEGylates demonstrated better potency than the native exenatide, evidenced by lower EC50. Db/db mouse experiments to evaluate in vivo hypoglycemic activity indicated that: (1) all mono-PEGylates resulted in improved glucose tolerance compared to the native exenatide, (2) the homodimer PEGylate demonstrated much stronger hypoglycemic activity, especially during the initial period, and (3) the H-shape and T-shape mono-PEGylates (40 kD) maintained hypoglycemia for up to ca. 168 and 140 h, representing approximately 12- and 14-fold increase, respectively, compared with the native exenatide. Our findings suggest that the exenatide mono-PEGylates in unclassical molecular structures can improve in vivo pharmacokinetics properties.

The pharmacological or toxicological activities of the degradation products of drug candidates have been unaddressed during the drug development process. Ischemic stroke accounts for 80% of all strokes and is responsible for considerable mortality and disability worldwide. Despite decades of research on neuroprotective agents, tissue plasminogen activators (t-PA), a thrombolytic agent, remains the only approved acute stroke pharmacological therapy. NXY-059, a free radical scavenger, exhibited striking neuroprotective properties in preclinical models and met all the criteria established by the Stroke Academic Industry Roundtable (STAIR) for a neuroprotective agent. In phase 3 clinical trials, NXY-059 exhibited significant neuroprotective effects in one trial (SAINT-I), but not in the second (SAINT-II). Some have hypothesized that N-t-butyl hydroxylamine (NtBHA), a breakdown product of NXY-059 was the actual neuroprotective agent in SAINT-I and that changes to the formulation of NXY-059 to prevent its breakdown to NtBHA in SAINT -II was the reason for the lack of efficacy. We evaluated the neuroprotective effect of NtBHA in N-methyl-D-aspartate (NMDA)-treated primary neurons and in rat focal cerebral ischemia. NtBHA significantly attenuated infarct volume in rat transient focal ischemia, and attenuated NMDA-induced cytotoxicity in primary cortical neurons. NtBHA also reduced free radical generation and exhibited mitochondrial protection.

The growing prevalence, the risk of disabilities, and the lack of effective causal or disease-modifying therapies make cerebrovascular disorders a serious burden. Carnosine, an evolutionarily conserved endogenous dipeptide and over-the-counter food supplement, has a wealth of evidence exhibiting potentially neuroprotective effects. It is a natural, multi-functional, non-toxic anti-glycating antioxidant found in high concentrations in human and mammalian muscle and brain tissues. The physiological presence of carnosine in the brain, ability to cross the blood-brain barrier, and evidence from in vitro, and in vivo studies all point to it as a promising neurotherapeutic agent for cerebrovascular disorders. This review is intended to provide a detailed overview of the neuroprotective role of carnosine in cerebrovascular diseases and summarize existing evidence from cell and animal studies.

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.

Isothiazolinone (IT) biocides are potent antibacterial substances used as preservatives and disinfectants. These biocides exert differing biocidal effects and display environmental stability based upon chemical structure. In agreement with our recent study reporting that 2-n-octyl-4-isothiazolin-3-one (OIT) induced dysfunction of the blood-brain barrier (BBB), the potential adverse health effects of two IT biocides 1,2-benzisothiazolin-3-one (BIT) and 4,5-dichloro-2-n-octyl-isothiazolin-3-one (DCOIT) were compared using brain endothelial cells (ECs) derived from murine brain endothelial cell line (bEND.3). BIT possesses an unchlorinated IT ring structure and used as a preservative in cleaning products. DCOIT contains a chlorinated IT ring structure and employed as an antifouling agent in paints. Data demonstrated that DCOIT altered cellular metabolism at a lower concentration than BIT. Both BIT and DCOIT increased reactive oxygen species (ROS) generation at the mitochondrial and cellular levels. However, the effect of DCOIT on glutathione (GSH) levels appeared to be greater than BIT. While mitochondrial membrane potential (MMP) was decreased in both BIT- and DCOIT-exposed cells, direct disturbance in mitochondrial bioenergetic flux was only observed in BIT-treated ECs. Taken together, IT biocides produced toxicity in brain EC and barrier dysfunction, but at different concentration ranges suggesting distinct differing mechanisms related to chemical structure.

