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Divergent effects of Tlr9 deletion in experimental late venous thrombosis resolution and vein wall injury.

ABSTRACT: Deep-vein thrombosis (DVT) resolves via a sterile inflammatory response. Defining the inflammatory response of DVT may allow for new therapies that do not involve anticoagulation. Previously, we have shown that Toll-like receptor 9 (Tlr9) gene deleted mice had impaired venous thrombosis (VT) resolution. Here, we further characterise the role of Tlr9 signalling and sterile inflammation in chronic VT and vein wall responses. First, we found a human precedent exists with Tlr9+ cells present in chronic post thrombotic intraluminal tissue. Second, in a stasis VT mouse model, endogenous danger signal mediators of uric acid, HMGB-1, and neutrophil extracellular traps marker of citrullinated histone-3 (and extracellular DNA) were greater in Tlr9-/- thrombi as compared with wild-type (WT), corresponding with larger VT at 8 and 21 days. Fewer M1 type (CCR2+) monocyte/macrophages (MØ) were present in Tlr9-/- thrombi than WT controls at 8 days, suggesting an impaired inflammatory cell influx. Using bone marrow-derived monocyte (BMMØ) cell culture, we found decreased fibrinolytic gene expression with exposure to several endogenous danger signals. Next, adoptive transfer of cultured Tlr9+/+ BMMØ to Tlr9-/- mice normalised VT resolution at 8 days. Lastly, although the VT size was larger at 21 days in Tlr9-/- mice and correlated with decreased endothelial antigen markers, no difference in fibrosis was found. These data suggest that Tlr9 signalling in MØ is critical for later VT resolution, is associated with necrosis clearance, but does not affect later vein wall fibrosis. These findings provide insight into the Tlr9 MØ mechanisms of sterile inflammation in this disease process.

SUBMITTER: Dewyer NA

PROVIDER: S-EPMC5131365 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Divergent effects of Tlr9 deletion in experimental late venous thrombosis resolution and vein wall injury.

Dewyer Nicholas A NA El-Sayed Osama M OM Luke Catherine E CE Elfline Megan M Kittan Nicolai N Allen Ron R Laser Adriana A Oostra Carson C Comerota Anthony A Hogaboam Cory C Kunkel Steven L SL Henke Peter K PK

Thrombosis and haemostasis 20150716 5

Deep-vein thrombosis (DVT) resolves via a sterile inflammatory response. Defining the inflammatory response of DVT may allow for new therapies that do not involve anticoagulation. Previously, we have shown that Toll-like receptor 9 (Tlr9) gene deleted mice had impaired venous thrombosis (VT) resolution. Here, we further characterise the role of Tlr9 signalling and sterile inflammation in chronic VT and vein wall responses. First, we found a human precedent exists with Tlr9+ cells present in chro ...[more]

