The oxylipin analog CS585 prevents platelet activation and thrombosis through activation of the prostacyclin receptor.

Cardiovascular disease remains the primary cause of morbidity and mortality globally. Platelet activation is critical for maintaining hemostasis and preventing the leakage of blood cells from the vessel. There has been a paucity in the development of new drugs to target platelet reactivity. Recently, the oxylipin 12(S)-hydroxy-eicosatrienoic acid (12-HETrE), which is produced in platelets, was shown to limit platelet reactivity by activating the prostacyclin receptor. Here, we demonstrated the synthesis of a novel analog of 12-HETrE, known as CS585. Human blood and mouse models of hemostasis and thrombosis were assessed for the ability of CS585 to attenuate platelet activation and thrombosis without increasing the risk of bleeding. Human platelet activation was assessed using aggregometry, flow cytometry, western blot analysis, total thrombus formation analysis system, microfluidic perfusion chamber, and thromboelastography. Hemostasis, thrombosis, and bleeding assays were performed in mice. CS585 was shown to potently target the prostacyclin receptor on the human platelet, resulting in a highly selective and effective mechanism for the prevention of platelet activation. Furthermore, CS585 was shown to inhibit platelet function in human whole blood ex vivo, prevent thrombosis in both small and large vessels in mouse models, and exhibit long-lasting prevention of clot formation. Finally, CS585 was not observed to perturb coagulation or increase the risk of bleeding in the mouse model. Hence, CS585 represents a new validated target for the treatment of thrombotic diseases without the risk of bleeding or off-target activation observed with other prostaglandin receptor agonists.

Regulatory workshop on challenge strain development and GMP manufacture - A stakeholder meeting report.

Within the Innovative Health Initiative (IHI) Inno4Vac CHIMICHURRI project, a regulatory workshop was organised on the development and manufacture of challenge agent strains for Controlled Human Infection Model (CHIM) studies. Developers are often uncertain about which GMP requirements or regulatory guidelines apply but should be guided by the 2022 technical white paper "Considerations on the Principles of Development and Manufacturing Qualities of Challenge Agents for Use in Human Infection Models" (published by hVIVO, Wellcome Trust, HIC-Vac consortium members). Where those recommendations cannot be met, regulators advise following the "Principles of GMP" until definitive guidelines are available. Sourcing wild-type virus isolates is a significant challenge for developers. Still, it is preferred over reverse genetics challenge strains for several reasons, including implications and regulations around genetically modified organisms (GMOs). Official informed consent guidelines for collecting isolates are needed, and the characterisation of these isolates still presents risks and uncertainty. Workshop topics included ethics, liability, standardised clinical endpoints, selection criteria, sharing of challenge agents, and addressing population heterogeneity concerning vaccine response and clinical course. The organisers are confident that the workshop discussions will contribute to advancing ethical, safe, and high-quality CHIM studies of influenza, RSV and C. difficile, including adequate regulatory frameworks.

Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults.

BACKGROUND: Testing of vaccine candidates to prevent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in an older population is important, since increased incidences of illness and death from coronavirus disease 2019 (Covid-19) have been associated with an older age. METHODS: We conducted a phase 1, dose-escalation, open-label trial of a messenger RNA vaccine, mRNA-1273, which encodes the stabilized prefusion SARS-CoV-2 spike protein (S-2P) in healthy adults. The trial was expanded to include 40 older adults, who were stratified according to age (56 to 70 years or ≥71 years). All the participants were assigned sequentially to receive two doses of either 25 µg or 100 µg of vaccine administered 28 days apart. RESULTS: Solicited adverse events were predominantly mild or moderate in severity and most frequently included fatigue, chills, headache, myalgia, and pain at the injection site. Such adverse events were dose-dependent and were more common after the second immunization. Binding-antibody responses increased rapidly after the first immunization. By day 57, among the participants who received the 25-µg dose, the anti-S-2P geometric mean titer (GMT) was 323,945 among those between the ages of 56 and 70 years and 1,128,391 among those who were 71 years of age or older; among the participants who received the 100-µg dose, the GMT in the two age subgroups was 1,183,066 and 3,638,522, respectively. After the second immunization, serum neutralizing activity was detected in all the participants by multiple methods. Binding- and neutralizing-antibody responses appeared to be similar to those previously reported among vaccine recipients between the ages of 18 and 55 years and were above the median of a panel of controls who had donated convalescent serum. The vaccine elicited a strong CD4 cytokine response involving type 1 helper T cells. CONCLUSIONS: In this small study involving older adults, adverse events associated with the mRNA-1273 vaccine were mainly mild or moderate. The 100-µg dose induced higher binding- and neutralizing-antibody titers than the 25-µg dose, which supports the use of the 100-µg dose in a phase 3 vaccine trial. (Funded by the National Institute of Allergy and Infectious Diseases and others; mRNA-1273 Study ClinicalTrials.gov number, NCT04283461.).

