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Purpose: Tissue ischemia triggers a bunch of cellular responses directed to restore blood flow and accelerate repair. Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, contribute extensively to tissue remodeling in a variety of normal and disease processes, including ischemic disorders associated to vascular dysfunction (e.g.: peripheral artery disease, PAD). High gelatinolytic activity is necessary for tissue remodeling in response to arterial occlusion in experimental ischemia, however, the contribution of other MMPs, such as; collagenases, or stromelysins, has not yet been investigated. MMP-10 or stromelysin 2 has been suggested to be involved in disorders associated to tissue remodeling and inflammation, although it remains unknown its involvement in ischemic diseases.Methods: we used a model of femoral artery occlusion in wild type (WT) and MMP-10 deficient (Mmp10-/-) mice to study the role of MMP-10 in skeletal muscle repair.Results: At the degenerative phase, MMP-10 deficiency reduced tissue perfusion and increased necrosis and inflammation. At the regenerative phase, MMP-10 deficiency resulted in reduced tissue regeneration. The injection of recombinant human MMP-10 (rhMMP10) in Mmp10-/- mice rescued the observed phenotype. Compared to WT, ischemic Mmp10-/- muscles had higher levels of Cxcl1 (Gro-alpha), suggesting the involvement of chemokine production in muscle regeneration by MMP-10.Conclusions: Our results demonstrate an important role of MMP-10 for proper muscle repair during degenerative and regenerative phases of limb ischemia, and open new pathways for specific manipulation of MMP-10 in ischemic conditions. [ABSTRACT FROM AUTHOR]

Purpose: Bleeding can be a complication of surgery (e.g. cardiac surgery) leading to blood transfusion requirements and substantial morbidity and mortality having a great health and econimical impact. Hyperfibrinolysis is an integral component of bleeding in this condition, therefore administration of antifibrinolytics like tranexamic acid (TXA), is the strategy of choice. However, side effects associated with venous or arterial thrombosis and seizures, limit its use.We hypothesized that inhibition of metalloproteinases (MMPs) may represent a new therapeutic approach to control bleeding, based on its role on hemostasis.Methods: Through the implemented drug discovery process, based on in-vitro MMPs activity biochemical test and a functional thromboelastometry assay, synthesized compounds from a novel chemical series were prioritized to be assayed in-vivo. In particular , the compounds that show a 50 % significant delay in blood lysis time were tested in a tail bleeding model in mouse, and after partial hepatectomy as compared with TXA. Moreover, pathological studies were performed with a dose 10 times higher than the effective in ensuring the safety of the compound and discard thrombotic events. Finally the pharmacokinetics and toxicity of selected candidate was further analyzed.Results: After some iterations a lead compound was achieved: CM-352 significantly reduced the bleeding time by 89% in the hyperfibrinolytic tail bleeding model at a dose of 10 ug/Kg - 30,000 times lower than TXA. Moreover, CM-352 was also effective at reducing blood loss after liver laceration, where TXA had no significant effect. CM-352 showed optimal ADME/Tox and pharmacokinetic profile as well as cardiovascular safety; in addition, anatomopathological analysis (at 10 mg/kg) showed no thrombus formation in lung, brain, kidney or liver.Conclusions: Our findings demonstrate that CM-352 inhibits fibrinolysis and reduces blood loss safely and efficiently through a new mechanism of action, which underscores its potential for patients suffering major bleeding due to various medical and surgical conditions. [ABSTRACT FROM PUBLISHER]

Purpose: Early reperfusion using tissue-type plasminogen activator (tPA) is the only therapeutic strategy to treat focal cerebral ischemia with proven efficacy in stroke patients. Moreover, efficacy of tPA is not sufficient since only a third of tPA-treated patients recover and are free from disability. tPA has also shown neurotoxicity in experimental models of cerebral ischemia. Therefore, a combination treatment that would reduce the deleterious effects of tPA, while maintaining or improving its efficacy, might extend its usability and efficacy.Aim: Considering the clinical need of new therapies for the ischemic brain treatment, and our previous results showing the profibrinolytic effect of MMP-10 in this context, we conducted further studies to assess the potential of MMP-10, not only as a clot-dissolving enzyme, but also as a cerebroprotective agent in combination with tPA. By using a transient stroke model we examined the therapeutic and protective effect of combining MMP-10 with tPA that might maintain the benefits of recanalization, but extending the efficacy of tPA with new vasculoprotection properties.Methods: The murine ischemic stroke model consisted in the local injection of thrombin into the middle cerebral artery and reperfusion with different tPA doses (1-10 mg/Kg) or tPA combined with recombinant MMP-10 (6.5 μg/Kg) to analyse infarct size, inflammation and neuronal degeneration. In vitro cultures were also performed to determine the effect of tPA/MMP-10 on endothelial vascular cells (permeability assay) and neurons (excitotoxicity assay).Results: We observed that 10 mg/Kg of tPA plus MMP-10 significantly reduced the infarct size in the thromboembolic ischemic stroke model compared with tPA alone. Moreover, a 10-fold reduction in tPA dose in the presence of MMP-10 produced the same effect on infarct size than full-dose of tPA+MMP-10 (p<0.05) without increasing haemorrhage. In vitro data showed that MMP-10 reduced the tPA endothelial permeability and preserved the expression of claudin-5. Moreover, combination of tPA/MMP10 improved neuronal excitotoxicity of tPA and this effect was reversed in the presence of a monoclonal antibody against MMP-10.Conclusions: These data suggest that the combination tPA/MMP-10 could be a new strategy for thrombolysis in ischemic stroke, allowing significant reduction of ischemic brain damage by decreasing inflammation, and protecting against cerebrovascular injury. [ABSTRACT FROM AUTHOR]

