Post-Translational Oxidative Modifications of Hemostasis Proteins: Structure, Function, and Regulation.

Reactive oxygen species (ROS) are constantly generated in a living organism. An imbalance between the amount of generated reactive species in the body and their destruction leads to the development of oxidative stress. Proteins are extremely vulnerable targets for ROS molecules, which can cause oxidative modifications of amino acid residues, thus altering structure and function of intra- and extracellular proteins. The current review considers the effect of oxidation on the structural rearrangements and functional activity of hemostasis proteins: coagulation system proteins such as fibrinogen, prothrombin/thrombin, factor VII/VIIa; anticoagulant proteins - thrombomodulin and protein C; proteins of the fibrinolytic system such as plasminogen, tissue plasminogen activator and plasminogen activator inhibitor-1. Structure and function of the proteins, oxidative modifications, and their detrimental consequences resulting from the induced oxidation or oxidative stress in vivo are described. Possible effects of oxidative modifications of proteins in vitro and in vivo leading to disruption of the coagulation and fibrinolysis processes are summarized and systematized, and the possibility of a compensatory mechanism in maintaining hemostasis under oxidative stress is analyzed.

Comparison of results obtained using clot-fibrinolysis waveform analysis and global fibrinolysis capacity assay with rotational thromboelastography.

Global fibrinolysis assays detect the fibrinolysis time of clot dissolution using tissue-type plasminogen activator (tPA). Two such assays, clot-fibrinolysis waveform analysis (CFWA) and global fibrinolysis capacity (GFC) assay, were recently developed. These were compared with rotational thromboelastography (ROTEM). Healthy donor blood samples were divided into four groups based on tPA-spiked concentrations: 0, 100, 500, and 1000 ng/mL. CFWA and GFC fibrinolysis times, including 4.1 µg/mL and 100 ng/mL tPA in the assays, were determined, denoted as CFWA-Lys and GFC-Lys, respectively. Statistical differences were recognized between tPA concentrations of 0 and 500/1000 ng/mL for CFWA-Lys, and 0 and 100/500/1000 ng/mL for GFC-Lys. The correlation coefficients with lysis onset time (LOT) of extrinsic pathway evaluation and intrinsic pathway evaluation in ROTEM were statistically significant at 0.610 and 0.590 for CFWA-Lys, and 0.939 and 0.928 for GFC-Lys, respectively (p-values < 0.0001 for all correlations). Both assays showed significant correlations with ROTEM; however, the GFC assay proved to have better agreement with ROTEM compared with the CFWA assay. These assays have the potential to reflect a hyperfibrinolysis status with high tPA concentrations.

Jasmonic acid improves barley photosynthetic efficiency through a possible regulatory module, MYC2-RcaA, under combined drought and salinity stress.

The combined stress of drought and salinity is prevalent in various regions of the world, affects several physiological and biochemical processes in crops, and causes their yield to decrease. Photosynthesis is one of the main processes that are disturbed by combined stress. Therefore, improving the photosynthetic efficiency of crops is one of the most promising strategies to overcome environmental stresses, making studying the molecular basis of regulation of photosynthesis a necessity. In this study, we sought a potential mechanism that regulated a major component of the combined stress response in the important crop barley (Hordeum vulgare L.), namely the Rubisco activase A (RcaA) gene. Promoter analysis of the RcaA gene led to identifying Jasmonic acid (JA)-responsive elements with a high occurrence. Specifically, a Myelocytomatosis oncogenes 2 (MYC2) transcription factor binding site was highlighted as a plausible functional promoter motif. We conducted a controlled greenhouse experiment with an abiotic stress-susceptible barley genotype and evaluated expression profiling of the RcaA and MYC2 genes, photosynthetic parameters, plant water status, and cell membrane damages under JA, combined drought and salinity stress (CS) and JA + CS treatments. Our results showed that applying JA enhances barley's photosynthetic efficiency and water relations and considerably compensates for the adverse effects of combined stress. Significant association was observed among gene expression profiles and evaluated physiochemical characteristics. The results showed a plausible regulatory route through the JA-dependent MYC2-RcaA module involved in photosynthesis regulation and combined stress tolerance. These findings provide valuable knowledge for further functional studies of the regulation of photosynthesis under abiotic stresses toward the development of multiple-stress-tolerant crops.