The present study aims to investigate the neuroprotective effects of carnosine-entrapped elastic liposomes (CAR-ELs) against cerebral ischemia. CAR-ELs were prepared by extrusion method using egg phosphatidylcholine (eggPC) as a phospholipid and Tween 80 (TW80) as an edge activator (eggPC:TW80 = 8:2, w/w). The prepared CAR-ELs were purified by centrifugal ultrafiltration followed by characterization for particle size, polydispersity index, zeta potential and entrapment efficiency. The elasticity of CAR-ELs, the most distinct feature of elastic liposomes, was determined using a stainless steel pressure filter and compared with that of conventional liposomes. In vivo neuroprotective effects of CAR-ELs were evaluated in cerebral ischemia induced by permanent middle cerebral artery occlusion (pMCAO) in rats. CAR-ELs (250 mg/kg of CAR) were intravenously administered 20 min before pMCAO and 6 h after pMCAO, respectively. The infarct volume in brain was measured by staining with 2,3,5-triphenyltetrazolium chloride after 24 h of cerebral ischemia. CAR-ELs showed nanometric particle size near 100 nm and homogeneous distribution with polydispersity index below 0.1. The elasticity of CAR-ELs was 2-fold higher than that of conventional liposomes. The brain ischemia was successfully developed with pMCAO as indicated by highly infarcted hemisphere (~ 50%) in saline-treated rats. The pre-treatment with CAR-ELs significantly reduced infarct volume (7.9%) compared with CAR solution (19.1%)- and saline (50.8%)-pretreated rats. CAR solution, however, showed better neuroprotective effects than CAR-ELs when administered 6 h after ischemia induction. The pre-treatment with CAR-ELs could be promising nanocarrier-based neuroprotective therapeutics against ischemic stroke.

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.

Disposable sanitary pads are a necessity for women's health, but safety concerns regarding the use of these products have created anxiety. The aim of this study was to conduct a risk assessment of 74 volatile organic compounds (VOCs), which were expected to be contained within sanitary pads. Of the 74 VOCs, 50 were found in sanitary pads retailed in Korea at concentrations ranging from 0.025 to 3548.09 µg/pad. In order to undertake a risk assessment of the VOCs, the toxicological database of these compounds in the United States Environmental Protection Agency (USEPA), Agency for Toxic Substances and Disease Registry (ATSDR), National Toxicology Program (NTP) and World Health Organization (WHO) was searched. Ethanol was found to exhibit the highest reference dose (RfD) while 1,2-dibromo-3-chloro-propane displayed the lowest RfD. Consequently, a worst-case exposure scenario was applied in this study. It was assumed that there was the use of 7.5 sanitary napkins/day for 7 days/month. In the case of panty liners or overnight sanitary napkins, the utilization of 90 panty liners/month or 21 overnight sanitary napkins/month was assumed, respectively. In addition, 43 kg, the body weight of 12 to 13-year-old young women, and 100% VOCs skin absorption were employed for risk assessment. The systemic exposure dose (SED) values were calculated ranging from 1.74 (1,1,2-trichloroethane) ng/kg/day to 144.4 (ethanol, absolute) µg/kg/day. Uncertainty factors (UFs) were applied ranging from 10 to 100,000 in accordance with the robustness of animal or human experiments. The margin of exposure (MOE) of 34 VOCs was more than 1 (acceptable MOE > 1). Applicable carcinogenic references reported that the cancer risk of five VOCs was below 10-6. Based on our findings, evidence indicates that the non-cancer and cancer risks associated with VOCs detected in sanitary pads currently used in South Korea do not pose an adverse health risk in women.