Introduction: Despite the key role of the endothelium in atherosclerosis, there are no direct techniques for its analysis. The study of extracellular vesicles of endothelial origin (EEVs), might lead to the identification of molecular signatures and early biomarkers of atherosclerosis. The aim of this work was to set up the methods for EEVs separation and transcriptomic analysis., Methods: We adapted an antibody-magnetic-bead based immunocapture protocol for plasma EEVs separation from control (G1), subclinical atherosclerosis (G2) and peripheral artery disease subjects (PAD) (G3), and modified an ultra-low input RNASeq method (n=5/group). By bioinformatics analysis we compared the transcriptome of plasma EEVs with that of human aortic endothelial cells (TeloHAECs), and then, searched for differentially expressed genes (DEG) among EEVs of G1, G2 and G3. From those DEG, UCP2 was selected for further validation in plasma EVs (qPCR), and in vitro, in stimulated TeloHAECs (IL-1β, TNFα, oxLDL and hypoxia)., Results: The RNASeq analysis of plasma EEVs rendered 1667 genes enriched in transcripts expressed by TeloHAECs (NES: 1.93, p adjust=1.4 e-73 ). One hundred seventy DEGs were identified between G2 vs G1, and 180 between G3 vs G1, of which 17 were similarly expressed in G2 and G3 vs control, including UCP2. IL-1β and TNFα (10ng/mL, p<0.05), hypoxia (1% O 2 , p=0.05) and oxLDL (100μg/mL, p=0.055) reduced UCP2 expression in TeloHAECs., Conclusions: We set up a protocol for EEVs separation and sequencing that might be useful for the identification of early markers of endothelial dysfunction in atherosclerosis., (Copyright © 2024 The Authors. Publicado por Elsevier España, S.L.U. All rights reserved.)

Background: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19, or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen, but only a few studies have compared some of these assays., Objectives: The International Society on Thrombosis and Haemostasis Scientific and Standardization Committee Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity, and reproducibility of these assays., Methods: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without lipopolysaccharide stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays., Results: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared with antigen assays. In addition, there was a large intra-assay and interassay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared with assays that isolated EVs by high-speed centrifugation., Conclusion: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody., Competing Interests: Declaration of competing interests F.D.-G. and R.L. filed a patent on microvesicle fibrinolytic activity licensed to Stago and obtained a common grant within the framework of the excellence program innovative tests to customize antiplatelet therapy in chronic kidney disease with acute coronary syndrome. The remaining authors declare no competing financial interests., (Copyright © 2024. Published by Elsevier Inc.)

Background and Aims: Peripheral arterial disease (PAD) is a leading cause of morbimortality worldwide. Lipocalin-2 (LCN2) has been associated with higher risk of amputation or mortality in PAD and might be involved in muscle regeneration. Our aim is to unravel the role of LCN2 in skeletal muscle repair and PAD., Methods and Results: WT and Lcn2 -/- mice underwent hindlimb ischemia. Blood and crural muscles were analyzed at the inflammatory and regenerative phases. At day 2, Lcn2 -/- male mice, but not females, showed increased blood and soleus muscle neutrophils, and elevated circulating pro-inflammatory monocytes (p < 0.05), while locally, total infiltrating macrophages were reduced (p < 0.05). Moreover, Lcn2 -/- soleus displayed an elevation of Cxcl1 (p < 0.001), and Cxcr2 (p < 0.01 in males), and a decrease in Ccl5 (p < 0.05). At day 15, Lcn2 deficiency delayed muscle recovery, with higher density of regenerating myocytes (p < 0.04) and arterioles (αSMA + , p < 0.025). Reverse target prediction analysis identified miR-138-5p as a potential regulator of LCN2, showing an inverse correlation with Lcn2 mRNA in skeletal muscles (rho = -0.58, p < 0.01). In vitro, miR-138-5p mimic reduced Lcn2 expression and luciferase activity in murine macrophages (p < 0.05). Finally, in human serum miR-138-5p was inversely correlated with LCN2 (p ≤ 0.001 adjusted, n = 318), and associated with PAD (Odds ratio 0.634, p = 0.02, adjusted, PAD n = 264, control n = 54)., Conclusions: This study suggests a possible dual role of LCN2 in acute and chronic conditions, with a probable role in restraining inflammation early after skeletal muscle ischemia, while being associated with vascular damage in PAD, and identifies miR-138-5p as one potential post-transcriptional regulator of LCN2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Predicting the progression of small aneurysms is a main challenge in abdominal aortic aneurysm (AAA) management. The combination of circulating biomarkers and image techniques might provide an alternative for risk stratification. We evaluated the association of plasma TAT complexes (TAT) and D-dimer with AAA severity in 3 groups of patients: group 1, without AAA (n = 52), group 2, AAA 40−50 mm (n = 51) and group 3, AAA > 50 mm (n = 50). TAT (p < 0.001) and D-dimer (p < 0.001) were increased in patients with AAA (groups 2 and 3) vs. group 1. To assess the association between baseline TAT and D-dimer concentrations, and AAA growth, aortic diameter and volume (volumetry) were measured by computed tomography angiography (CTA) in group 2 at recruitment (baseline) and 1-year after inclusion. Baseline D-dimer and TAT levels were associated with AAA diameter and volume variations at 1-year independently of confounding factors (p ≤ 0.044). Additionally, surgery incidence, recorded during a 4-year follow-up in group 2, was associated with larger aneurysms, assessed by aortic diameter and volumetry (p ≤ 0.036), and with elevated TAT levels (sub-hazard ratio 1.3, p ≤ 0.029), while no association was found for D-dimer. The combination of hemostatic parameters and image techniques might provide valuable tools to evaluate AAA growth and worse evolution.