PMID: 26179893

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Project description:BackgroundVascular adventitia contains progenitor cells and is shown to participate in vascular remolding. Progenitor cells are recruited into the venous thrombi in mice to promote neovascularization. We hypothesized that the adventitial progenitor cells of human great saphenous vein (HGSV-AdPC) enhance the resolution of venous thrombosis via neovascularization.MethodsHuman great saphenous vein (HGSV) was harvested from the patients with great saphenous vein varicose and sectioned for immunohistochemistry, or minced for progenitor cell primary culture, or placed in sodium dodecyl sulfate solution for decellularization. Human venous thrombi were collected from patients with great saphenous vein varicose and superficial thrombophlebitis. Infrarenal abdominal aorta of New Zealand white rabbits was replaced with interposing decellularized vessel, and the patency of the grafts was confirmed by ultrasonic examination. Animal venous thrombi in the left infrarenal vena cava of mice were produced with Prolene suture ligation and ophthalmic force clipping of this portion. After HGSVs were digested by collagenase, the CD34+CD117+ HGSV-AdPC were isolated on FACS system, labelled with CM-Dil, and transplanted into the adventitia of infrarenal vena cava of nude mice. The percentage of thrombus organization area to the thrombus area was calculated as the organization rate. The thrombus cell, endothelial cells, and macrophages in the thrombi were counted in sections. Cell smears and frozen sections of human saphenous veins and venous thrombi were labeled with Sca1, CD34, CD117, Flk1, CD31, and F4/80 antibodies. The CD34+CD117+ HGSV-AdPC were cultured in endothelial growth medium with vascular endothelial growth factor (VEGF) to induce endothelial cell differentiation and analyzed with real time-PCR, Western blotting, and tube formation assays.ResultsImmunohistochemical staining showed that the CD34+CD117+ cells were located within the adventitia of HGSVs, and many CD34+ and CD117+ cells have emerged in the human venous thrombi. The number of progenitor cells within the marginal area of 7 days mice thrombi was shown to be Sca1+ ≈21%, CD34+ ≈12%, CD117+ ≈9%, and Flk1+ ≈5%. Many CD34+adventitial progenitor cells have migrated into the decellularized vessels. FACS showed that the number of CD34+CD117+ HGSV-AdPC in primary cultured cells as 1.2 ± 0.07%. After CD34+CD117+HGSV-AdPC were transplanted into the adventitia of nude mice vena cava with venous thrombi, the organization rate, nucleate cell count, endothelial cells, and macrophage cells of thrombi were shown to be significantly increased. The transplanted CD34+CD117+ HGSV-AdPC at the adventitia have crossed the vein wall, entered the venous thrombi, and differentiated into endothelial cells. The CD34+CD117+ HGSV-AdPC in the culture medium in the presence of VEGF-promoted gene and protein expression of endothelial cell markers in vitro and induced tube formation.ConclusionsHGSV-AdPC could cross the vein wall and migrate from the adventitia into the venous thrombi. Increased HGSV-AdPC in the adventitia has enhanced the resolution of venous thrombi via differentiating into endothelial cells of neovascularization.

Project description:BackgroundThe postthrombotic syndrome is a common, often morbid sequela of venous thrombosis (VT) that arises from thrombus persistence and inflammatory scarring of juxtaposed vein walls and valves. Noninvasive 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) imaging can measure neutrophil inflammation in VT. Here, we hypothesized (1) early fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) VT inflammation can predict subsequent vein wall scarring (VWS) and (2) statin therapy can reduce FDG-PET VT inflammation and subsequent VWS.MethodsC57BL/6J mice (n=75) underwent induction of stasis-induced VT of the inferior vena cava or jugular vein. Inferior vena cava VT mice (n=44) were randomized to daily oral rosuvastatin 5 mg/kg or saline starting at day -1. Subgroups of mice then underwent FDG-PET/CT 2 days after VT induction. On day 14, a subset of mice was euthanized, and VWS was assessed via histology. In vitro studies were further performed on bone marrow-derived neutrophils.ResultsStatin therapy reduced early day 2 FDG-PET VT inflammation, thrombus neutrophil influx, and plasma IL (interleukin)-6 levels. At day 14, statin therapy reduced VWS but did not affect day 2 thrombus mass, cholesterol, or white blood counts, nor reduce day 2 glucose transporter 1 or myeloperoxidase expression in thrombus or in isolated neutrophils. In survival studies, the day 2 FDG-PET VT inflammation signal as measured by mean and maximum standardized uptake values predicted the extent of day 14 VWS (area under the receiver operating characteristic curve =0.82) with a strong correlation coefficient (r) of r=0.73 and r=0.74, respectively. Mediation analyses revealed that 40% of the statin-induced VWS reduction was mediated by reductions in VT inflammation as quantified by FDG-PET.ConclusionsEarly noninvasive FDG-PET/CT imaging of VT inflammation predicts the magnitude of subsequent VWS and may provide a new translatable approach to identify individuals at risk for postthrombotic syndrome and to assess anti-inflammatory postthrombotic syndrome therapies, such as statins.