Mechanical induction of osteoanabolic Wnt1 promotes osteoblast differentiation via Plat.

Physical activity-induced mechanical stimuli play a crucial role in preserving bone mass and structure by promoting bone formation. While the Wnt pathway is pivotal for mediating the osteoblast response to loading, the exact mechanisms are not fully understood. Here, we found that mechanical stimulation induces osteoblastic Wnt1 expression, resulting in an upregulation of key osteogenic marker genes, including Runx2 and Sp7, while Wnt1 knockdown using siRNA prevented these effects. RNAseq analysis identified Plat as a major target through which Wnt1 exerts its osteogenic influence. This was corroborated by Plat depletion using siRNA, confirming its positive role in osteogenic differentiation. Moreover, we demonstrated that mechanical stimulation enhances Plat expression, which, in turn leads to increased expression of osteogenic markers like Runx2 and Sp7. Notably, Plat depletion by siRNA prevented this effect. We have established that Wnt1 regulates Plat expression by activating ß-Catenin. Silencing Wnt1 impairs mechanically induced ß-Catenin activation, subsequently reducing Plat expression. Furthermore, our findings showed that Wnt1 is essential for osteoblasts to respond to mechanical stimulation and induce Runx2 and Sp7 expression, in part through the Wnt1/ß-Catenin/Plat signaling pathway. Additionally, we observed significantly reduced Wnt1 and Plat expression in bones from ovariectomy (OVX)-induced and age-related osteoporotic mouse models compared with non-OVX and young mice, respectively. Overall, our data suggested that Wnt1 and Plat play significant roles in mechanically induced osteogenesis. Their decreased expression in bones from OVX and aged mice highlights their potential involvement in post-menopausal and age-related osteoporosis, respectively.

The effect of plasmin-mediated degradation on fibrinolysis and tissue plasminogen activator diffusion.

We modify a three-dimensional multiscale model of fibrinolysis to study the effect of plasmin-mediated degradation of fibrin on tissue plasminogen activator (tPA) diffusion and fibrinolysis. We propose that tPA is released from a fibrin fiber by simple kinetic unbinding, as well as by "forced unbinding," which occurs when plasmin degrades fibrin to which tPA is bound. We show that, if tPA is bound to a small-enough piece of fibrin that it can diffuse into the clot, then plasmin can increase the effective diffusion of tPA. If tPA is bound to larger fibrin degradation products (FDPs) that can only diffuse along the clot, then plasmin can decrease the effective diffusion of tPA. We find that lysis rates are fastest when tPA is bound to fibrin that can diffuse into the clot, and slowest when tPA is bound to FDPs that can only diffuse along the clot. Laboratory experiments confirm that FDPs can diffuse into a clot, and they support the model hypothesis that forced unbinding of tPA results in a mix of FDPs, such that tPA bound to FDPs can diffuse both into and along the clot. Regardless of how tPA is released from a fiber, a tPA mutant with a smaller dissociation constant results in slower lysis (because tPA binds strongly to fibrin), and a tPA mutant with a larger dissociation constant results in faster lysis.

Parabiosis Discriminates the Circulating, Endothelial, and Parenchymal Contributions of Endogenous Tissue-Type Plasminogen Activator to Stroke.

BACKGROUND: Intravenous injection of alteplase, a recombinant tPA (tissue-type plasminogen activator) as a thrombolytic agent has revolutionized ischemic stroke management. However, tPA is a more complex enzyme than expected, being for instance able to promote thrombolysis, but at the same time, also able to influence neuronal survival and to affect the integrity of the blood-brain barrier. Accordingly, the respective impact of endogenous tPA expressed/present in the brain parenchyma versus in the circulation during stroke remains debated. METHODS: To address this issue, we used mice with constitutive deletion of tPA (tPANull [tPA-deficient mice]) or conditional deletion of endothelial tPA (VECad [vascular endothelial-Cadherin-Cre-recombinase]-Cre∆tPA). We also developed parabioses between tPANull and wild-type mice (tPAWT), anticipating that a tPAWT donor would restore levels of tPA to normal ones, in the circulation but not in the brain parenchyma of a tPANull recipient. Stroke outcomes were investigated by magnetic resonance imaging in a thrombo-embolic or a thrombotic stroke model, induced by local thrombin injection or FeCl3 application on the endothelium, respectively. RESULTS: First, our data show that endothelial tPA, released into the circulation after stroke onset, plays an overall beneficial role following thrombo-embolic stroke. Accordingly, after 24 hours, tPANull/tPANull parabionts displayed less spontaneous recanalization and reperfusion and larger infarcts compared with tPAWT/tPAWT littermates. However, when associated to tPAWT littermates, tPANull mice had similar perfusion deficits, but less severe brain infarcts. In the thrombotic stroke model, homo- and hetero-typic parabionts did not differ in the extent of brain damages and did not differentially recanalize and reperfuse. CONCLUSIONS: Together, our data reveal that during thromboembolic stroke, endogenous circulating tPA from endothelial cells sustains a spontaneous recanalization and reperfusion of the tissue, thus, limiting the extension of ischemic lesions. In this context, the impact of endogenous parenchymal tPA is limited.