Objective: Peripheral arterial disease (PAD) is the most prevalent cardiovascular (CV) condition globally. Despite the high CV risk of PAD patients, no reliable predictors of adverse clinical evolution are yet available. In this regard, previous transcriptomic analyses revealed increased expression of calprotectin (S100A8/A9) and lipocalin-2 (LCN2) in circulating extracellular vesicles (EVs) of patients with PAD. The aim of this study was to determine the prognostic value of LCN2 and calprotectin for CV risk assessment in PAD., Methods: LCN2 and the S100A9 subunit of calprotectin were examined in human femoral plaques by immunohistochemistry and qPCR. LCN2 and calprotectin were determined by ELISA in PAD (CHN cohort, n = 331, Fontaine II-IV, serum), and PAD diagnosed by population based screening (VIVA trial, n = 413, the majority Fontaine 0-I, plasma). Patients were followed up for a mean of four years, recording the primary outcomes; CV death or amputation in the CHN cohort and CV death or major lower limb events (MALE) in the VIVA population. Secondary outcomes were all cause death or amputation, and all cause death or MALE, respectively., Results: LCN2 and S100A9 were detected in human plaques in regions rich in inflammatory cells. LCN2 and calprotectin levels were 70% and 64% lower in plasma than in serum. In the CHN cohort, high serum levels of LCN2 and calprotectin increased the risk of primary and secondary outcomes 5.6 fold (p < .001) and 1.8 fold (p = .034), respectively, after covariable adjustment. Similarly, elevated plasma levels of LCN2 and calprotectin increased by three fold the risk of primary and secondary outcomes (p < .001) in the VIVA cohort. Moreover, addition of the combined variable to basal models, considering clinically relevant risk factors, improved reclassification for the primary outcome in both cohorts (p ≤ .024)., Conclusion: Combined assessment of the inflammatory biomarkers LCN2 and calprotectin might be useful for risk stratification in advanced and early PAD., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Peripheral arterial disease (PAD) of the lower extremities is a chronic illness predominantly of atherosclerotic aetiology, associated to traditional cardiovascular (CV) risk factors. It is one of the most prevalent CV conditions worldwide in subjects >65 years, estimated to increase greatly with the aging of the population, becoming a severe socioeconomic problem in the future. The narrowing and thrombotic occlusion of the lower limb arteries impairs the walking function as the disease progresses, increasing the risk of CV events (myocardial infarction and stroke), amputation and death. Despite its poor prognosis, PAD patients are scarcely identified until the disease is advanced, highlighting the need for reliable biomarkers for PAD patient stratification, that might also contribute to define more personalized medical treatments. In this review, we will discuss the usefulness of inflammatory molecules, matrix metalloproteinases (MMPs), and cardiac damage markers, as well as novel components of the liquid biopsy, extracellular vesicles (EVs), and non-coding RNAs for lower limb PAD identification, stratification, and outcome assessment. We will also explore the potential of machine learning methods to build prediction models to refine PAD assessment. In this line, the usefulness of multimarker approaches to evaluate this complex multifactorial disease will be also discussed.

Background: Actual clinical management of ischemic stroke (IS) is based on restoring cerebral blood flow using tissue plasminogen activator (tPA) and/or endovascular treatment (EVT). Mechanical thrombectomy has permitted the analysis of thrombus structural and cellular classic components. Nevertheless, histological assessment of hemostatic parameters such as thrombin-activatable fibrinolysis inhibitor (TAFI) and matrix metalloproteinase 10 (MMP-10) remains unknown, although their presence could determine thrombus stability and its response to thrombolytic treatment, improving patient's outcome. Methods: We collected thrombi ( n = 45) from large vessel occlusion (LVO) stroke patients ( n = 53) and performed a histological analysis of different hemostatic parameters [TAFI, MMP-10, von Willebrand factor (VWF), and fibrin] and cellular components (erythrocytes, leukocytes, macrophages, lymphocytes, and platelets). Additionally, we evaluated the association of these parameters with plasma levels of MMP-10, TAFI and VWF activity and recorded clinical variables. Results: In this study, we report for the first time the presence of MMP-10 and TAFI in all thrombi collected from LVO patients. Both proteins were localized in regions of inflammatory cells, surrounded by erythrocyte and platelet-rich areas, and their content was significantly associated ( r = 0.41, p < 0.01). Thrombus TAFI was lower in patients who died during the first 3 months after stroke onset [odds ratio (OR) (95%CI); 0.59 (0.36-0.98), p = 0.043]. Likewise, we observed that thrombus MMP-10 was inversely correlated with the amount of VWF ( r = -0.30, p < 0.05). Besides, VWF was associated with the presence of leukocytes ( r = 0.37, p < 0.05), platelets ( r = 0.32, p < 0.05), and 3 months mortality [OR (95%CI); 4.5 (1.2-17.1), p = 0.029]. Finally, plasma levels of TAFI correlated with circulating and thrombus platelets, while plasma MMP-10 was associated with cardiovascular risk factors and functional dependence at 3 months. Conclusions: The present study suggests that the composition and distribution of thrombus hemostatic components might have clinical impact by influencing the response to pharmacological and mechanical therapies as well as guiding the development of new therapeutic strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Marta-Enguita, Navarro-Oviedo, Muñoz, Olier-Arenas, Zalba, Lecumberri, Mendioroz, Paramo, Roncal and Orbe.)