Background Silver nanoparticles (AgNP) are widely used in medical practices owing to their distinct antibacterial, antiviral and anticancer activities. However, with increasing use of AgNP, concerns over its potential toxicity are also escalating. Here, we demonstrated the potential thrombotic effect of AgNP which was mediated by the procoagulant activity of red blood cells (RBCs). Results In freshly isolated human RBCs, AgNP, but not silver microparticles (AgMP), elicited morphological changes, phosphatidylserine (PS) exposure and microvesicles (MV) generation, the key indicators of procoagulant activity in RBCs at concentration ranges (≤ 100 μg/mL) that were free of significant hemolysis. In line with this, AgNP potentiated thrombin generation and adherence of RBCs to endothelial cells, while AgMP did not. Oxidative stress, intracellular calcium increase and ATP depletion were found to underlie the procoagulant effects of AgNP, which led to altered activity of membrane aminophospholipid translocases. These in vitro findings were well reproduced in rat in vivo, where intravenously exposure to AgNP promoted venous thrombosis significantly. Of note, RBCs isolated from cancer patients, who inherently convey the risk of thrombogenesis, were more sensitive to the procoagulant effects of AgNP. In addition, AgNP significantly potentiated the procoagulant effects of a chemotherapeutic drug, paclitaxel. Conclusion Collectively, these results suggest that AgNP may have prothrombotic risks by promoting procoagulant activity of RBCs and caution shall be taken for its use in the population sensitive to thrombosis like cancer patients. Electronic supplementary material The online version of this article (10.1186/s12989-019-0292-6) contains supplementary material, which is available to authorized users.

Arsenic-associated carcinogenesis and related mortality are a major public health concern worldwide; however, the underlying mechanism of action remains unclear. Here, we demonstrated that arsenic promotes tumor metastasis by stimulating tumor cell-platelet aggregation (TCPA), which can ultimately increase cancer-related mortality. In freshly isolated human platelets in vitro, arsenic potentiated TCPA prompted by diverse cancer cell lines, which was attributable to increased platelet reactivity to TCPA with respect to thrombin generation and P-selectin, GPIIb/IIIa expression. Consistently, the co-existence of platelets and arsenic significantly enhanced tumor cell adhesion, extravasation and invasion along with increased metastasis-related markers like metallo-matrix proteinase-2 and -9 in vitro, which was attenuated by platelet activation blockers. Importantly, the exposure to arsenic-contaminated drinking water (2 ppm, 3 weeks) in mice in vivo significantly increased the metastasis of intravenously injected melanoma cells into lung. Furthermore, the exposure to arsenic-contaminated drinking water significantly reduced the survival of melanoma cell-injected mice, which was attenuated by the pretreatment of platelet-activation blockers; aspirin and eptifibatide. All these results provide an important clue to understand the mechanism underlying arsenic-associated cancer mortality and its prevention.

Isothiazolinone (IT) biocides are potent antibacterial substances commonly used as preservatives or disinfectants, and 2-n-Octyl-4-isothiazolin-3-one (OIT, octhilinone) is a common IT biocide that is present in leather products, glue, paints, and cleaning products. Although humans are exposed to OIT through personal and industrial use, the potentially deleterious effects of OIT on human health are still unknown. To investigate the effects of OIT on the vascular system, which is continuously exposed to xenobiotics through systemic circulation, we treated brain endothelial cells with OIT. OIT treatment significantly activated caspase-3-mediated apoptosis and reduced the bioenergetic function of mitochondria in a bEnd.3 cell-based in vitro blood–brain barrier (BBB) model. Interestingly, OIT significantly altered the thiol redox status, as evidenced by reduced glutathione levels and protein S-nitrosylation. The endothelial barrier function of bEnd.3 cells was significantly impaired by OIT treatment. OIT affected mitochondrial dynamics through mitophagy and altered mitochondrial morphology in bEnd.3 cells. N-acetyl cysteine significantly reversed the effects of OIT on the metabolic capacity and endothelial function of bEnd.3 cells. Taken together, we demonstrated that the alteration of the thiol redox status and mitochondrial damage contributed to OIT-induced BBB dysfunction, and we hope that our findings will improve our understanding of the potential hazardous health effects of IT biocides.

