Transcriptional signatures in human macrophage-like cells infected by Leishmania infantum, Leishmania major and Leishmania tropica.

BACKGROUND: In the Mediterranean basin, three Leishmania species have been identified: L. infantum, L. major and L. tropica, causing zoonotic visceral leishmaniasis (VL), zoonotic cutaneous leishmaniasis (CL) and anthroponotic CL, respectively. Despite animal models and genomic/transcriptomic studies provided important insights, the pathogenic determinants modulating the development of VL and CL are still poorly understood. This work aimed to identify host transcriptional signatures shared by cells infected with L. infantum, L. major, and L. tropica, as well as specific transcriptional signatures elicited by parasites causing VL (i.e., L. infantum) and parasites involved in CL (i.e., L. major, L. tropica). METHODOLOGY/PRINCIPAL FINDINGS: U937 cells differentiated into macrophage-like cells were infected with L. infantum, L. major and L. tropica for 24h and 48h, and total RNA was extracted. RNA sequencing, performed on an Illumina NovaSeq 6000 platform, was used to evaluate the transcriptional signatures of infected cells with respect to non-infected cells at both time points. The EdgeR package was used to identify differentially expressed genes (fold change > 2 and FDR-adjusted p-values < 0.05). Then, functional enrichment analysis was employed to identify the enriched ontology terms in which these genes are involved. At 24h post-infection, a common signature of 463 dysregulated genes shared among all infection conditions was recognized, while at 48h post-infection the common signature was reduced to 120 genes. Aside from a common transcriptional response, we evidenced different upregulated functional pathways characterizing L. infantum-infected cells, such as VEGFA-VEGFR2 and NFE2L2-related pathways, indicating vascular remodeling and reduction of oxidative stress as potentially important factors for visceralization. CONCLUSIONS: The identification of pathways elicited by parasites causing VL or CL could lead to new therapeutic strategies for leishmaniasis, combining the canonical anti-leishmania compounds with host-directed therapy.

Platelet distribution width (PDW) as a significant correlate of COVID-19 infection severity and mortality.

SARS-CoV-2 infection may cause a wide spectrum of symptoms, from asymptomatic, to mild respiratory symptoms and life-threatening sepsis. Among the clinical laboratory biomarkers analyzed during COVID-19 pandemic, platelet indices have raised great interest, due to the critical involvement of platelets in COVID-19-related thromboinflammation. Through an electronic literature search on MEDLINE, CINAHL, PubMed, EMBASE, Web of Science, and preprint servers we performed and updated a systematic review aimed at providing a detailed analysis of studies addressing the potential clinical utility of platelet distribution width, platelet distribution width (PDW), in laboratory medicine, exploring the possible association between increased PDW levels, disease severity, and mortality in COVID-19. Our systematic review revealed a wide heterogeneity of COVID-19 cohorts examined and a lack of homogenous expression of platelet indices. We found that 75 % of studies reported significantly elevated PDW values in COVID-19 infected cohorts compared to healthy/non-COVID-19 controls, and 40 % of studies reported that patients with severe COVID-19 showed increased PDW values than those with less-than-severe illness. Interestingly, 71.4 % of studies demonstrated significant increased PDW values in non survivors vs. survivors. Overall, these results suggest that platelets are critically involved as major players in the process of immunothrombosis in COVID-19, and platelet reactivity and morphofunctional alterations are mirrored by PDW, as indicator of platelet heterogeneity. Our results confirm that the use of PDW as prognostic biomarkers of COVID-19 sepsis still remains debated due to the limited number of studies to draw a conclusion, but new opportunities to investigate the crucial role of platelets in thrombo-inflammation are warranted.

The influence of redox modulation on hypoxic endothelial cell metabolic and proteomic profiles through a small thiol-based compound tuning glutathione and thioredoxin systems.