Intranasal Live Influenza Vaccine Priming Elicits Localized B Cell Responses in Mediastinal Lymph Nodes.

Pandemic live attenuated influenza vaccines (pLAIV) prime subjects for a robust neutralizing antibody response upon subsequent administration of a pandemic inactivated subunit vaccine (pISV). However, a difference was not detected in H5-specific memory B cells in the peripheral blood between pLAIV-primed and unprimed subjects prior to pISV boost. To investigate the mechanism underlying pLAIV priming, we vaccinated groups of 12 African green monkeys (AGMs) with H5N1 pISV or pLAIV alone or H5N1 pLAIV followed by pISV and examined immunity systemically and in local draining lymph nodes (LN). The AGM model recapitulated the serologic observations from clinical studies. Interestingly, H5N1 pLAIV induced robust germinal center B cell responses in the mediastinal LN (MLN). Subsequent boosting with H5N1 pISV drove increases in H5-specific B cells in the axillary LN, spleen, and circulation in H5N1 pLAIV-primed animals. Thus, H5N1 pLAIV primes localized B cell responses in the MLN that are recalled systemically following pISV boost. These data provide mechanistic insights for the generation of robust humoral responses via prime-boost vaccination.IMPORTANCE We have previously shown that pandemic live attenuated influenza vaccines (pLAIV) prime for a rapid and robust antibody response on subsequent administration of inactivated subunit vaccine (pISV). This is observed even in individuals who had undetectable antibody (Ab) responses following the initial vaccination. To define the mechanistic basis of pLAIV priming, we turned to a nonhuman primate model and performed a detailed analysis of B cell responses in systemic and local lymphoid tissues following prime-boost vaccination with pLAIV and pISV. We show that the nonhuman primate model recapitulates the serologic observations from clinical studies. Further, we found that pLAIVs induced robust germinal center B cell responses in the mediastinal lymph node. Subsequent boosting with pISV in pLAIV-primed animals resulted in detection of B cells in the axillary lymph nodes, spleen, and peripheral blood. We demonstrate that intranasally administered pLAIV elicits a highly localized germinal center B cell response in the mediastinal lymph node that is rapidly recalled following pISV boost into germinal center reactions at numerous distant immune sites.

Sensitivity of tropical carbon to climate change constrained by carbon dioxide variability.

The release of carbon from tropical forests may exacerbate future climate change, but the magnitude of the effect in climate models remains uncertain. Coupled climate-carbon-cycle models generally agree that carbon storage on land will increase as a result of the simultaneous enhancement of plant photosynthesis and water use efficiency under higher atmospheric CO(2) concentrations, but will decrease owing to higher soil and plant respiration rates associated with warming temperatures. At present, the balance between these effects varies markedly among coupled climate-carbon-cycle models, leading to a range of 330 gigatonnes in the projected change in the amount of carbon stored on tropical land by 2100. Explanations for this large uncertainty include differences in the predicted change in rainfall in Amazonia and variations in the responses of alternative vegetation models to warming. Here we identify an emergent linear relationship, across an ensemble of models, between the sensitivity of tropical land carbon storage to warming and the sensitivity of the annual growth rate of atmospheric CO(2) to tropical temperature anomalies. Combined with contemporary observations of atmospheric CO(2) concentration and tropical temperature, this relationship provides a tight constraint on the sensitivity of tropical land carbon to climate change. We estimate that over tropical land from latitude 30° north to 30° south, warming alone will release 53 ± 17 gigatonnes of carbon per kelvin. Compared with the unconstrained ensemble of climate-carbon-cycle projections, this indicates a much lower risk of Amazon forest dieback under CO(2)-induced climate change if CO(2) fertilization effects are as large as suggested by current models. Our study, however, also implies greater certainty that carbon will be lost from tropical land if warming arises from reductions in aerosols or increases in other greenhouse gases.