Stroke is one of the leading causes of death and disability worldwide. However, treatment options for ischemic stroke remain limited. Matrix-metalloproteinases (MMPs) contribute to brain damage during ischemic strokes by disrupting the blood-brain barrier (BBB) and causing brain edemas. Carnosine, an endogenous dipeptide, was found by us and others to be protective against ischemic brain injury. In this study, we investigated whether carnosine influences MMP activity. Brain MMP levels and activity were measured by gelatin zymography after permanent occlusion of the middle cerebral artery (pMCAO) in rats and in vitro enzyme assays. Carnosine significantly reduced infarct volume and edema. Gelatin zymography and in vitro enzyme assays showed that carnosine inhibited brain MMPs. We showed that carnosine inhibited both MMP-2 and MMP-9 activity by chelating zinc. Carnosine also reduced the ischemia-mediated degradation of the tight junction proteins that comprise the BBB. In summary, our findings show that carnosine inhibits MMP activity by chelating zinc, an essential MMP co-factor, resulting in the reduction of edema and brain injury. We believe that our findings shed new light on the neuroprotective mechanism of carnosine against ischemic brain damage. Ministry of Health & Welfare, Republic of Korea ; National Research Foundation of Korea ; Ministry of Science, ICT & Future Planning ; NIHR Sheffield Biomedical Research Centre (BRC)

During the past two decades, the clinical use of biopharmaceutical products has markedly increased because of their obvious advantages over conventional small-molecule drug products. These advantages include better specificity, potency, targeting abilities, and reduced side effects. Despite the substantial clinical and commercial success, the macromolecular structure and intrinsic instability of biopharmaceuticals make their formulation and administration challenging and render parenteral delivery as the only viable option in most cases. The use of nanocarriers for efficient delivery of biopharmaceuticals is essential due to their practical benefits such as protecting from degradation in a hostile physiological environment, enhancing plasma half-life and retention time, facilitating absorption through the epithelium, providing site-specific delivery, and improving access to intracellular targets. In the current review, we highlight the clinical and commercial success of biopharmaceuticals and the overall applications and potential of nanocarriers in biopharmaceuticals delivery. Effective applications of nanocarriers for biopharmaceuticals delivery via invasive and noninvasive routes (oral, pulmonary, nasal, and skin) are presented here. The presented data undoubtedly demonstrate the great potential of combining nanocarriers with biopharmaceuticals to improve healthcare products in the future clinical landscape. In conclusion, nanocarriers are promising delivery tool for the hormones, cytokines, nucleic acids, vaccines, antibodies, enzymes, and gene- and cell-based therapeutics for the treatment of multiple pathological conditions.

Acute kidney injury (AKI) is a serious threat to human health. Clinically, ischemia-reperfusion (I/R) injury is considered one of the most common contributors to AKI. Emodin has been reported to alleviate I/R injury in the heart, brain, and small intestine in rats and mice through its anti-inflammatory effects. The present study investigated whether emodin improved AKI induced by I/R and elucidated the molecular mechanisms. We used a mouse model of renal I/R injury and human renal tubular epithelial cell model of hypoxia/reoxygenation (H/R) injury. Ischemia/reperfusion resulted in renal dysfunction. Pretreatment with emodin ameliorated renal injury in mice following I/R injury. Emodin reduced mitochondrial-mediated apoptosis, suppressed the overproduction of mitochondrial reactive oxygen species and accelerated the recovery of adenosine triphosphate both in vivo and in vitro. Emodin prevented mitochondrial fission and restored the balance of mitochondrial dynamics. The phosphorylation of dynamin-related protein 1 (DRP1) at Ser616, a master regulator of mitochondrial fission, was upregulated in both models of I/R and H/R injury, and this upregulation was blocked by emodin. Using computational cognate protein kinase prediction and specific kinase inhibitors, we found that emodin inhibited the phosphorylation of calcium/calmodulin-dependent protein kinase II (https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1554), thereby inhibiting its kinase activity and reducing the phosphorylation of DRP1 at Ser616. The results demonstrated that emodin pretreatment could protect renal function by improving mitochondrial dysfunction induced by I/R.