Peripheral arterial disease (PAD) is associated with a high risk of cardiovascular events and death and is postulated to be a critical socioeconomic cost in the future. Extracellular vesicles (EVs) have emerged as potential candidates for new biomarker discovery related to their protein and nucleic acid cargo. In search of new prognostic and therapeutic targets in PAD, we determined the prothrombotic activity, the cellular origin and the transcriptomic profile of circulating EVs. This prospective study included control and PAD patients. Coagulation time (Procoag-PPL kit), EVs cellular origin and phosphatidylserine exposure were determined by flow cytometry in platelet-free plasma (n = 45 PAD). Transcriptomic profiles of medium/large EVs were generated using the MARS-Seq RNA-Seq protocol (n = 12/group). The serum concentration of the differentially expressed gene S100A9, in serum calprotectin (S100A8/A9), was validated by ELISA in control (n = 100) and PAD patients (n = 317). S100A9 was also determined in EVs and tissues of human atherosclerotic plaques (n = 3). Circulating EVs of PAD patients were mainly of platelet origin, predominantly Annexin V positive and were associated with the procoagulant activity of platelet-free plasma. Transcriptomic analysis of EVs identified 15 differentially expressed genes. Among them, serum calprotectin was elevated in PAD patients ( p  < 0.05) and associated with increased amputation risk before and after covariate adjustment (mean follow-up 3.6 years, p  < 0.01). The combination of calprotectin with hs-CRP in the multivariate analysis further improved risk stratification ( p  < 0.01). Furthermore, S100A9 was also expressed in femoral plaque derived EVs and tissues. In summary, we found that PAD patients release EVs, mainly of platelet origin, highly positive for AnnexinV and rich in transcripts related to platelet biology and immune responses. Amputation risk prediction improved with calprotectin and was significantly higher when combined with hs-CRP. Our results suggest that EVs can be a promising component of liquid biopsy to identify the molecular signature of PAD patients., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.)

Peripheral artery disease (PAD) is a major cause of acute and chronic illness, with extremely poor prognosis that remains underdiagnosed and undertreated. Trimethylamine-N-Oxide (TMAO), a gut derived metabolite, has been associated with atherosclerotic burden. We determined plasma levels of TMAO by mass spectrometry and evaluated their association with PAD severity and prognosis. 262 symptomatic PAD patients (mean age 70 years, 87% men) categorized in intermittent claudication (IC, n = 147) and critical limb ischemia (CLI, n = 115) were followed-up for a mean average of 4 years (min 1-max 102 months). TMAO levels were increased in CLI compared to IC (P < 0.001). Receiver operating characteristic (ROC) curves for severity (CLI) rendered a cutoff of 2.26 µmol/L for TMAO (62% sensitivity, 76% specificity). Patients with TMAO > 2.26 µmol/L exhibited higher risk of cardiovascular death (sub-hazard ratios ≥2, P < 0.05) that remained significant after adjustment for confounding factors. TMAO levels were associated to disease severity and CV-mortality in our cohort, suggesting an improvement of PAD prognosis with the measurement of TMAO. Overall, our results indicate that the intestinal bacterial function, together with the activity of key hepatic enzymes for TMA oxidation (FMO3) and renal function, should be considered when designing therapeutic strategies to control gut-derived metabolites in vascular patients.

Introduction: Wound healing after myocardial infarction (MI) is mediated by different cell types, secreted proteins, components of the extracellular matrix (ECM) and, as increasing evidences suggest, extracellular vesicles (EVs). We aim to determine the dynamics of release and origin of EVs after MI, as well as their biological activity on endothelial cells (ECs)., Methods: MI was induced in WT mice and blood and tissues collected at baseline, 3, 15 and 30 days post-ligation for cardiac function (echocardiography) and histological evaluation. Circulating EVs subpopulations were measured by flow cytometry in mouse, and in a small cohort of patients with ST-segment elevation MI (STEMI, n = 6). In vitro, EVs were isolated from a cardiomyocyte cell line (HL1) and their function assayed on ECs., Results: Leukocyte and endothelial EVs increased concomitant to inflammatory and angiogenic processes triggered by ischemia. More strikingly, cardiomyocyte EVs (connexin43 + ) were detected in STEMI patients and in murine MI, where a significant increase in their levels was reported at day 15 post-ischemia (p < 0.05 vs baseline). In vitro, HL1EVs induced ECs migration (p = 0.05) and proliferation (p < 0.05), but impaired tube formation. These apparent contradictory results could be partially explained by the upregulation of MMP3, and the apoptosis and senescence genes, p53 and p16, induced by HL1EVs on ECs (p < 0.05)., Conclusions: MI induces the release of different EVs subpopulations, including those of cardiac origin, in a preclinical model of MI and STEMI patients. In vitro, cardiomyocyte derived EVs are able to modulate endothelial function, suggesting their active role in heart repair after ischemia., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Chemotherapy is considered the most appropriate treatment for metastatic uterine sarcoma, despite its limited efficacy. No other treatment has been conclusively proved to be a real alternative, but some reports suggest that anti-hormonal therapy could be active in a small subset of patients. We report the case of a patient with metastatic uterine carcinosarcoma with positive hormonal receptors and a complete pathological response., Case Presentation: A 54-year-old white woman presented to our emergency room with hypovolemic shock and serious vaginal bleeding. After stabilization, she was diagnosed as having a locally advanced uterine carcinosarcoma with lymph nodes and bone metastatic disease. In order to control the bleeding, palliative radiotherapy was administered. Based on the fact that positive hormone receptors were found in the biopsy, non-steroidal aromatase inhibitor therapy with letrozole was started. In the following weeks, her general status improved and restaging imaging tests demonstrated a partial response of the primary tumor. Ten months after initiating aromatase inhibitor therapy, she underwent a radical hysterectomy and the pathological report showed a complete response. After completing 5 years of treatment, aromatase inhibitor therapy was stopped. She currently continues free of disease, without further therapy, and maintains a normal and active life., Conclusions: This case shows that patients with uterine carcinosarcoma and positive hormone receptors may benefit from aromatase inhibitor therapy. A multidisciplinary strategy that includes local therapies such as radiation and/or surgery should be considered the mainstay of treatment. Systemic therapies such as hormone inhibitors should be taken into consideration and deserve further clinical research in the era of precision medicine.