Reduction in oxygen levels is a key feature in the physiology of the bone marrow (BM) niche where hematopoiesis occurs. The BM niche is a highly vascularized tissue and endothelial cells (ECs) support and regulate blood cell formation from hematopoietic stem cells (HSCs). While in vivo studies are limited, ECs when cultured in vitro at low O2 (<5%), fail to support functional HSC maintenance due to oxidative environment. Therefore, changes in EC redox status induced by antioxidant molecules may lead to alterations in the cellular response to hypoxia likely favoring HSC self-renewal. To evaluate the impact of redox regulation, HUVEC, exposed for 1, 6, and 24 h to 3% O2 were treated with N-(N-acetyl-l-cysteinyl)-S-acetylcysteamine (I-152). Metabolomic analyses revealed that I-152 increased glutathione levels and influenced the metabolic profiles interconnected with the glutathione system and the redox couples NAD(P)+/NAD(P)H. mRNA analysis showed a lowered gene expression of HIF-1α and VEGF following I-152 treatment whereas TRX1 and 2 were stimulated. Accordingly, the proteomic study revealed the redox-dependent upregulation of thioredoxin and peroxiredoxins that, together with the glutathione system, are the main regulators of intracellular ROS. Indeed, a time-dependent ROS production under hypoxia and a quenching effect of the molecule were evidenced. At the secretome level, the molecule downregulated IL-6, MCP-1, and PDGF-bb. These results suggest that redox modulation by I-152 reduces oxidative stress and ROS level in hypoxic ECs and may be a strategy to fine-tune the environment of an in vitro BM niche able to support functional HSC maintenance.

Monocyte distribution width alterations and cytokine storm are modulated by circulating histones.

OBJECTIVES: Extracellular histone levels are associated with the severity of many human pathologies, including sepsis and COVID-19. This study aimed to investigate the role of extracellular histones on monocyte distribution width (MDW), and their effect on the release of cytokines by blood cells. METHODS: Peripheral venous blood was collected from healthy subjects and treated with different doses of a histone mixture (range 0-200 µg/mL) to analyze MDW modifications up-to 3 h and digital microscopy of blood smears. Plasma obtained after 3 h of histone treatment were assayed to evaluate a panel of 24 inflammatory cytokines. RESULTS: MDW values significantly increased in a time- and dose-dependent manner. These findings are associated with the histone-induced modifications of cell volume, cytoplasmic granularity, vacuolization, and nuclear structure of monocytes, promoting their heterogeneity without affecting their count. After 3 h of treatment almost all cytokines significantly increased in a dose-dependent manner. The most relevant response was shown by the significantly increased G-CSF levels, and by the increase of IL-1ß, IL-6, MIP-1ß, and IL-8 at the histone doses of 50, 100, and 200 µg/mL. VEGF, IP-10, GM-CSF, TNF-α, Eotaxin, and IL-2 were also up-regulated, and a lower but significant increase was observed for IL-15, IL-5, IL-17, bFGF, IL-10, IFN-γ, MCP-1, and IL-9. CONCLUSIONS: Circulating histones critically induce functional alterations of monocytes mirrored by MDW, monocyte anisocytosis, and hyperinflammation/cytokine storm in sepsis and COVID-19. MDW and circulating histones may be useful tools to predict higher risks of worst outcomes.

Deciphering the role of monocyte and monocyte distribution width (MDW) in COVID-19: an updated systematic review and meta-analysis.

The SARS-CoV-2 infection is characterized by both systemic and organ hyper-thromboinflammation, with a clinical course ranging from mild up-to critical systemic dysfunction and death. In patients with coronavirus disease 2019 (COVID-19) the monocyte/macrophage population is deeply involved as both trigger and target, assuming the value of useful diagnostic/prognostic marker of innate cellular immunity. Several studies correlated morphological and immunophenotypic alterations of circulating monocytes with clinical outcomes in COVID-19 patients, concluding that monocyte distribution width (MDW) may retain clinical value in stratifying the risk of disease worsening. Through an electronic search in Medline and Scopus we performed an updated literature review and meta-analysis aimed to explore the association between increased MDW levels and illness severity in COVID-19 patients, deciphering role(s) and function(s) of monocytes in the harmful network underlining SARS-CoV-2 infection. We found that significantly elevated MDW values were frequently present in COVID-19 patients who developed unfavorable clinical outcomes, compounded by a significant association between monocyte anisocytosis and SARS-CoV-2 outcomes. These findings suggest that blood MDW index and its scatter plot could represent useful routine laboratory tools for early identification of patients at higher risk of unfavorable COVID-19 and for monitoring the progression of viral infection, clinical outcomes, and therapeutic efficacy throughout hospitalization. According to this evidence, therapeutic decisions in patients with SARS-CoV-2 infection could benefit from monitoring MDW value, with administration of drugs limiting thrombo-inflammation due to monocyte hyper-activation in patients with severe/critical COVID-19 disease.