Methylglyoxal (MG) is a dicarbonyl compound, the level of which is increased in the blood of diabetes patients. MG is reported to be involved in the development of cerebrovascular complications in diabetes, but the exact mechanisms need to be elucidated. Here, we investigated the possible roles of oxidative stress and mitophagy in MG-induced functional damage in brain endothelial cells (ECs). Treatment of MG significantly altered metabolic stress as observed by the oxygen-consumption rate and barrier-integrity as found in impaired trans-endothelial electrical resistance in brain ECs. The accumulation of MG adducts and the disturbance of the glyoxalase system, which are major detoxification enzymes of MG, occurred concurrently. Reactive oxygen species (ROS)-triggered oxidative damage was observed with increased mitochondrial ROS production and the suppressed Akt/hypoxia-inducible factor 1 alpha (HIF-1&alpha, ) pathway. Along with the disturbance of mitochondrial bioenergetic function, parkin-1-mediated mitophagy was increased by MG. Treatment of N-acetyl cysteine significantly reversed mitochondrial damage and mitophagy. Notably, MG induced dysregulation of tight junction proteins including occludin, claudin-5, and zonula occluden-1 in brain ECs. Here, we propose that diabetic metabolite MG-associated oxidative stress may contribute to mitochondrial damage and autophagy in brain ECs, resulting in the dysregulation of tight junction proteins and the impairment of permeability.

l-carnosine is an attractive therapeutic agent for acute ischemic stroke based on its robust preclinical cerebroprotective properties and wide therapeutic time window. However, large doses are needed for efficacy because carnosine is rapidly degraded in serum by carnosinases. The need for large doses could be particularly problematic when translating to human studies, as humans have much higher levels of serum carnosinases. We hypothesized that d-carnosine, which is not a substrate for carnosinases, may have a better pharmacological profile and may be more efficacious at lower doses than l-carnosine. To test our hypothesis, we explored the comparative pharmacokinetics and neuroprotective properties of d- and L-carnosine in acute ischaemic stroke in mice. We initially investigated the pharmacokinetics of d- and L-carnosine in serum and brain after intravenous (IV) injection in mice. We then investigated the comparative efficacy of d- and l-carnosine in a mouse model of transient focal cerebral ischemia followed by in vitro testing against excitotoxicity and free radical generation using primary neuronal cultures. The pharmacokinetics of d- and l-carnosine were similar in serum and brain after IV injection in mice. Both d- and l-carnosine exhibited similar efficacy against mouse focal cerebral ischemia. In vitro studies in neurons showed protection against excitotoxicity and the accumulation of free radicals. d- and l-carnosine exhibit similar pharmacokinetics and have similar efficacy against experimental stroke in mice. Since humans have far higher levels of carnosinases, d-carnosine may have more favorable pharmacokinetics in future human studies.

Background Paclitaxel is one of the most widely used anti-cancer drugs, but numerous case reports of thrombotic events in the cancer patients using paclitaxel raise concern over its pro-thrombotic risk. Materials and Methods We investigated whether paclitaxel can elicit pro-thrombotic properties in red blood cells (RBCs) through phosphatidylserine (PS) exposure and microvesicle (MV) release. Results In freshly isolated human RBCs, paclitaxel induced thrombin generation through PS exposure and MV release, whereas either coagulation factors or platelets were unaffected. Paclitaxel-induced PS exposure in RBC was mediated by scramblase activation which was induced by calcium-independent protein kinase C (PKC)ζ activation. Paclitaxel also increased RBC-endothelial cell adhesion and RBC aggregate formation which can also contribute to thrombosis. Indeed, intravenous administration of paclitaxel to rats induced PS exposure and PKCζ activation in RBCs in vivo which ultimately promoted venous thrombus formation. Conclusion These results demonstrated that paclitaxel may elicit pro-thrombotic properties in RBCs through PS exposure and MV release, which can ultimately promote thrombus formation.