Background and Purpose: The debate over the fact that experimental drugs proposed for the treatment of stroke fail in the translation to the clinical situation has attracted considerable attention in the literature. In this context, we present a retrospective pooled analysis of a large data set from preclinical studies, to examine the effects of early versus late administration of intravenous recombinant tissue-type plasminogen activator., Methods: We collected data from 26 individual studies from 9 international centers (13 researchers; 716 animals) that compared recombinant tissue-type plasminogen activator with controls, in a unique mouse model of thromboembolic stroke induced by an in situ injection of thrombin into the middle cerebral artery. Studies were classified into early (<3 hours) versus late (≥3 hours) drug administration. Final infarct volumes, assessed by histology or magnetic resonance imaging, were compared in each study, and the absolute differences were pooled in a random-effect meta-analysis. The influence of time of administration was tested., Results: When compared with saline controls, early recombinant tissue-type plasminogen activator administration was associated with a significant benefit (absolute difference, -6.63 mm(3); 95% confidence interval, -9.08 to -4.17; I(2)=76%), whereas late recombinant tissue-type plasminogen activator treatment showed a deleterious effect (+5.06 mm(3); 95% confidence interval, +2.78 to +7.34; I(2)=42%; Pint<0.00001). Results remained unchanged after subgroup analyses., Conclusions: Our results provide the basis needed for the design of future preclinical studies on recanalization therapies using this model of thromboembolic stroke in mice. The power analysis reveals that a multicenter trial would require 123 animals per group instead of 40 for a single-center trial., (© 2016 American Heart Association, Inc.)

Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) plays an important role in coagulation and fibrinolysis. Whereas TAFI deficiency may lead to a haemorrhagic tendency, data from TAFI knockout mice (TAFI-/-) are controversial and no differences have been reported in these animals after ischemic stroke. There are also no data regarding the role of circulating microparticles (MPs) in TAFI-/-., Objectives: to examine the effect of tPA on the rate of intracranial haemorrhage (ICH) and on MPs generated in a model of ischemic stroke in TAFI-/- mice., Methods: Thrombin was injected into the middle cerebral artery (MCA) to analyse the effect of tPA (10mg/Kg) on the infarct size and haemorrhage in the absence of TAFI. Immunofluorescence for Fluoro-Jade C was performed on frozen brain slides to analyse neuronal degeneration after ischemia. MPs were isolated from mouse blood and their concentrations calculated by flow cytometry., Results: Compared with saline, tPA significantly increased the infarct size in TAFI-/- mice (p<0.05). Although plasma fibrinolytic activity (fibrin plate assay) was higher in these animals, no macroscopic or microscopic ICH was detected. A positive signal for apoptosis and degenerating neurons was observed in the infarct area, being significantly higher in tPA treated TAFI-/- mice (p<0.05). Interestingly, higher numbers of MPs were found in TAFI-/- plasma as compared to wild type, after stroke (p<0.05)., Conclusions: TAFI deficiency results in increased brain damage in a model of thrombolysis after ischemic stroke, which was not associated with bleeding but with neuronal degeneration and MP production., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background & Aims: Radioembolization may rarely induce liver disease resulting in a syndrome that is similar to veno-occlusive disease complicating bone marrow transplantation where inflammation, endothelial cell activation and thrombosis are likely involved. We hypothesized that similar mechanisms could be implicated in radioembolization-induced liver disease (REILD). Moreover, lobar radioembolization may induce hypertrophy of the non-treated hemiliver most probably by inducing liver regeneration., Methods: In patients with hepatocellular carcinoma, we prospectively studied serum levels of markers of liver regeneration, oxidative stress, pro-inflammatory pathways, endothelial activation and coagulation parameters over 2 months after radioembolization., Results: Although REILD did not occur among 14 treated patients, a decrease in effective liver blood flow was observed. Radioembolization was followed by a persistent increase in pro-inflammatory (interleukin 6 and 8) and oxidative stress (malondyaldehide) markers, an induction of endothelial injury markers (vW factor and PAI-1) and an activation of the coagulation cascade (factor VIII, PAI-1, D-Dimer) as well as a significant increase in factors related to liver regeneration (FGF-19 and HGF)., Conclusion: Radioembolization activates liver regeneration, produces oxidative stress, activates inflammatory cytokines and induces endothelial injury with partial activation of the coagulation cascade. These findings may have implications in the pathogenesis, prevention and therapy of REILD and in the development of new therapies to enhance hypertrophy with a surgical perspective., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We studied the role of matrix metalloproteinase-10 (MMP-10) during skeletal muscle repair after ischemia using a model of femoral artery excision in wild-type (WT) and MMP-10 deficient (Mmp10(-/-)) mice. Functional changes were analyzed by small animal positron emission tomography and tissue morphology by immunohistochemistry. Gene expression and protein analysis were used to study the molecular mechanisms governed by MMP-10 in hypoxia. Early after ischemia, MMP-10 deficiency resulted in delayed tissue reperfusion (10%, P < 0.01) and in increased necrosis (2-fold, P < 0.01), neutrophil (4-fold, P < 0.01), and macrophage (1.5-fold, P < 0.01) infiltration. These differences at early time points resulted in delayed myotube regeneration in Mmp10(-/-) soleus at later stages (regenerating myofibers: 30 ± 9% WT vs. 68 ± 10% Mmp10(-/-), P < 0.01). The injection of MMP-10 into Mmp10(-/-) mice rescued the observed phenotype. A molecular analysis revealed higher levels of Cxcl1 mRNA (10-fold, P < 0.05) and protein (30%) in the ischemic Mmp10(-/-) muscle resulting from a lack of transcriptional inhibition by MMP-10. This was further confirmed using siRNA against MMP-10 in vivo. Our results demonstrate an important role of MMP-10 for proper muscle repair after ischemia, and suggest that chemokine regulation such as Cxcl1 by MMP-10 is involved in muscle regeneration., (© FASEB.)