Circulating histones contribute to monocyte and MDW alterations as common mediators in classical and COVID-19 sepsis.

OBJECTIVE: Histone proteins are physiologically involved in DNA packaging and gene regulation but are extracellularly released by neutrophil/monocyte extracellular traps and mediate thrombo-inflammatory pathways, associated to the severity of many human pathologies, including bacterial/fungal sepsis and COVID-19. Prominent and promising laboratory features in classic and viral sepsis emphasize monocyte distribution width (MDW), due to its ability to distinguish and stratify patients at higher risk of critical conditions or death. No data are available on the roles of histones as MDW modifiers. DESIGN: Comparison of MDW index was undertaken by routine hematology analyzer on whole blood samples from patients with COVID-19 and Sepsis. The impact of histones on the MDW characteristics was assessed by the in vitro time-dependent treatment of healthy control whole blood with histones and histones plus lipopolysaccharide to simulate viral and classical sepsis, respectively. MEASUREMENTS AND MAIN RESULTS: We demonstrated the breadth of early, persistent, and significant increase of MDW index in whole blood from healthy subject treated in vitro with histones, highlighting changes similar to those found in vivo in classic and viral sepsis patients. These findings are mechanistically associated with the histone-induced modifications of cell volume, cytoplasmic granularity and vacuolization, and nuclear structure alterations of the circulating monocyte population. CONCLUSIONS: Histones may contribute to the pronounced and persistent monocyte alterations observed in both acute classical and viral sepsis. Assessment of the biological impact of circulating histone released during COVID-19 and sepsis on these blood cells should be considered as key factor modulating both thrombosis and inflammatory processes, as well as the importance of neutralization of their cytotoxic and procoagulant activities by several commercially available drugs (e.g., heparins and heparinoids).

What is the impact of circulating histones in COVID-19: a systematic review.

The infectious respiratory condition COVID-19 manifests a clinical course ranging from mild/moderate up-to critical systemic dysfunction and death linked to thromboinflammation. During COVID-19 infection, neutrophil extracellular traps participating in cytokine storm and coagulation dysfunction have emerged as diagnostic/prognostic markers. The characterization of NET identified that mainly histones, have the potential to initiate and propagate inflammatory storm and thrombosis, leading to increased disease severity and decreased patient survival. Baseline assessment and serial monitoring of blood histone concentration may be conceivably useful in COVID-19. We performed a literature review to explore the association among increased circulating levels of histones, disease severity/mortality in COVID-19 patients, and comparison of histone values between COVID-19 and non-COVID-19 patients. We carried out an electronic search in Medline and Scopus, using the keywords "COVID-19" OR "SARS-CoV-2" AND "histone" OR "citrullinated histones" OR "hyperhistonemia", between 2019 and present time (i.e., June 07th, 2022), which allowed to select 17 studies, totaling 1,846 subjects. We found that substantially elevated histone values were consistently present in all COVID-19 patients who developed unfavorable clinical outcomes. These findings suggest that blood histone monitoring upon admission and throughout hospitalization may be useful for early identification of higher risk of unfavorable COVID-19 progression. Therapeutic decisions in patients with SARS-CoV-2 based on the use of histone cut-off values may be driven by drugs engaging histones, finally leading to the limitation of cytotoxic, inflammatory, and thrombotic effects of circulating histones in viral sepsis.

Do Circulating Histones Represent the Missing Link among COVID-19 Infection and Multiorgan Injuries, Microvascular Coagulopathy and Systemic Hyperinflammation?