Ginseng and its major active ingredients, ginsenosides are widely consumed for various health benefits including the prevention or treatment of cancer without any special precaution. Previously, we demonstrated that Rg3, one of ginsenosides with a potent anti-cancer activity, may cause contractile dysfunction, and structural damage against normal vascular smooth muscle through apoptotic cell death. However, mechanism underlying the vascular toxicity of Rg3 was not fully elucidated. Here we demonstrated that the vascular toxicity of Rg3 occurs through actin disruption and Bmf-initiated mitochondrial apoptotic pathway, the mechanism which may be shared with its anti-cancer effects. In normal rat primary vascular smooth muscle cells, Rg3 induced apoptosis and contractile dysfunction as determined by caspase-3 activation, TUNEL staining, and insufficient myosin light chain phosphorylation. Rg3 induced actin degradation with RhoA inactivation, followed by mitochondrial translocation of Bmf and dissociation of mitochondrial membrane potential (φ). Pre-treatment of jasplakinolide, an inhibitor of actin depolymerization, significantly blocked Rg3-induced φ dissipation and apoptosis. Notably, Rg3 also induced actin disruption in hepatoma cell line, HepG2, which was reversed by jasplakinolide, confirming that the anti-cancer effects of Rg3 shares the mechanism at least in part. Collectively, these results demonstrated that Rg3 may induce vascular toxicity as well as anti-cancer effects through disrupting actin, suggesting the potential toxicity associated with inadvertent use of ginseng and its products.

The global burden of neurological diseases including stroke has significantly increased,1 and an urgent need exists to develop new treatment strategies. Impairment of autophagic regulation has been observed in diseases including neurodegenerative diseases and ischemic stroke, suggesting that modulation of autophagy could be a potential therapeutic target.2 Endothelial cells (ECs) maintain homeostasis by regulating the vascular tone and permeability and endothelial dysfunction is associated with diverse cardiovascular diseases (CVDs). The integrity of the blood–brain barrier (BBB), which shows selective permeability for substances into the brain, is significantly impaired under ischemic stroke. This review focuses on autophagy in endothelial dysfunction in the context of ischemic stroke and potential targets for therapeutic manipulation. Autophagy or autophagocytosis is an evolutionarily conserved mechanism for the degradation and recycling of cellular organelles and protein.2 It occurs continually at basal levels in cells and contributes to the maintenance of cellular homeostasis. When external nutrient supplies are limited, cells attempt to generate their energy by degrading and recycling macromolecules and cellular organelles by autophagy.3 Autophagy is also an important defense mechanism against stress including oxidative stress and infection, enabling cellular repair, or clearance of pathogens.1,2 Defects in autophagy flux may lead to the accumulation of damaged or senescent proteins and abnormal protein aggregates, and this is closely associated with human diseases including neurodegenerative, cardiovascular, and metabolic diseases, as well as cancer.2,4 In contrast to the protective role of autophagy in maintaining basal cellular homeostasis, excessive autophagy may also cause dysregulation of catabolic activity and maladaptation to cellular stress, leading to autophagic cell death.5 Accumulating evidence shows that modulating the level of autophagy by targeting specific regulatory molecules in the autophagy machinery may impact disease onset or disease …

Endothelial cells (ECs) maintain the structure and function of blood vessels and are readily exposed to exogenous and endogenous toxic substances in the circulatory system. Bone marrow-derived endothelial progenitor cells (EPCs) circulate in the blood and differentiate to EC, which are known to participate in angiogenesis and regeneration of injured vessels. Dysfunction in EPC contributes to cardiovascular complications in patients with diabetes, but the precise molecular mechanisms underlying diabetic EPC abnormalities are not completely understood. The aim of this study was to investigate the mechanisms underlying diabetic EPC dysfunction using methylglyoxal (MG), an endogenous toxic diabetic metabolite. Data demonstrated that MG decreased cell viability and protein expression of vascular endothelial growth factor receptor (VEGFR)-2 associated with functional impairment of tube formation in EPC. The generation of advanced glycation end (AGE) products was increased in EPC following exposure to MG. Blockage of receptor for AGE (RAGE) by FPS-ZM1, a specific antagonist for RAGE, significantly reversed the decrease of VEGFR-2 protein expression and angiogenic dysfunction in MG-incubated EPC. Taken together, data demonstrated that MG induced angiogenic impairment in EPC via alterations in the AGE/RAGE-VEGFR-2 pathway which may be utilized in the development of potential therapeutic and preventive targets for diabetic vascular complications.

One new flavone glycoside, nomaminoside (3), containing a threono-1,4-lactone moiety, together with three known compounds, isoorientin 2′′-O-rhamnoside (1), coatline B (2), and myrcetin (4), were isolated from the n-butanol fraction of Cassia nomame methanolic extract. The chemical structure of the new compound was elucidated by spectroscopic analysis including HR-ESI-MS, 1D and 2D NMR techniques.