Internal fibrinolysis of fibrin clots is driven by pore expansion.

Blood clots, which are composed of blood cells and a stabilizing mesh of fibrin fibers, are critical in cessation of bleeding following injury. However, their action is transient and after performing their physiological function they must be resolved through a process known as fibrinolysis. Internal fibrinolysis is the degradation of fibrin by the endogenous or innate presence of lytic enzymes in the bloodstream; under healthy conditions, this process regulates hemostasis and prevents bleeding or clotting. Fibrin-bound tissue plasminogen activator (tPA) converts nearby plasminogen into active plasmin, which is bound to the fibrin network, breaking it down into fibrin degradation products and releasing the entrapped blood cells. It is poorly understood how changes in the fibrin structure and lytic protein ratios influence the biochemical regulation and behavior of internal fibrinolysis. We used turbidity kinetic tracking and microscopy paired with mathematical modeling to study fibrin structure and lytic protein ratios that restrict internal fibrinolysis. Analysis of simulations and experiments indicate that fibrinolysis is driven by pore expansion of the fibrin network. We show that this effect is strongly influenced by the ratio of fibrin:tPAwhen compared to absolute tPA concentration. Thus, it is essential to consider relative protein concentrations when studying internal fibrinolysis both experimentally and in the clinic. An improved understanding of effective internal lysis can aid in development of better therapeutics for the treatment of bleeding and thrombosis.

Identification and characterization of calreticulin as a novel plasminogen receptor.

Calreticulin (CRT) was originally identified as a key calcium-binding protein of the endoplasmic reticulum. Subsequently, CRT was shown to possess multiple intracellular functions, including roles in calcium homeostasis and protein folding. Recently, several extracellular functions have been identified for CRT, including roles in cancer cell invasion and phagocytosis of apoptotic and cancer cells by macrophages. In the current report, we uncover a novel function for extracellular CRT and report that CRT functions as a plasminogen-binding receptor that regulates the conversion of plasminogen to plasmin. We show that human recombinant or bovine tissue-derived CRT dramatically stimulated the conversion of plasminogen to plasmin by tissue plasminogen activator or urokinase-type plasminogen activator. Surface plasmon resonance analysis revealed that CRT-bound plasminogen (KD = 1.8 µM) with moderate affinity. Plasminogen binding and activation by CRT were inhibited by ε-aminocaproic acid, suggesting that an internal lysine residue of CRT interacts with plasminogen. We subsequently show that clinically relevant CRT variants (lacking four or eight lysines in carboxyl-terminal region) exhibited decreased plasminogen activation. Furthermore, CRT-deficient fibroblasts generated 90% less plasmin and CRT-depleted MDA MB 231 cells also demonstrated a significant reduction in plasmin generation. Moreover, treatment of fibroblasts with mitoxantrone dramatically stimulated plasmin generation by WT but not CRT-deficient fibroblasts. Our results suggest that CRT is an important cellular plasminogen regulatory protein. Given that CRT can empower cells with plasmin proteolytic activity, this discovery may provide new mechanistic insight into the established role of CRT in cancer.

ALDH1A3 promotes invasion and metastasis in triple-negative breast cancer by regulating the plasminogen activation pathway.