A Live Attenuated Chimeric West Nile Virus Vaccine, rWN/DEN4Δ30, Is Well Tolerated and Immunogenic in Flavivirus-Naive Older Adult Volunteers.

West Nile virus (WNV) is a major cause of mosquito-borne illness in the United States. Human disease ranges from mild febrile illness to severe fatal neurologic infection. Adults aged >60 years are more susceptible to neuroinvasive disease accompanied by a high mortality rate or long-lasting neurologic sequelae. A chimeric live attenuated West Nile virus vaccine, rWN/DEN4Δ30, was shown to be safe and immunogenic in healthy adults aged 18-50 years. This study evaluated rWN/DEN4Δ30 in flavivirus-naive adults aged 50-65 years and found it to be safe and immunogenic. Outbreaks of WNV infection tend to be unpredictable, and a safe and effective vaccine will be an important public health tool.

Evaluation of the Safety and Immunogenicity of a Candidate Pandemic Live Attenuated Influenza Vaccine (pLAIV) Against Influenza A(H7N9).

BACKGROUND: We evaluated a candidate A/Anhui/2013(H7N9) pandemic live attenuated influenza vaccine (pLAIV) in healthy adults, and assessed the ability of 1 or 2 doses to induce immune memory. METHODS: Healthy subjects in 2 age groups (18-49 years and 50-70 years) with undetectable hemagglutination-inhibiting (HAI) antibody to H7N9 were enrolled. Younger subjects received either 1 or 2 intranasal doses of 10(7.0) fluorescent focus units of A/Anhui/1/2013 pLAIV, while older subjects received a single dose. All subjects received a single 30-µg dose of unadjuvanted, antigenically matched A/Shanghai2/2013(H7N9) pandemic inactivated influenza vaccine (pIIV) 12 weeks after their first dose of pLAIV. RESULTS: Both vaccines were well tolerated. Serum HAI antibody responses were detected in 0 of 32 younger subjects and 1 of 17 older subjects after 1 dose of pLAIV and in 2 of 16 younger subjects after a second dose. Strong serum antibody responses were detected after a single subsequent dose of pIIV that was broadly reactive against H7 influenza viruses. CONCLUSIONS: An A(H7N9) pLAIV candidate was safe in both age groups. Priming with pLAIV resulted in responses to subsequent pIIV that exceeded those seen in naive subjects in previous reports. The A(H7N9) pLAIV induces strong immune memory that can be demonstrated by exposure to subsequent antigenic challenge. CLINICAL TRIALS REGISTRATION: NCT01995695 and NCT02274545.

Generation and Protective Ability of Influenza Virus-Specific Antibody-Dependent Cellular Cytotoxicity in Humans Elicited by Vaccination, Natural Infection, and Experimental Challenge.

BACKGROUND: Nonneutralizing antibodies (Abs) involved in antibody-dependent cellular cytotoxicity (ADCC) may provide some protection from influenza virus infection. The ability of influenza vaccines to induce ADCC-mediating Abs (ADCC-Abs) in adults and children is unclear. METHODS: We quantified ADCC-Abs in serum samples from adults who received a dose of inactivated subunit vaccine (ISV) targeting monovalent 2009 pandemic influenza A(H1N1) virus or live-attenuated influenza vaccine (LAIV) or who had laboratory-confirmed influenza A(H1N1) virus infection. We also measured ADCC-Abs in children who either received a dose of trivalent seasonal ISV followed by trivalent seasonal LAIV or 2 doses of LAIV. Finally, we assessed the ability of low and high ADCC-Ab titers to protect adults from experimental challenge with influenza A/Wisconsin/67/131/2005(H3N2) virus. RESULTS: Adults and children who received a dose of ISV had a robust increase in ADCC-Ab titers to both recombinant hemagglutinin (rHA) protein and homologous virus-infected cells. There was no detectable increase in titers of ADCC-Abs to rHA or virus-infected cells in adults and children who received LAIV. Higher titers (≥320) of preexisting ADCC-Abs were associated with lower virus replication and a significant reduction in total symptom scores in experimentally infected adults. CONCLUSIONS: ADCC-Ab titers increased following experimental influenza virus infection in adults and after ISV administration in both children and adults.