Several studies shed light on the interplay among inflammation, thrombosis, multi-organ failures and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Increasing levels of both free and/or circulating histones have been associated to coronavirus disease 2019 (COVID-19), enhancing the risk of heart attack and stroke with coagulopathy and systemic hyperinflammation. In this view, by considering both the biological and clinical rationale, circulating histones may be relevant as diagnostic biomarkers for stratifying COVID-19 patients at higher risk for viral sepsis, and as predictive laboratory medicine tool for targeted therapies.

Recent Updates and Advances in Winiwarter-Buerger Disease (Thromboangiitis Obliterans): Biomolecular Mechanisms, Diagnostics and Clinical Consequences.

Thromboangiitis obliterans (TAO) or Buerger's disease is a segmental inflammatory, thrombotic occlusive peripheral vascular disease with unknown aetiology that usually involves the medium and small-sized vessels of young male smokers. Due to its unknown aetiology and similarities with atherosclerosis and vasculitis, TAO diagnosis is still challenging. We aimed to review the status of biomolecular and laboratory para-clinical markers in TAO compared to atherosclerosis and vasculitis. We reported that, although some biomarkers might be common in TAO, atherosclerosis, and vasculitis, each disease occurs through a different pathway and, to our knowledge, there is no specific and definitive marker for differentiating TAO from atherosclerosis or vasculitis. Our review highlighted that pro-inflammatory and cell-mediated immunity cytokines, IL-33, HMGB1, neopterin, MMPs, ICAM1, complement components, fibrinogen, oxidative stress, NO levels, eNOS polymorphism, adrenalin and noradrenalin, lead, cadmium, and homocysteine are common markers. Nitric oxide, MPV, TLRs, MDA, ox-LDL, sST2, antioxidant system, autoantibodies, and type of infection are differential markers, whereas platelet and leukocyte count, haemoglobin, lipid profile, CRP, ESR, FBS, creatinine, d-dimer, hypercoagulation activity, as well as protein C and S are controversial markers. Finally, our study proposed diagnostic panels for laboratory differential diagnosis to be considered at first and in more advanced stages.

Why Venous Leg Ulcers Have Difficulty Healing: Overview on Pathophysiology, Clinical Consequences, and Treatment.

Venous leg ulcers (VLUs) are one of the most common ulcers of the lower extremity. VLU affects many individuals worldwide, could pose a significant socioeconomic burden to the healthcare system, and has major psychological and physical impacts on the affected individual. VLU often occurs in association with post-thrombotic syndrome, advanced chronic venous disease, varicose veins, and venous hypertension. Several demographic, genetic, and environmental factors could trigger chronic venous disease with venous dilation, incompetent valves, venous reflux, and venous hypertension. Endothelial cell injury and changes in the glycocalyx, venous shear-stress, and adhesion molecules could be initiating events in VLU. Increased endothelial cell permeability and leukocyte infiltration, and increases in inflammatory cytokines, matrix metalloproteinases (MMPs), reactive oxygen and nitrogen species, iron deposition, and tissue metabolites also contribute to the pathogenesis of VLU. Treatment of VLU includes compression therapy and endovenous ablation to occlude the axial reflux. Other interventional approaches such as subfascial endoscopic perforator surgery and iliac venous stent have shown mixed results. With good wound care and compression therapy, VLU usually heals within 6 months. VLU healing involves orchestrated processes including hemostasis, inflammation, proliferation, and remodeling and the contribution of different cells including leukocytes, platelets, fibroblasts, vascular smooth muscle cells, endothelial cells, and keratinocytes as well as the release of various biomolecules including transforming growth factor-ß, cytokines, chemokines, MMPs, tissue inhibitors of MMPs (TIMPs), elastase, urokinase plasminogen activator, fibrin, collagen, and albumin. Alterations in any of these physiological wound closure processes could delay VLU healing. Also, these histological and soluble biomarkers can be used for VLU diagnosis and assessment of its progression, responsiveness to healing, and prognosis. If not treated adequately, VLU could progress to non-healed or granulating VLU, causing physical immobility, reduced quality of life, cellulitis, severe infections, osteomyelitis, and neoplastic transformation. Recalcitrant VLU shows prolonged healing time with advanced age, obesity, nutritional deficiencies, colder temperature, preexisting venous disease, deep venous thrombosis, and larger wound area. VLU also has a high, 50-70% recurrence rate, likely due to noncompliance with compression therapy, failure of surgical procedures, incorrect ulcer diagnosis, progression of venous disease, and poorly understood pathophysiology. Understanding the molecular pathways underlying VLU has led to new lines of therapy with significant promise including biologics such as bilayer living skin construct, fibroblast derivatives, and extracellular matrices and non-biologic products such as poly-N-acetyl glucosamine, human placental membranes amnion/chorion allografts, ACT1 peptide inhibitor of connexin 43, sulodexide, growth factors, silver dressings, MMP inhibitors, and modulators of reactive oxygen and nitrogen species, the immune response and tissue metabolites. Preventive measures including compression therapy and venotonics could also reduce the risk of progression to chronic venous insufficiency and VLU in susceptible individuals.