Aldehyde dehydrogenase 1A3 (ALDH1A3) is a cancer stem cell marker that promotes metastasis. Triple-negative breast cancer (TNBC) progression has been linked to ALDH1A3-induced gene expression changes. To investigate the mechanism of ALDH1A3-mediated breast cancer metastasis, we assessed the effect of ALDH1A3 on the expression of proteases and the regulators of proteases that degrade the extracellular matrix, a process that is essential for invasion and metastasis. This revealed that ALDH1A3 regulates the plasminogen activation pathway; it increased the levels and activity of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). This resulted in a corresponding increase in the activity of serine protease plasmin, the enzymatic product of tPA and uPA. The ALDH1A3 product all-trans-retinoic acid similarly increased tPA and plasmin activity. The increased invasion of TNBC cells by ALDH1A3 was plasminogen-dependent. In patient tumours, ALDH1A3 and tPA are co-expressed and their combined expression correlated with the TNBC subtype, high tumour grade and recurrent metastatic disease. Knockdown of tPA in TNBC cells inhibited plasmin generation and lymph node metastasis. These results identify the ALDH1A3-tPA-plasmin axis as a key contributor to breast cancer progression.

Crosstalk between Nrf2 signaling pathway and inflammation in ischemic stroke: Mechanisms of action and therapeutic implications.

One of the major causes of long-term disability and mortality is ischemic stroke that enjoys limited treatment approaches. On the one hand, oxidative stress, induced by excessive generation of reactive oxygen species (ROS), plays a critical role in post-stroke inflammatory response. Increased ROS generation is one of the basic factors in the progression of stroke-induced neuroinflammation. Moreover, intravenous (IV) thrombolysis using recombinant tissue plasminogen activator (rtPA) as the only medication approved for patients with acute ischemic stroke who suffer from some clinical restrictions it could not cover the complicated episodes that happen after stroke. Thus, identifying novel therapeutic targets is crucial for successful preparation of new medicines. Recent evidence indicates that the transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) contributes significantly to regulating the antioxidant production in cytosol, which causes antiinflammatory effects on neurons. New findings have shown a relationship between activation of the Nrf2 and glial cells, nuclear factor kappa B (NF-κB) pathway, the nucleotide-binding domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling, and expression of inflammatory markers, suggesting induction of Nrf2 activation can represent a promising therapeutic alternative as the modulators of Nrf2 dependent pathways for targeting inflammatory responses in neural tissue. Hence, this review addresses the relationship of Nrf2 signaling with inflammation and Nrf2 activators' potential as therapeutic agents. This review helps to improve required knowledge for focused therapy and the creation of modern and improved treatment choices for patients with ischemic stroke.

Site-specific PEGylation of recombinant tissue-type plasminogen activator.

Tissue-type plasminogen activator (tPA) is the gold standard for emergency treatment of ischemic stroke, which is the third leading cause of death worldwide. Major challenges of tPA therapy are its rapid elimination by plasminogen activator inhibitor-1 (PAI-1) and hepatic clearance, leading to the use of high doses and consequent serious side effects, including internal bleeding, swelling and low blood pressure. In this regard, we developed three polyethylene glycol (PEG)ylated tPA bioconjugates based on the recombinant human tPA drug Alteplase using site-specific conjugation strategies. The first bioconjugate with PEGylation at the N-terminus of tPA performed by reductive alkylation showed a reduced proteolytic activity of 68 % compared to wild type tPA. PEGylation at the single-free cysteine of tPA with linear and branched PEG revealed similar proteolytic activities as the wild-type protein. Moreover, both bioconjugates with PEG-cysteine-modification showed 2-fold slower inhibition kinetics by PAI-1. All bioconjugates increased in hydrodynamic size as a critical requirement for half-life extension.

Quercetin Attenuates Atherosclerosis via Modulating Apelin Signaling Pathway Based on Plasma Metabolomics.