A 12-Month-Interval Dosing Study in Adults Indicates That a Single Dose of the National Institute of Allergy and Infectious Diseases Tetravalent Dengue Vaccine Induces a Robust Neutralizing Antibody Response.

UNLABELLED: The ideal dengue vaccine will provide protection against all serotypes of dengue virus and will be economical and uncomplicated in its administration. To determine the ability of a single dose of the live attenuated tetravalent dengue vaccine TV003 to induce a suitable neutralizing antibody response, a placebo-controlled clinical trial was performed in 48 healthy adults who received 2 doses of vaccine or placebo administered 12 months apart. Evaluation of safety, vaccine viremia, and neutralizing antibody response after each dose indicated that the first dose of vaccine was capable of preventing infection with the second dose, thus indicating that multiple doses are unnecessary. CLINICAL TRIALS REGISTRATION: NCT01782300.

Gram-Negative Pneumonia Alters Large-Vein Cell-Adhesion Molecule Profile and Potentiates Experimental Stasis Venous Thrombosis.

BACKGROUND/AIMS: Pneumonia is a significant risk factor for the development of venous thrombosis (VT). Cell-adhesion molecules (CAMs) are linked to the pathogenesis of both pneumonia and VT. We hypothesized that remote infection would confer a prothrombogenic milieu via systemic elevation of CAMs. METHODS: Lung injury was induced in wild-type (C57BL/6) mice by lung contusion or intratracheal inoculation with Klebsiella pneumoniae or saline controls. K. pneumoniae-treated mice and controls additionally underwent inferior vena cava (IVC) ligation to generate VT. RESULTS: Lung-contusion mice demonstrated no increase in E-selectin or P-selectin whereas mice infected with K. pneumoniae demonstrated increased circulating P-selectin, ICAM-1, VCAM-1 and thrombin-antithrombin (TAT) complexes. Mice with pneumonia formed VT 3 times larger than controls, demonstrated significantly more upregulation of vein-wall and systemic CAMs, and formed erythrocyte-rich thrombi. CONCLUSION: Elevated CAM expression was identified in mice with pneumonia, but not lung contusion, indicating that the type of inflammatory stimulus and the presence of infection drive the vein-wall response. Elevation of CAMs was associated with amplified VT and may represent an alternate mechanism by which to target the prevention of VT.

Intact Toll-like receptor 9 signaling in neutrophils modulates normal thrombogenesis in mice.

BACKGROUND: Deletion of Toll-like receptor 9 (Tlr9) signaling, which is important for sterile inflammatory processes, results in impaired resolution of venous thrombosis (VT) in mice. The purpose of this study was to determine if deletion of Tlr9 affected sterile necrosis, apoptosis, and neutrophil extracellular trap (NET) production in VT. METHODS: Stasis and nonstasis murine models of VT were used in wild-type (WT) and Tlr9-/- mice, with assessment of thrombus size and determination of NETs, necrosis, and apoptosis markers. Anti-polymorphonuclear neutrophil (PMN) and antiplatelet antibody strategies were used to determine the cellular roles and their roles in WT and Tlr9-/- mice. RESULTS: At 2 days, stasis thrombi in Tlr9-/- mice were 62% larger (n = 6-10), with 1.4-fold increased uric acid levels, 1.7-fold more apoptotic cells, 2-fold increased citrullinated histones, 2-fold increased peptidylarginine deiminase 4 (PAD4), and 1.5-fold increased elastase and a 2.4-fold reduction in tissue factor pathway inhibitor compared with WT mice (all n = 4-7; P < .05). In contrast, the sizes of nonstasis thrombi were not significantly different in Tlr9-/- mice (n = 4-6), and they did not have elevated necrosis or NET markers. Stasis thrombus size was not reduced at the 2-day time point in WT or Tlr9-/- mice that received treatment with deoxyribonuclease I or in PAD4-/- mice, which are incapable of forming NETs. In Tlr9-/- mice undergoing PMN depletion (n = 8-10), stasis thrombus size was reduced 18% and was associated with 29-fold decreased citrullinated histones, 1.3-fold decreased elastase, and 1.5-fold increased tissue factor pathway inhibitor (all n = 6; P < .05). Last, platelet depletion (>90% reduction) did not significantly reduce stasis thrombus size in Tlr9-/- mice. CONCLUSIONS: These data suggest that the thrombogenic model affects Tlr9 thrombogenic mechanisms and that functional Tlr9 signaling in PMNs, but not in platelets or NETs, is an important mechanism in early stasis experimental venous thrombogenesis.