Amorphous carbon (a-C) nanoclusters were synthesized by the implantation of carbon ions (C-) into thermally grown silicon dioxide film (-500 nm thick) on a Si (100) wafer and processed by high temperature thermal annealing. The carbon ions were implanted with an energy of 70 keV at a fluence of 5 x 10(17) atoms/cm2. The implanted samples were annealed at 1100 degrees C for different time periods in a gas mixture of 96% Ar+4% H2. Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and High Resolution Transmission Electron Microscopy (HRTEM) were used to study the structural properties of both the as-implanted and annealed samples. HRTEM reveals the formation of nanostructures in the annealed samples. The Raman spectroscopy also confirms the formation of carbon nano-clusters in the samples annealed for 10 min, 30 min, 60 min and 90 min. No Raman features originating from the carbon-clusters are observed for the sample annealed further to 120 min, indicating a complete loss of implanted carbon from the SiO2 layer. The loss of the implanted carbon in the 120 min annealed sample from the SiO2 layer was also observed in the XPS depth profile measurements. Room temperature photoluminescence (PL) spectroscopy revealed visible emissions from the samples pointing to carbon ion induced defects as the origin of a broad 2.0-2.4 eV band, and the intrinsic defects in SiO2 as the possible origin of the -2.9 eV bands. In low temperature photoluminescence spectra, two sharp and intense photoluminescence lines at -3.31 eV and -3.34 eV appear for the samples annealed for 90 min and 120 min, whereas no such bands are observed in the samples annealed for 10 min, 30 min, and 60 min. The Si nano-clusters forming at the Si-SiO2 interface could be the origin of these intense peaks.

Objective: Our aim was to analyze the regulation of CC Chemokine ligand 20 (CCL20) by LDL in human vascular smooth muscle cells (VSMC)., Methods and Results: In asymptomatic subjects, circulating CCL20 levels were higher in patients with hypercholesterolemia (18.5±3.2 versus 9.1±1.3 pg/mL; P<0.01). LDL induced the expression of CCL20 in VSMC in a dose- and time-dependent manner. Increased levels of CCL20 secreted by LDL-treated VSMC significantly induced human lymphocyte migration, an effect reduced by CCL20 silencing. The upregulation of CCL20 by LDL was dependent on the activation of kinase signaling pathways and NF-κB. By site-directed mutagenesis, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we identified a NF-κB site (-80/-71) in CCL20 promoter critical for LDL responsiveness. Lysophosphatidic acid mimicked the upregulation of CCL20 induced by LDL, and minimal oxidation of LDL increased the ability of LDL to induce CCL20 through a mechanism that involves lysophosphatidic acid receptors. CCL20 was overexpressed in atherosclerotic lesions from coronary artery patients, colocalizing with VSMC. CCL20 was detected in conditioned media from healthy human aorta and its levels were significantly higher in secretomes from carotid endarterectomy specimens., Conclusion: This study identifies CCL20 in atherosclerotic lesions and recognizes this chemokine as a mediator highly sensitive to the inflammatory response elicited by LDL.