Recent Updates and Advances in the Use of Glycated Albumin for the Diagnosis and Monitoring of Diabetes and Renal, Cerebro- and Cardio-Metabolic Diseases.

Diabetes mellitus is a heterogeneous and dysmetabolic chronic disease in which the laboratory plays a fundamental role, from diagnosis to monitoring therapy and studying complications. Early diagnosis and good glycemic control should start as early as possible to delay and prevent metabolic and cardio-vascular complications secondary to this disease. Glycated hemoglobin is currently used as the reference parameter. The accuracy of the glycated hemoglobin dosage may be compromised in subjects suffering from chronic renal failure and terminal nephropathy, affected by the reduction in the survival of erythrocytes, with consequent decrease in the time available for glucose to attach to the hemoglobin. In the presence of these renal comorbidities as well as hemoglobinopathies and pregnancy, glycated hemoglobin is not reliable. In such conditions, dosage of glycated albumin can help. Glycated albumin is not only useful for short-term diagnosis and monitoring but predicts the risk of diabetes, even in the presence of euglycemia. This protein is modified in subjects who do not yet have a glycemic alteration but, as a predictive factor, heralds the risk of diabetic disease. This review summarizes the importance of glycated albumin as a biomarker for predicting and stratifying the cardiovascular risk linked to multiorgan metabolic alterations.

Redox homeostasis as a target for new antimycobacterial agents.

Despite early treatment with antimycobacterial combination therapy, drug resistance continues to emerge. Maintenance of redox homeostasis is essential for Mycobacterium avium (M. avium) survival and growth. The aim of the present study was to investigate the antimycobacterial activity of two pro-glutathione (pro-GSH) drugs that are able to induce redox stress in M. avium and to modulate cytokine production by macrophages. Hence, we investigated two molecules shown to possess antiviral and immunomodulatory properties: C4-GSH, an N-butanoyl GSH derivative; and I-152, a prodrug of N-acetyl-cysteine (NAC) and ß-mercaptoethylamine (MEA). Both molecules showed activity against replicating M. avium, both in the cell-free model and inside macrophages. Moreover, they were even more effective in reducing the viability of bacteria that had been kept in water for 7 days, proving to be active both against replicating and non-replicating bacteria. By regulating the macrophage redox state, I-152 modulated cytokine production. In particular, higher levels of interferon-gamma (IFN-γ), interleukin 1 beta (IL-1ß), IL-18 and IL-12, which are known to be crucial for the control of intracellular pathogens, were found after I-152 treatment. Our results show that C4-GSH and I-152, by inducing perturbation of redox equilibrium, exert bacteriostatic and bactericidal activity against M. avium. Moreover, I-152 can boost the host response by inducing the production of cytokines that serve as key regulators of the Th1 response.

Impact of Glucose-Lowering Medications on Cardiovascular and Metabolic Risk in Type 2 Diabetes.