Live attenuated and inactivated influenza vaccines in children.

BACKGROUND: Live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine (IIV) are available for children. Local and systemic immunity induced by LAIV followed a month later by LAIV and IIV followed by LAIV were investigated with virus recovery after LAIV doses as surrogates for protection against influenza on natural exposure. METHODS: Fifteen children received IIV followed by LAIV, 13 an initial dose of LAIV, and 11 a second dose of LAIV. The studies were done during autumn 2009 and autumn 2010 with the same seasonal vaccine (A/California/07/09 [H1N1], A/Perth/16/09 [H3N2], B/Brisbane/60/08). RESULTS: Twenty-eight of 39 possible influenza viral strains were recovered after the initial dose of LAIV. When LAIV followed IIV, 21 of 45 viral strains were identified. When compared to primary LAIV infection, the decreased frequency of shedding with the IIV-LAIV schedule was significant (P = .023). With LAIV-LAIV, the fewest viral strains were recovered (3/33)--numbers significantly lower (P < .001) than shedding after initial LAIV and after IIV-LAIV (P < .001). Serum hemagglutination inhibition antibody responses were more frequent after IIV than LAIV (P = .02). In contrast, more mucosal immunoglobulin A responses were seen with LAIV. CONCLUSIONS: LAIV priming induces greater inhibition of virus recovery on LAIV challenge than IIV priming. The correlate(s) of protection are the subject of ongoing analysis. CLINICAL TRIALS REGISTRATION: NCT01246999.

Robust and Balanced Immune Responses to All 4 Dengue Virus Serotypes Following Administration of a Single Dose of a Live Attenuated Tetravalent Dengue Vaccine to Healthy, Flavivirus-Naive Adults.

BACKGROUND: The 4 serotypes of dengue virus, DENV-1-4, are the leading cause of arboviral disease globally. The ideal dengue vaccine would provide protection against all serotypes after a single dose. METHODS: Two randomized, placebo-controlled trials were performed with 168 flavivirus-naive adults to demonstrate the safety and immunogenicity of a live attenuated tetravalent dengue vaccine (TV003), compared with those of a second tetravalent vaccine with an enhanced DENV-2 component (TV005), and to evaluate the benefit of a booster dose at 6 months. Safety data, viremia, and neutralizing antibody titers were evaluated. RESULTS: A single dose of TV005 elicited a tetravalent response in 90% of vaccinees by 3 months after vaccination and a trivalent response in 98%. Compared with TV003, the higher-dose DENV-2 component increased the observed frequency of immunogenicity to DENV-2 in the TV005 trial. Both the first and second doses were well tolerated. Neither vaccine viremia, rash, nor a significant antibody boost were observed following a second dose. CONCLUSIONS: A single subcutaneous dose of TV005 dengue vaccine is safe and induces a tetravalent antibody response at an unprecedented frequency among vaccinees. A second dose has limited benefit and appears to be unnecessary. Studies to confirm these findings and assess vaccine efficacy will now move to populations in regions where DENV transmission is endemic. CLINICAL TRIALS REGISTRATION: NCT01072786 and NCT01436422.