OBJECTIVE: To interpret the pharmacology of quercetin in treatment of atherosclerosis (AS). METHODS: Fourteen apolipoprotein E-deficient (ApoE-/-) mice were divided into 2 groups by a random number table: an AS model (ApoE-/-) group and a quercetin treatment group (7 in each). Seven age-matched C57 mice were used as controls (n=7). Quercetin [20 mg/(kg·d)] was administered to the quercetin group intragastrically for 8 weeks for pharmacodynamic evaluation. Besides morphological observation, the distribution of CD11b, F4/80, sirtuin 1 (Sirt1) and P21 was assayed by immunohistochemistry and immunofluorescence to evaluate macrophage infiltration and tissue senescence. Ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MSC/MS) was performed to study the pharmacology of quercetin against AS. Then, simultaneous administration of an apelin receptor antagonist (ML221) with quercetin was conducted to verify the possible targets of quercetin. Key proteins in apelin signaling pathway, such as angiotensin domain type 1 receptor-associated proteins (APJ), AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), tissue plasminogen activator (TPA), uncoupling protein 1 (UCP1) and angiotensin II receptor 1 (AT1R), were assayed by Western blot. RESULTS: Quercetin administration decreased lipid deposition in arterial lumen and improved the morphology of ApoE-/- aortas in vivo. Quercetin decreased the densities of CD11b, F4/80 and P21 in the aorta and increased the level of serum apelin and the densities of APJ and Sirt1 in the aorta in ApoE-/- mice (all P<0.05). Plasma metabolite profiling identified 118 differential metabolites and showed that quercetin affected mainly glycerophospholipids and fatty acyls. Bioinformatics analysis suggested that the apelin signaling pathway was one of the main pathways. Quercetin treatment increased the protein expressions of APJ, AMPK, PGC-1α, TPA and UCP1, while decreased the AT1R level (all P<0.05). After the apelin pathway was blocked by ML221, the effect of quercetin was abated significantly, confirming that quercetin attenuated AS by modulating the apelin signaling pathway (all P<0.05). CONCLUSION: Quercetin alleviated AS lesions by up-regulation the apelin signaling pathway.

Intracellular tPA-PAI-1 interaction determines VLDL assembly in hepatocytes.

Apolipoprotein B (apoB)-lipoproteins initiate and promote atherosclerotic cardiovascular disease. Plasma tissue plasminogen activator (tPA) activity is negatively associated with atherogenic apoB-lipoprotein cholesterol levels in humans, but the mechanisms are unknown. We found that tPA, partially through the lysine-binding site on its Kringle 2 domain, binds to the N terminus of apoB, blocking the interaction between apoB and microsomal triglyceride transfer protein (MTP) in hepatocytes, thereby reducing very-low-density lipoprotein (VLDL) assembly and plasma apoB-lipoprotein cholesterol levels. Plasminogen activator inhibitor 1 (PAI-1) sequesters tPA away from apoB and increases VLDL assembly. Humans with PAI-1 deficiency have smaller VLDL particles and lower plasma levels of apoB-lipoprotein cholesterol. These results suggest a mechanism that fine-tunes VLDL assembly by intracellular interactions among tPA, PAI-1, and apoB in hepatocytes.

Association of pro-fibrinolytic receptor AnnexinA2 with tissue plasminogen activator/Inhibitor-1 in pre-eclampsia.

Background: The clinical manifestations of pre-eclampsia are related to placental anti-angiogenic factor alteration. These variations are mainly due to the alteration of plasminolytic components. The study aims to compare the expression of plasminolytic components in the placenta of women with and without pre-eclampsia. Material and Methods: The study included pregnant women with pre-eclampsia as PE group (n = 30) and without pre-eclampsia as a control group (n = 30). Placental bed biopsy tissues were collected. AnxA2, tPA, PAI-1 expression in the placental villous tissue was quantitatively evaluated using immunohistochemistry, western blot, and real time-PCR analysis. Results: The results of the study showed a significant decrease in the expression of ANXA2 and increased expression of tPA and PAI-1 in PE group compared to control group (p<0.005). AnxA2 expression showed positive correlation with tPA (r=+0.895, p=0.002) and negative correlation with PAI-1(r=-0.905, p=0.020) in control group whereas in the PE group AnxA2 expression was negatively correlated with tPA ((r=-0.801, p=0.016) and PAI-1 (R=-0.831, P=0.010). Conclusion: Decreased AnxA2 with increased expression of PAI-1 and tPA may be responsible for the altered fibrinolytic activity and play a significant role in pre-eclampsia pathogenesis.

Macrophage Function Modulated by tPA Signaling in Mouse Experimental Kidney Disease Models.