Genetic predisposition to hereditary hemochromatosis (HH) is associated with primary hypertriglyceridemia (HTG). If iron overload influences the development of HTG, the management of these patients could be different. However, the metabolic syndrome in primary HTG is frequent; and it could partially confuse the association. The objective was to determine whether periodic bloodletting could decrease triglyceride concentrations in subjects with HH and iron overload. We retrospectively studied 155 genetically defined HH patients (C282Y homozygotes and compound heterozygotes C282Y/H63D) with iron overload and under periodic therapeutic phlebotomy. Hypertriglyceridemia (triglycerides ≥150 mg/dL) was present in 49 subjects at baseline (31.6%). Phlebotomies significantly decreased triglycerides, especially in subjects with basal HTG (from 287 mg/dL at baseline to 133 mg/dL after phlebotomies, P < .001). Blood glucose and total cholesterol did not change with phlebotomies. The triglyceride-lowering effect was obtained until ferritin concentration decreased to less than 200 μg/L and transferrin saturation to less than 40%. The triglyceride-lowering effect was obtained for glucose levels both less than and greater than 100 mg/dL. In summary, HH subjects frequently have HTG that improves after therapeutic phlebotomy, independently of basal blood glucose. Our results suggest that therapeutic phlebotomy could be a useful therapeutic approach in patients with HTG and iron overload., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Venous and combined malformations are slow-flow haemodynamically inactive lesions that are present at birth and worsen slowly with advancing age, showing no tendency towards involution. The pathogenesis of vascular anomalies has not been fully elucidated, but their formation and progression are closely related to angiogenesis. Localized intravascular coagulation associated with venous or combined malformations is characterized by low fibrinogen, high D-dimers, and normal platelet count., Objectives: To assess the relationship of angiogenic factors with prothrombotic and endothelial damage/dysfunction markers in patients with extensive slow-flow vascular malformations., Methods: A 2-year study (2005-2007) included 31 consecutive patients with extensive slow-flow vascular malformations from one centre., Results: Serum levels of the endothelial receptor tyrosine kinase TIE-2, matrix metalloproteinase (MMP)-9 and angiopoietin (Ang)-2 and plasma levels of D-dimer, plasminogen activator inhibitor type 1 (PAI-1), tissue-type plasminogen activator and von Willebrand factor (vWf) were significantly increased in patients compared with healthy controls, whereas serum levels of vascular endothelial growth factor (VEGF)-C, VEGF-D, MMP-2, Ang-1, platelet-derived growth factor (PDGF)-AB and PDGF-BB were significantly decreased in patients compared with controls. A strong positive correlation was present between Ang-1 and PDGF-AB levels (r = 0.63, P < 0.001), between PDGF-AB and PDGF-BB levels (r = 0.67, P < 0.001), and between fibrinogen and PAI-1 levels (r = 0.41, P = 0.031). A strong negative correlation was present between Ang-1 and vWf levels (r = -0.48, P = 0.006), between D-dimer and fibrinogen levels (r = -0.71, P < 0.001), and between PDGF-AB and vWf levels (r = -0.42, P = 0.017)., Conclusions: These findings suggest that angiogenic, coagulation and endothelial damage/dysfunction markers are possibly linked in pathogenesis of extensive slow-flow vascular malformations, and might have therapeutic implications.

Adipose tissue development is associated with angiogenesis, adipogenesis and extracellular matrix degradation. The class of matrix metalloproteinases contributes to these processes, but little information is available on the role of individual proteinases. We report that stromelysin-2 (MMP-10) deficiency has no significant effect on total body weight or on subcutaneous (SC) or gonadal (GON) adipose tissue mass of mice kept on a high fat diet for 15 weeks. The adipocyte size and density in SC and GON adipose tissues were also comparable in MMP-10 deficient and wild-type control mice. Similarly, blood vessel size and density in obese SC and GON adipose tissues was not affected by MMP-10 deficiency. Metabolic parameters and blood cell composition were similar for both genotypes. Stromelysin-1 (MMP-3) expression was significantly reduced in adipose tissues of the deficient mice as compared to the wild-type controls. These data indicate that MMP-10 does not significantly contribute to adipose tissue development and associated angiogenesis in a mouse model of nutritionally induced obesity.

Atherosclerosis is the common pathophysiological substrate of ischemic vascular diseases and their thrombotic complications. The unbalance between matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) has been hypothesized to be involved in the growth, destabilization, and eventual rupture of atherosclerotic lesions. Different MMPs have been assigned relevant roles in the pathology of vascular diseases and MMP-10 (stromelysin-2) has been involved in vascular development and atherogenesis. This article examines the pathophysiological role of MMPs, particularly MMP-10, in the onset and progression of vascular diseases and their regulation by pro-inflammatory stimuli. MMP-10 over-expression has been shown to compromise vascular integrity and it has been associated with aortic aneurysms. MMP-10 is induced by C-reactive protein in endothelial cells, and it is over-expressed in atherosclerotic lesions. Additionally, higher MMP-10 serum levels are associated with inflammatory markers, increased carotid intima-media thickness and the presence of atherosclerotic plaques. We have cloned the promoter region of the MMP-10 gene and studied the effect of inflammatory stimuli on MMP-10 transcriptional regulation, providing evidences further supporting the involvement of MMP-10 in the pathophysiology of atherothrombosis.

Postoperative changes related to coagulation and fibrinolysis and their correlation with the incidence of deep venous thrombosis (DVT) were studied in 30 patients undergoing total hip replacement. Pre- and postoperative measurements of fibrinogen, factor Xa, VIII:C, VIIIR:Ag and its electrophoretic mobility, antifactor Xa activity, antithrombin III (AT III) and its electrophoretic mobility in plasma and serum, fibrin monomers, euglobulin lysis time, fibrinogen degradation products (FDP), alpha 2-antiplasmin and plasmin-antiplasmin complexes were determined. DVT was detected by 125I-fibrinogen leg scanning in 11 patients. There was a significant and progressive increase in fibrinogen, VIII:C, VIIIR:Ag, fibrin monomers, FDP and alpha 2-anti-plasmin levels after operation and likewise a prolongation of euglobulin lysis time. There were changes in electrophoretic mobility of AT III in plasma and serum in 12 patients. The presence of plasmin-antiplasmin complexes was demonstrated in 9 patients. No correlation between the changes in coagulation and fibrinolysis and the incidence of postoperative DVT was found. We conclude that important changes occur in several parameters of coagulation and fibrinolysis after total hip replacement. Such changes are not related to the development of postoperative DVT.