A live attenuated influenza A(H5N1) vaccine induces long-term immunity in the absence of a primary antibody response.

BACKGROUND: Highly pathogenic avian influenza A(H5N1) causes severe infections in humans. We generated 2 influenza A(H5N1) live attenuated influenza vaccines for pandemic use (pLAIVs), but they failed to elicit a primary immune response. Our objective was to determine whether the vaccines primed or established long-lasting immunity that could be detected by administration of inactivated subvirion influenza A(H5N1) vaccine (ISIV). METHODS: The following groups were invited to participate in the study: persons who previously received influenza A(H5N1) pLAIV; persons who previously received an irrelevant influenza A(H7N3) pLAIV; and community members who were naive to influenza A(H5N1) and LAIV. LAIV-experienced subjects received a single 45-µg dose of influenza A(H5N1) ISIV. Influenza A(H5N1)- and LAIV-naive subjects received either 1 or 2 doses of ISIV. RESULTS: In subjects who had previously received antigenically matched influenza A(H5N1) pLAIV followed by 1 dose of ISIV compared with those who were naive to influenza A(H5N1) and LAIV and received 2 doses of ISIV, we observed an increased frequency of antibody response (82% vs 50%, by the hemagglutination inhibition assay) and a significantly higher antibody titer (112 vs 76; P = .04). The affinity of antibody and breadth of cross-clade neutralization was also enhanced in influenza A(H5N1) pLAIV-primed subjects. CONCLUSIONS: ISIV administration unmasked long-lasting immunity in influenza A(H5N1) pLAIV recipients, with a rapid, high-titer, high-quality antibody response that was broadly cross-reactive across several influenza A(H5N1) clades. CLINICAL TRIALS REGISTRATION: NCT01109329.

A single dose of any of four different live attenuated tetravalent dengue vaccines is safe and immunogenic in flavivirus-naive adults: a randomized, double-blind clinical trial.

BACKGROUND: Dengue virus (DENV) causes hundreds of millions of infections annually. Four dengue serotypes exist, and previous infection with one serotype increases the likelihood of severe disease with a second, heterotypic DENV infection. METHODS: In a randomized, placebo-controlled study, the safety and immunogenicity of 4 different admixtures of a live attenuated tetravalent (LATV) dengue vaccine were evaluated in 113 flavivirus-naive adults. Serum neutralizing antibody levels to all 4 dengue viruses were measured on days 0, 28, 42, and 180. RESULTS: A single dose of each LATV admixture induced a trivalent or better neutralizing antibody response in 75%-90% of vaccinees. There was no significant difference in the incidence of adverse events between vaccinees and placebo-recipients other than rash. A trivalent or better response correlated with rash and with non-black race (P < .0001). Black race was significantly associated with a reduced incidence of vaccine viremia. CONCLUSIONS: TV003 induced a trivalent or greater antibody response in 90% of flavivirus-naive vaccinees and is a promising candidate for the prevention of dengue. Race was identified as a factor influencing the infectivity of the LATV viruses, reflecting observations of the effect of race on disease severity in natural dengue infection.

Rodent Inferior Vena Cava Venoplasty Balloon Model.

Balloon venoplasty is a commonly used clinical technique to treat deep vein stenosis and occlusion as a consequence of trauma, congenital anatomic abnormalities, acute deep vein thrombosis (DVT), or stenting. Chronic deep venous obstruction is histopathologically characterized by thrombosis, fibrosis, or both. Currently, no direct treatment is available to target these pathological processes. Therefore, a reliable in vivo animal model to test novel interventions is necessary. The rodent survival inferior vena cava (IVC) venoplasty balloon model (VBM) allows the study of balloon venoplasty in non-thrombotic and post-thrombotic conditions across multiple time points. The local and systemic effect of coated and uncoated venoplasty balloons can be quantified via tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent assay (ELISA), zymography, vein wall and thrombus cellular analysis, whole blood and plasma assays, and histological analysis. The VBM is reproducible, replicates surgical human interventions, can identify local vein wall-thrombi protein changes, and allows multiple analyses from the same sample, decreasing the number of animals required per group.