Despite the increasing attention on the therapeutic potential of Curcuma longa (turmeric), the biological activities of curcuminoids other than curcumin are not well understood. Here, we investigated antivasoconstrictive activities of C. longa extract and its ingredients using freshly isolated rat aortic rings. C. longa extract significantly suppressed agonist-stimulated vasoconstriction, and cyclocurcumin was found to be the most potent (IC50 against phenylephrine-induced vasoconstriction: 14.9 ± 1.0 μM) among the 10 tested ingredients including four curcuminoids. Cyclocurcumin significantly inhibited contraction of vascular smooth muscle, which was mediated by the suppression of myosin-light-chain phosphorylation and calcium influx via the L-type calcium channel. The inhibitory effect of cyclocurcumin was observed to be reversible and without cytotoxicity. Taken together, we demonstrated that cyclocurcumin, a bioactive ingredient in C. longa, may have a therapeutic potential as a novel antivasoconstrict...

In this study, we investigated the effects of antibiotics on the pharmacological effects of aspirin. The antithrombotic activity of aspirin was evaluated after antibiotic treatment using tail bleeding assay. The pyrosequencing analysis and selective medium culture assay were performed to investigate the alterations in gut microbiota. In addition, the in vitro metabolism assay with fecal suspension and in vivo pharmacokinetic experiments with antibiotic treatment were conducted. Ampicillin treatment significantly prolonged the bleeding time in aspirin-dosed rats. Oral administration of ampicillin significantly reduced gut microbial aspirin-metabolizing activity by 67.0% in rats. Furthermore, systemic exposure to aspirin and its primary metabolite (M1) was significantly increased in ampicillin-treated rats. The results from the pyrosequencing and selective medium culture with rat fecal samples revealed that ampicillin treatment led to the changes of the amounts and composition profile of gut microbiota. These findings suggest that co-administration of antibiotics can modulate the metabolism and pharmacokinetics of aspirin via suppression of metabolic activity of gut microbiota, which could potentiate the therapeutic potency of aspirin.

Stroke is one of the commonest causes of death with limited treatment options. L-Carnosine has shown great promise as a neuroprotective agent in experimental stroke, but translation to the clinic is impeded by the large doses needed. We developed and evaluated the therapeutic potential of a novel delivery vehicle which encapsulated carnosine in lipoprotein receptor related protein-1 (LRP-1)-targeted functionalized polymersomes in experimental ischemic stroke. We found that following ischemic stroke, polymersomes encapsulating carnosine exhibited remarkable neuroprotective effects with a dose of carnosine 3 orders of magnitude lower than free carnosine. The LRP-1-targeted functionalization was essential for delivery of carnosine to the brain, as non-targeted carnosine polymersomes did not exhibit neuroprotection. Using Cy3 fluorescence in vivo imaging, we showed that unlike non-targeted carnosine polymersomes, LRP-1-targeted carriers accumulated in brain in a time dependent manner. Our findings suggest that these novel carriers have the ability to deliver neuroprotective cargo effectively to the brain.

The skin is an important physiological barrier against external stimuli, such as ultraviolet radiation (UV), xenobiotics, and bacteria. Dermal inflammatory reactions are associated with various skin disorders, including chemical-induced irritation and atopic dermatitis. Modulation of skin inflammatory response is a therapeutic strategy for skin diseases. Here, we synthesized chrysin-derivatives and identified the most potent derivative of Compound 6 (CPD 6). We evaluated its anti-inflammatory effects in vitro cells of macrophages and keratinocytes, and in vivo dermatitis mouse models. In murine macrophages stimulated by lipopolysaccharide (LPS), CPD 6 significantly attenuated the release of inflammatory mediators such as nitric oxide (NO) (IC50 for NO inhibition: 3.613 &mu, M) and other cytokines. In cultured human keratinocytes, CPD 6 significantly attenuated the release of inflammatory cytokines induced by the combination of IFN-&gamma, and TNF-&alpha, UV irradiation, or chemical irritant stimulation. CPD 6 inhibited NF&kappa, B and JAK2/STAT1 signaling pathways, and activated Nrf2/HO-1 signaling. In vivo relevancy of anti-inflammatory effects of CPD 6 was observed in acute and chronic skin inflammation models in mice. CPD 6 showed significant anti-inflammatory properties both in vitro cells and in vivo dermatitis animal models, mediated by the inhibition of the NF&kappa, B and JAK2-STAT1 pathways and activation of Nrf2/HO-1 signaling. We propose that the novel chrysin-derivative CPD 6 may be a potential therapeutic agent for skin inflammation.