Project description:ObjectiveVenous thrombosis (VT) damages the vein wall, both physically by prolonged distension and from inflammation. These factors contribute to post-thrombotic syndrome (PTS). Interleukin (IL)-6 might play a role in experimental PTS and vein wall responses. Previous assessments of post-thrombotic vein wall injury used static measures such as histologic examination and immunologic assays. The purpose of the present study was to use myography to quantify the changes in contraction and relaxation of murine vessels exposed to an acute VT.MethodsWild-type (WT) C57BL/6 mice were used to determine the baseline vein wall passive tension on a DMT 610m myograph (DMT-USA, Inc., Ann Arbor, Mich), including dosing concentrations of phenylephrine (Phe) and acetylcholine (Ach). WT and IL-6-/- mice underwent VT using inferior vena cava (IVC) ligation (complete stasis) and stenosis (partial stasis), with no-surgery mice used as controls. The mice were harvested at 2 days (2D) and analyzed using a myograph. The vessels were stimulated with Phe and Ach to stimulate a contraction and relaxation response. The endothelial responses to VT were quantified by CD31 immunohistochemistry, Greiss assay, polymerase chain reaction, and Evans blue assay.ResultsOptimal passive tension was determined to be 2 mN, with an optimal concentration of Phe and Ach of 7E-3M and 1E-5M, respectively. No significant differences were found in the contractions when exposed to Phe between the WT control, WT 2D ligation, and WT 2D stenosis IVC segments and the IL-6-/- mice with and without thrombus (P > .05 for all). When treated with Ach, significantly more relaxation was found in the nonthrombosed control IVC segments than in those IVC segments that had had a 2D thrombus from either ligation- or stenosis-derived thrombotic mechanisms in both WT and IL-6-/- mice. CD31 staining showed ?20% less luminal endothelium after stasis thrombosis (P ? .01) but no loss in the controls (P > .05). Evans blue staining showed a trend toward increased leakiness in post-thrombotic vein walls. No significant difference in the endothelial gene markers or nitric oxide production was found.ConclusionsCompared with the controls, acute thrombosis in the total or partial stasis models did not impair IVC contractile responses, suggesting no effect on the medial vascular smooth muscle response. The relaxation response was significantly reduced in the post-thrombotic groups, likely from direct endothelial injury. These findings suggest, at acute points, that VT impairs the endothelial function of a vein wall while retaining the vascular smooth muscle cell function and might be a mechanism that promotes PTS.

Project description:BackgroundUpper extremity venous thrombosis (UEVT) represents about 10% of venous thrombo-embolic disease. This is mainly explained by the increasing use of central venous line, for oncologic or nutritional care. The factors associated with venous recanalization are not known.ObjectiveThe aim of this study was to investigate prognosis factor associated with venous recanalization after UEVT.MethodsThis study included patients with UEVT diagnosed with duplex ultra-sonography (DUS) from January 2015 to December 2017 with DUS evaluations during follow-up. A multivariate Cox proportional-hazards-model analysis was performed to identify predictive factors of UEVT complete recanalization.ResultsThis study included 494 UEVT, 304 proximal UEVT and 190 distal UEVT. The median age was 58 years, 39.5% were women. Clinical context was: hematological malignancy (40.7%), solid cancer (14.2%), infectious or inflammatory context (49.9%) and presence of venous catheters or pacemaker leads in 86.4%. The rate of recanalization without sequelae of UEVT was 38%. For all UEVT, in multivariate analysis, factors associated with complete vein recanalization were: thrombosis associated with central venous catheter (CVC) (HR:2.40, [1.45;3.95], p<0.001), UEVT limited to a venous segment (HR:1.94, [1.26;3.00], p = 0.003), occlusive thrombosis (HR:0.48 [0.34;0.67], p<0.0001), the presence of a PICC Line (HR:2.29, [1.48;3.52], p<0.001), a thrombosis of deep and distal topography (HR:1.70, [1.10;2.63], p = 0.02) or superficial thrombosis of the forearm (HR:2.79, [1.52;5.12], p<0.001). For deep and proximal UEVT, non-occlusive UEVT (HR:2.23, [1.49;3.33], p<0.0001), thrombosis associated with CVC (HR:1.58, [1.01;2.47], p = 0.04) and infectious or inflammatory context (HR:1.63, [1.10;2.41], p = 0.01) were factors associated with complete vein recanalization.ConclusionIn this study, factors associated with UEVT recanalization were UEVT limited to a venous segment, thrombosis associated with CVC, a thrombosis of deep and distal thrombosis topography and superficial thrombosis of the forearm. Occlusive thrombosis was associated with the absence of UEVT recanalization.

Dataset Information

Divergent effects of Tlr9 deletion in experimental late venous thrombosis resolution and vein wall injury.

Publications

Divergent effects of Tlr9 deletion in experimental late venous thrombosis resolution and vein wall injury.

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