The effect of matrix metalloproteinase 2 and matrix metalloproteinase 2/9 deletion in experimental post-thrombotic vein wall remodeling.

BACKGROUND: Vein wall fibrotic injury following deep venous thrombosis (VT) is associated with elevated matrix metalloproteinases (MMPs). Whether and by what mechanism MMP2 contributes to vein wall remodeling after VT is unknown. METHODS: Stasis VT was produced by ligation of the inferior vena cava and tissue was harvested at 2, 8, and 21 days in MMP2 -/- and genetic wild type (WT) mice. Tissue analysis by immunohistochemistry, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and zymography was performed. RESULTS: Thrombus resolution was less at 8 days in MMP2 -/- compared with WT, evidenced by a 51% increase in VT size (P < .01), and threefold fewer von Willebrand's factor positive channels (P < .05). In MMP2 -/- mice, the main phenotypic fibrotic differences occurred at 8 days post-VT, with significantly less vein wall collagen content (P = .013), fourfold lower procollagen III gene expression (P < .01), but no difference in procollagen I compared with WT. Decreased inflammation in MMP2 -/- vein walls was suggested by ∼ threefold reduced TNFα and IL-1ß at 2 days and 8 days post-VT (P < .05). A fourfold increase in vein wall monocytes (P = .03) with threefold decreased apoptosis (P < .05), but no difference in cellular proliferation at 8 days was found in MMP2 -/- compared with WT. As increased compensatory MMP9 activity was observed in the MMP2 -/-mice, MMP2/9 double null mice had thrombus induced with VT harvest at 8 days. Consistently, twofold larger VT, a threefold decrease in vein wall collagen, and a threefold increase in monocytes were found (all P < .05). Similar findings were observed in MMP9 -/- mice administered an exogenous MMP2 inhibitor. CONCLUSIONS: In stasis VT, deletion of MMP2 was associated with less midterm vein wall fibrosis and inflammation, despite an increase in monocytes. Consideration that VT resolution was impaired with MMP2 (and MMP2/9) deletion suggests direct inhibition will likely also require anticoagulant therapy.

Experimental venous thrombus resolution is driven by IL-6 mediated monocyte actions.

Deep venous thrombosis and residual thrombus burden correlates with circulating IL-6 levels in humans. To investigate the cellular source and role of IL-6 in thrombus resolution, Wild type C57BL/6J (WT), and IL-6-/- mice underwent induction of VT via inferior vena cava (IVC) stenosis or stasis. Vein wall (VW) and thrombus were analyzed by western blot, immunohistochemistry, and flow cytometry. Adoptive transfer of WT bone marrow derived monocytes was performed into IL6-/- mice to assess for rescue. Cultured BMDMs from WT and IL-6-/- mice underwent quantitative real time PCR and immunoblotting for fibrinolytic factors and matrix metalloproteinase activity. No differences in baseline coagulation function or platelet function were found between WT and IL-6-/- mice. VW and thrombus IL-6 and IL-6 leukocyte-specific receptor CD126 were elevated in a time-dependent fashion in both VT models. Ly6Clo Mo/MØ were the predominant leukocyte source of IL-6. IL-6-/- mice demonstrated larger, non-resolving stasis thrombi with less neovascularization, despite a similar number of monocytes/macrophages (Mo/MØ). Adoptive transfer of WT BMDM into IL-6-/- mice undergoing stasis VT resulted in phenotype rescue. Human specimens of endophlebectomized tissue showed co-staining of Monocyte and IL-6 receptor. Thrombosis matrix analysis revealed significantly increased thrombus fibronectin and collagen in IL-6-/- mice. MMP9 activity in vitro depended on endogenous IL-6 expression in Mo/MØ, and IL-6-/- mice exhibited stunted matrix metalloproteinase activity. Lack of IL-6 signaling impairs thrombus resolution potentially via dysregulation of MMP-9 leading to impaired thrombus recanalization and resolution. Restoring or augmenting monocyte-mediated IL-6 signaling in IL-6 deficient or normal subjects, respectively, may represent a non-anticoagulant target to improve thrombus resolution.