Type 2 Diabetes Mellitus (T2DM) is associated with a high risk of atherosclerotic cardiovascular (CV) disease. Among the well-known pathophysiologic factors, crucial roles are played by endothelial dysfunction (caused by oxidative stress and inflammation hyperglycemia-linked), increased activity of nuclear factor kB, altered macrophage polarization, and reduced synthesis of resident endothelial progenitor cells. As consequence, a potentially rapid progression of the atherosclerotic disease with a higher propensity to unstable plaque is arguable, finally leading to significantly increased cardiovascular mortality. Main managements are focused on both prevention and early diagnosis, by targeted treatment of hyperglycemia and vascular complications. Innovative therapeutic approaches for T2DM seek to customize the antidiabetic treatment to each patient in order to optimize glucose-lowering effects, minimize hypoglycemia and adverse effects, and prevent cardiovascular events. The newer drugs (e.g., Glucagon Like Peptide-1 Receptor Agonists, GLP-1 RAs; Sodium GLucose coTransporter-2 inhibitors, SGLT2is; DiPeptidyl Peptidase-4 inhibitors, and DPP4is) impact body weight, lipid parameters, and blood pressure, as well as endothelial (dys)functions, inflammatory markers, biomarkers of both oxidative stress, and subclinical atherosclerosis. The present review summarizes the results of the main trials focused on the cardiovascular safety of these drugs from the CV standpoint.

The Imbalance among Oxidative Biomarkers and Antioxidant Defense Systems in Thromboangiitis Obliterans (Winiwarter-Buerger Disease).

(1) Background: Thromboangiitis obliterans or Winiwarter-Buerger disease (WBD), is an inflammatory, thrombotic occlusive, peripheral vascular disease, usually occurring in young smokers. The pathophysiological mechanisms underlying the disease are not clearly understood. The aim of this study is to investigate the imbalance between oxidants and antioxidants occurring in these patients. (2) Patients and Methods: In this cross-sectional study, 22 male patients with WBD and 20 healthy male smoking habit matched control group were included. To evaluate the possible sources of oxidative stress, the antioxidant biomarkers, and the markers of lipid peroxidation and protein oxidation, serum samples were analyzed for total oxidative status (TOS), total antioxidant capacity (TAC), myeloperoxidase (MPO), coenzyme Q10 (CoQ10), superoxide dismutase (SOD), glutathione reductase (GR), malondialdehyde (MDA), and protein carbonyl (PC) activity and/or content. (3) Results: The circulating levels of TOS, TAC, and CoQ10 were significantly higher in WBD patients, with respect to healthy smokers as controls. No significant difference was found among the serum level of PC, total cholesterol, MPO, and GR activity in WBD patients and healthy smoker controls. The activity of SOD and the mean serum level of MDA were significantly lower in WBD patients, with respect to healthy smoker controls. (4) Conclusion: Considerably high levels of oxidative stress were detected in WBD patients, which were greater than the antioxidant capacity. The low level of MDA may be associated with the enzymatic degradation of lipid peroxidation products. High levels of CoQ10 and low levels of SOD may be related to a harmful oxidative cooperation, leading to the vasoconstriction of WBD, representing a promising tool to discern possible different clinical risks of this poorly understood peripheral occlusive disease.

MMP-2 and MMP-9 in Human Peripheral Blood: Optimizing Gelatinase Calibrator for Degradome Research and Discovering a Novel Gelatinolytic Enzyme.

Matrix metalloprotease-2 and -9 (gelatinase A and B, respectively) are enzymes crucially involved in a plethora of physiopathological conditions. Gelatin zymography is considered one of the major qualitative/semiquantitative assays for simultaneously determining zymogenic, active, and complexed forms of gelatinases. Critical steps are represented by variations in sample collection methods, molecular weight standard calibrators, and different zymography assay protocols. A normalization of these aspects is required for reducing discrepancies in technical procedures and interpreting results among different laboratories. In this study, we describe a novel protocol for gelatin zymography with increased pore size, which improves the separation of gelatinases with different molecular weights. A new method for obtaining gelatinase calibrator for gelatin zymography, by extracting MMP-2 and MMP-9 from peripheral blood, is also reported. Our method provides a gelatinase calibrator with enhanced stability both at room temperature and during multiple freeze-thaw cycles. This calibrator preparation is also suitable for in vitro post-translational modifications. For the first time, the improved zymography protocol allowed us to reveal in human peripheral blood samples new gelatinolytic bands resolved at very high molecular weight, likely complexes of MMP-9, undetectable with classical zymography protocols.

Validation of a Gravitational Model to Study Local Endogenous Biomarkers in Chronic Venous Insufficiency.