A family with a new congenital dysprothrombinemia is presented. The propositus is a 21-yr-old man who presented simultaneously with hemartrosis of the left knee and an extensive hematoma following a minor trauma. Prothrombin time and activated partial thromboplastin time were prolonged. Prothrombin activity was very low when measured by biological assay using physiological activators (7% by one-stage method and 20% by two-stage method) or a Russel's viper venom-cephalin mixture (23%), Notechis scutatus scutatus venom (15%) and Echis carinatus venom (17%); in contrast, the level was found to be borderline to normal using Taipan viper venom (64%) and normal by both staphylocoagulase and immunologic methods. Family studies revealed consanguinity between the propositus' mother and father and both presented a 50% reduced prothrombin level when physiological activators or Echis carinatus viper venom were used. A line of identity between normal and abnormal prothrombin was observed on immunodiffusion. The migration of the abnormal prothrombin was less anodic and was not changed by the addition of calcium. The patient's serum showed 3 bands in the bidimensional immunoelectrophoresis system, whereas normal serum showed only 2 bands. The term prothrombin Segovia is proposed to define this new prothrombin abnormality.

The influence of endotoxin-induced elevated plasma levels of the fast-acting inhibitor of plasminogen activator (PA-inhibitor) on thrombolysis was investigated in rabbits with a jugular vein thrombus. Infusion of human tissue-type plasminogen activator (t-PA) produced similar degrees of thrombolysis in control and endotoxin-treated rabbits, although no free t-PA could be demonstrated in plasma of endotoxin-treated animals. Infusion of t-PA in an extracorporeal arteriovenous shunt resulted in loss of thrombolytic activity in endotoxin-treated animals but not in control animals. Blood clots superfused in vitro with mixtures of t-PA and normal plasma lysed in contrast to clots superfused with t-PA and PA-inhibitor-rich plasma. However, addition of rabbit lung slices to the plasma surrounding the blood clot, reversed the inhibition of thrombolysis by PA-inhibitor-rich plasma. This indicates that tissue-derived factor(s) are involved in the regulation of in vivo thrombolysis. These hypothetical factor(s) are, however, very unstable in plasma, which has thus far precluded their further characterization.

Postoperative changes related to platelets and their correlation with the incidence of deep venous thrombosis (DVT) were studied in 30 patients undergoing total hip replacement. Levels of platelet count, platelet-crit, mean platelet volume, spontaneous platelet aggregation, platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) were measured before operation and on the 1st, 3rd and 7th postoperative days. DVT was detected by 125I-fibrinogen leg scanning in 11 of the patients. After the operation there was a significant and progressive increase (p less than 0.01) in PF4 and beta-TG, and the presence of circulating platelet aggregates was demonstrated. Platelet count levels and platelet-crit were decreased on the 1st and 3rd postoperative days followed by recovery on the 7th day. The changes observed following total hip replacement were not related to the development of postoperative DVT.

The inhibition of one-chain and two-chain molecular forms of human tissue-type plasminogen activator (t-PA) by the fast-acting inhibitor of plasminogen activator (PA-inhibitor) present in plasma was studied in vitro and in vivo in rabbits. In vitro, both one-chain and two-chain forms of t-PA were neutralized very rapidly in rabbit plasma with high levels of PA-inhibitor. The rate constant of the interaction between two-chain t-PA and PA-inhibitor was estimated to be 3.10(7) L/mol/sec. The presence of CNBr-digested fibrinogen, which mimics the effect of fibrin on the activation of plasminogen by t-PA, did not influence the rate constant. Moreover, PA-inhibitor-rich plasma inhibited in a very similar way in vitro thrombolysis by one-chain or two-chain t-PA incorporated into the clot. Injection of one-chain or two-chain t-PA into rabbits with increased levels of PA-inhibitor, induced by endotoxin, resulted in very rapid inhibition of t-PA activity. Within 30 seconds after injection, no residual free t-PA could be demonstrated. Gel filtration analysis showed that the disappearance of t-PA activity was associated with the generation of t-PA-PA-inhibitor complex with an apparent Mr of 100,000. This enzyme-inhibitor complex, like free t-PA, was cleared from the circulation with a half-life of approximately 2 minutes, mainly via the liver. It is concluded that PA-inhibitor neutralizes one-chain and two-chain molecular forms of t-PA in plasma at very similar rates, both in vitro and in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Endotoxin producing bacteria cause disseminated intravascular coagulation (DIC); however, the mechanism of endotoxin action in man is still unclear. Impairment of the fibrinolytic system has been suggested as a contributing mechanism. A single injection of Escherichia coli lipopolysaccharide in rabbits resulted in a marked and prolonged increase of the levels of a fast-acting inhibitor of plasminogen activator (PA-inhibitor) in plasma (from 3.9 +/- 0.7 to 41 +/- 13.2 U/ml after 3 h). Gel filtration studies indicated that inhibition of human tissue-type plasminogen activator (t-PA) by rabbit plasma is accompanied by a change in the elution profile of the activator compatible with the formation of an enzyme-inhibitor complex with an apparent molecular weight of 100,000. Injection of human t-PA (1,500 IU/kg body wt) in endotoxin treated animals resulted in very fast inhibition of t-PA and formation of a similar complex. The half-life of circulating PA-inhibitor activity in rabbits was about 7 min as estimated by donor receiver plasma transfusion experiments. Stimulation of cultured human endothelial cells with endotoxin resulted in enhanced rate of accumulation of PA-inhibitor activity in the culture medium (two- to sevenfold increase). In five patients with septicemia, markedly increased levels of PA-inhibitor (14.3 +/- 15.5 U/ml) as compared with control subjects (1.3 +/- 0.7 U/ml) were observed in plasma. A very strong correlation (r = 0.98) was found between inhibition of t-PA and of urokinase in all conditions, suggesting that this fast-acting inhibitor reacts with both plasminogen activators. These data suggest that the appearance of this fast-acting PA-inhibitor is very sensitive to endotoxin stimulation. The marked increase in the level of PA-inhibitor in blood may contribute to the pathogenesis of DIC in septicemia.