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.

Exenatide (Ex) is a 39-amino acid peptide of glucagon-like peptide-1 (GLP-1) receptor agonist that was approved by the FDA in 2005 as a Type II diabetes treatment. It shows a 53% homology with GLP-1 but has an extended half-life (ca. 2.4 h) relative to GLP-1 (ca. 2–3 min). In this study, to further extend its in vivo half-life, we constructed a fusion protein (Ex-(EBP)10-6xHis) using a biocompatible and inert elastin-based polypeptide (EBP) as a fusion partner. Valine was inserted into the guest position of the pentapeptide (VPGXG), no linker sequence was inserted in between the EBPs, and (EBP)10-6xHis tag was attached to the C-terminus of exenatide. By using a recombinant Saccharomyces cerevisiae expression system, the fusion protein was expressed and secreted to the broth and purified by Ni-NTA affinity chromatography. Compared with the native exenatide, the physical half-life of the fusion protein was ca. 3.7-fold extended while approximately 72% of the in-vitro insulin secreting activity was maintained. However, the biological half-life measured by a glucose tolerance test (GTT) and the hypoglycemic test in mice was not significantly different from that of the native form. The effects of EBPylation on bioactivity and half-life of the fusion protein are similar to those of PEGylation. The result suggests that the bioactivity and half-life should be carefully balanced to obtain optimal fusion proteins. We expect that EBPylation using an optimal repeat number of EBP can be an alternative to chemical modification for therapeutic biobetters with extended half-life.

A concise synthesis of sericetin (1) was performed in four steps from readily available 3- O-benzylgalangin (4), featuring electrocyclization to produce the tricyclic core and a sequential aromatic Claisen/Cope rearrangement to incorporate the 8-prenyl group of 1. In addition, the therapeutic potential of sericetin (1), isosericetin (2), and three prenylated tetracyclic synthetic intermediates (11, 12, and 14) against cisplatin-induced nephrotoxicity using renal tubular cells were evaluated. Compound 14 showed therapeutic potential against cisplatin-induced kidney damage.

Volatile organic compounds (VOCs) threaten the health of women who use disposable menstrual products (DMPs). In this study, we aimed to develop a simple and reliable analytical method using a cryogenic mill and static headspace–gas chromatography–mass spectrometry (HS-GC-MS) for the simultaneous determination of twelve toxic VOCs in DMPs, and estimate the measurement uncertainty of the method using the bottom-up approach to ensure the quality of analytical results. Static HS conditions for the direct thermal extraction of the target VOCs from the cryogenically ground sample powders were optimized in terms of extraction temperature and equilibrium time. The separation was achieved using a GC-MS equipped with a VF-624ms column. Cryogenic mill homogenized DMP into powder with 100-μm particles in scanning electron microscopy study. Sample powder was demonstrated to be homogenous as assessed by the homogeneity test. Validation studies showed that the method has reliable performance characteristics: linearity (R2 ≥ 0.9994), limit of quantification (LOQ, 8.4–50.5 ng g−1), precision (relative standard deviation ≤ 18.7%), and accuracy (recovery = 83.4–114.2%) for all the analytes. The proposed method was successfully applied to the commercial products of disposable sanitary pads and tampons. The content of styrene was the highest, followed by those of toluene, hexane, and ethylbenzene. The quantitative data exhibited acceptable precision, as evaluated using the intra-laboratory Horwitz ratio. The expanded uncertainties estimated at three concentration levels ranged from 6.4% to 77.9%. Our simple and reliable method will be useful for quality control testing or quantitative risk assessment and will contribute to resolving the safety issues associated with the risk of human exposure to trace volatile residues in DMPs.