OBJECTIVE/BACKGROUND: Unlike most systemic chronic diseases, chronic venous insufficiency (CVI) is ideal to study using endogenous biomarkers. The stimulus causing damage can be turned on and off with gravitational positioning and venous blood samples can be taken locally. Annexin V (apoptosis) and microparticles (cell membrane debris) were used as markers of cell destruction, with matrix metalloproteinases (MMPs) as markers of tissue remodelling. The aim of this proof of concept study was to validate a gravitational model by investigating whether standing induced biochemical stress and whether recovery occurs on lying and after compression. METHODS: Fourteen patients (C4a-b) and 14 volunteers (C0-1) were tested under three supervised laboratory conditions for 1 h on separate days: (i) stationary standing on a small paper square; (ii) lying with both legs elevated 20°; (iii) compression standing using a 23-32 mmHg below knee stocking. Immediately after each condition, venous blood was withdrawn from the ankle. Commercial enzyme linked immunosorbent assay kits were used for batch analysis of the plasma samples. RESULTS: Median (interquartile range [IQR]) values of annexin V (AU/mL) and microparticles (nM) standing were as follows: volunteers 2.9 (2 - 3.4) and 10.2 (8.8 - 13.8), and patients 2.2 (1.3 - 6) and 11.3 (7.7 - 20), respectively. Significant reductions were observed lying: volunteers 2.1 (1.5 - 2.7; p = .019) and 8.5 (7.4 - 9.4; p = .041), patients 1.7 (1.2 - 2.7; p = .004) and 8.5 (7.0 - 11.4; p = .041), respectively. Globally, all median MMP values in the patients reduced with lying and with compression versus standing (p = .004). Individually, significant reductions occurred in MMPs 2 and 13 with compression and MMPs 3, 7, 8, 9, 10, and 12 on lying. Lying was more effective at reducing MMP levels than compression. CONCLUSION: Annexin V and microparticle concentrations are responsive to elevation and compression after 1 h. In the patients, all the tested MMPs decreased after lying and with compression versus standing. This model provides evidence supporting gravitational protection in the treatment of CVI.

Electronegative LDL induces MMP-9 and TIMP-1 release in monocytes through CD14 activation: Inhibitory effect of glycosaminoglycan sulodexide.

OBJECTIVE: Electronegative LDL (LDL(-)) is involved in atherosclerosis through the activation of the TLR4/CD14 inflammatory pathway in monocytes. Matrix metalloproteinases (MMP) and their inhibitors (tissue inhibitors of metalloproteinase [TIMP]) are also crucially involved in atherosclerosis, but their modulation by LDL(-) has never been investigated. The aim of this study was to examine the ability of LDL(-) to release MMPs and TIMPs in human monocytes and to determine whether sulodexide (SDX), a glycosaminoglycan-based drug, was able to affect their secretion. APPROACH AND RESULTS: Native LDL (LDL(+)) and LDL(-) separated by anion-exchange chromatography were added to THP1-CD14 monocytes in the presence or absence of SDX for 24 h. A panel of 9 MMPs and 4 TIMPs was analyzed in cell supernatants with multiplex immunoassays. The gelatinolytic activity of MMP-9 was assessed by gelatin zymography. LDL(-) stimulated the release of MMP-9 (13-fold) and TIMP-1 (4-fold) in THP1-CD14 monocytes, as well as the gelatinolytic activity of MMP-9. Co-incubation of monocytes with LDL(-) and SDX for 24 h significantly reduced both the release of MMP-9 and TIMP-1 and gelatinase activity. In THP1 cells not expressing CD14, no effect of LDL(-) on MMP-9 or TIMP-1 release was observed. The uptake of DiI-labeled LDL(-) was higher than that of DiI-LDL(+) in THP1-CD14 but not in THP1 cells. This increase was inhibited by SDX. Experiments in microtiter wells coated with SDX demonstrated a specific interaction of LDL(-) with SDX. CONCLUSIONS: LDL(-) induced the release of MMP-9 and TIMP-1 in monocytes through CD14. SDX affects the ability of LDL(-) to promote TIMP-1 and MMP-9 release by its interaction with LDL(-).