Macrophage infiltration and accumulation is a hallmark of chronic kidney disease. Tissue plasminogen activator (tPA) is a serine protease regulating the homeostasis of blood coagulation, fibrinolysis, and matrix degradation, and has been shown to act as a cytokine to trigger various receptor-mediated intracellular signal pathways, modulating macrophage function in response to kidney injury. In this review, we discuss the current understanding of tPA-modulated macrophage function and underlying signaling mechanisms during kidney fibrosis and inflammation.

Disruption of the Clock Component Bmal1 in Mice Promotes Cancer Metastasis through the PAI-1-TGF-ß-myoCAF-Dependent Mechanism.

The circadian clock in animals and humans plays crucial roles in multiple physiological processes. Disruption of circadian homeostasis causes detrimental effects. Here, it is demonstrated that the disruption of the circadian rhythm by genetic deletion of mouse brain and muscle ARNT-like 1 (Bmal1) gene, coding for the key clock transcription factor, augments an exacerbated fibrotic phenotype in various tumors. Accretion of cancer-associated fibroblasts (CAFs), especially the alpha smooth muscle actin positive myoCAFs, accelerates tumor growth rates and metastatic potentials. Mechanistically, deletion of Bmal1 abrogates expression of its transcriptionally targeted plasminogen activator inhibitor-1 (PAI-1). Consequently, decreased levels of PAI-1 in the tumor microenvironment instigate plasmin activation through upregulation of tissue plasminogen activator and urokinase plasminogen activator. The activated plasmin converts latent TGF-ß into its activated form, which potently induces tumor fibrosis and the transition of CAFs into myoCAFs, the latter promoting cancer metastasis. Pharmacological inhibition of the TGF-ß signaling largely ablates the metastatic potentials of colorectal cancer, pancreatic ductal adenocarcinoma, and hepatocellular carcinoma. Together, these data provide novel mechanistic insights into disruption of the circadian clock in tumor growth and metastasis. It is reasonably speculated that normalization of the circadian rhythm in patients provides a novel paradigm for cancer therapy.

Endothelial-derived extracellular vesicles associated with electronic cigarette use impair cerebral microvascular cell function.

The aim of this study was to determine the effect of circulating endothelial cell-derived microvesicles (EMVs) isolated from e-cigarette users on human cerebral microvascular endothelial cells (hCMECs) nitric oxide (NO) and endothelin (ET)-1 production and tissue-type plasminogen activator (t-PA) release. Circulating EMVs (CD144-PE) were isolated (flow cytometry) from 27 young adults (19-25 yr): 10 nonsmokers (6 M/4 F), 10 e-cigarette users (6 M/4 F), and 7 tobacco cigarette smokers (4 M/3 F). hCMECs were cultured and treated with isolated EMVs for 24 h. EMVs from e-cigarette users and cigarette smokers induced significantly higher expression of p-eNOS (Thr495; 28.4 ± 4.6 vs. 29.1 ± 2.8 vs. 22.9 ± 3.8 AU), Big ET-1 (138.8 ± 19.0 vs. 141.7 ± 19.1 vs. 90.3 ± 18.8 AU) and endothelin converting enzyme (107.6 ± 10.1 and 113.5 ± 11.8 vs. 86.5 ± 13.2 AU), and significantly lower expression of p-eNOS (Ser1177; 7.4 ± 1.7 vs. 6.5 ± 0.5 vs. 9.7 ± 1.6 AU) in hCMECs than EMVs from nonsmokers. NO production was significantly lower and ET-1 production was significantly higher in hCMECs treated with EMVs from e-cigarette (5.7 ± 0.8 µmol/L; 33.1 ± 2.9 pg/mL) and cigarette smokers (6.3 ± 0.7 µmol/L; 32.1 ± 3.9 pg/mL) than EMVs from nonsmokers (7.6 ± 1.2 µmol/L; 27.9 ± 3.1 pg/mL). t-PA release in response to thrombin was significantly lower in hCMECs treated with EMVs from e-cigarette users (from 38.8 ± 6.3 to 37.4 ± 8.3 pg/mL) and cigarette smokers (31.5 ± 5.5 to 34.6 ± 8.4 pg/mL) than EMVs from nonsmokers (38.9 ± 4.3 to 48.4 ± 7.9 pg/mL). There were no significant differences in NO, ET-1, or t-PA protein expression or production in hCMECs treated with EMVs from e-cigarette users and smokers. Circulating EMVs associated with e-cigarette use adversely affects brain microvascular endothelial cells and may contribute to reported cerebrovascular dysfunction with e-cigarette use.NEW & NOTEWORTHY In the present study, we determined the effect of circulating endothelial cell-derived microvesicles (EMVs) isolated from e-cigarette users on human cerebral microvascular endothelial cells (hCMECs) nitric oxide (NO) and endothelin (ET)-1 production and tissue-type plasminogen activator (t-PA) release. EMVs from e-cigarette users reduced brain microvascular endothelial cell NO production, enhanced ET-1 production, and impaired endothelial t-PA release. EMVs are a potential mediating factor in the increased risk of stroke associated with e-cigarette use.

Antibody-mediated inhibition of tissue-type plasminogen activator binding to the low-density lipoprotein receptor-related protein 1 as a potential beneficial modulator for stroke therapy.

The acute ischemic stroke therapy of choice is the application of Alteplase, a drug containing the enzyme tissue-type plasminogen activator (tPa) which rapidly destabilizes blood clots. A central hallmark of stroke pathology is blood-brain barrier (BBB) breakdown associated with tight junction (TJ) protein degradation, which seems to be significantly more severe under therapeutic conditions. The exact mechanisms how tPa facilitates BBB breakdown are not entirely understood. There is evidence that an interaction with the lipoprotein receptor-related protein 1 (LRP1), allowing tPa transport across the BBB into the central nervous system, is necessary for this therapeutic side effect. Whether tPa-mediated disruption of BBB integrity is initiated directly on microvascular endothelial cells or other brain cell types is still elusive. In this study we could not observe any changes of barrier properties in microvascular endothelial cells after tPa incubation. However, we present evidence that tPa causes changes in microglial activation and BBB breakdown after LRP1-mediated transport across the BBB. Using a monoclonal antibody targeting the tPa binding sites of LRP1 decreased tPa transport across an endothelial barrier. Our results indicate that limiting tPa transport from the vascular system into the brain by coapplication of a LRP1-blocking monoclonal antibody might be a novel approach to minimize tPa-related BBB damage during acute stroke therapy.

Application of Nanotechnology-Based Products in Stroke.

Stroke is a disease with high disability and high mortality in the world. Due to the existence of the blood-brain barrier (BBB), complex brain structure, and numerous neural signal pathways, the treatment methods are limited, so new drugs and new treatments need to be developed urgently. Thankfully, the advent of nanotechnology offered a new opportunity for biomedical development because of the unique properties of nanoparticles that give them the ability to traverse the BBB and accumulate in relevant regions of the brain. More importantly, nanoparticles could be modified on the surface to meet a variety of specific properties that people need. Some could be used for effective drug delivery, including tissue plasminogen activator (tPA), neuroprotective agents, genes, and cytokines; some nanoparticles were used as contrast agents and biosensors in medical imaging for further diagnosis of stroke; some were used to track target cells for prognosis of stroke; and some were used to detect pathological markers of stroke that appear at different stages. This Review looks at the application and research progress of nanoparticles in the diagnosis and treatment of stroke, hoping to bring some help to researchers.

A novel recombinant human microplasminogen induced complete posterior vitreous detachment without morphological change of retina in juvenile rabbits.

Vitreomacular traction syndrome results from persistent vitreoretinal adhesions in the setting of partial posterior vitreous detachment (PVD). Vitrectomy and reattachment of retina is an effective therapeutic approach. The adhesion between vitreous cortex and internal limiting membrane (ILM) of the retina is stronger in youth, which brings difficulties to induce PVD in vitrectomy. Several clinical investigations demonstrated that intravitreous injection of plasmin before vitrectomy could reduce the risk of detachment. In our study, a novel recombinant human microplasminogen (rhµPlg) was expressed by Pichia pastoris. Molecular docking showed that the binding of rhµPlg with tissue plasminogen activator (t-PA) was similar to plasminogen, suggesting rh µPlg could be activated by t-PA to generate microplasmin (µPlm). Moreover, rhµPlg had higher catalytic activity than plasminogen in amidolytic assays. Complete PVD was found at vitreous posterior pole of 125 µg rhµPlg-treated eyes without morphological change of retina in juvenile rabbits via intraocular injection. Our results demonstrate that rhµPlg has a potential value in the treatment of vitreoretinopathy.