Venous thrombosis resolution is a complex process involving interplay between the interstitial matrix and cells. A critical enzyme for venous thrombus (VT) stability and compaction is factor XIII (FXIII). Prior experimental work has shown that global deletion of FXIII in mice resulted in a marked reduction of VT size and incidence. However, it is unknown how this deletion may affect VT resolution. Littermate FXIII+/+, FXIII–/+, and FXIII–/– mice on a C57BL/6 background were used. Mice were subjected to either a complete inferior vena cava ligation stasis or an inferior vena cava stenosis to create VT. Tissues and thrombi were harvested at 8 days. Tissues were evaluated by immunohistochemistry, trichrome analyses, weight-to-length data, and immunoblotting. Whereas FXIII+/+ and FXIII+/– mice had similar VT size 8 days after thrombus induction, FXIII-/- mice had significantly smaller VT than FXIII+/+ mice (10.3 mg/mm vs 18.0 mg/mm; n = 9-18; P = .001). The thrombus architecture was markedly different in the FXIII–/– stasis mice, with little interstitial matrix (Figure). Immunoblotting showed fibrin (0.15 vs 0.44, ratio to B-actin; n = 6; P = .004), laminin (0.0 vs 0.21; n = 6; P = .002), and fibronectin (0.0 vs 1.25; n = 6; P = .004), and CitH3 (a NETs marker) (0.02 vs 4.7; n = 6; P < .001) were significantly reduced compared with controls. Intra-thrombus proinflammatory monocyte (CCR2+) counts were reduced by 54% (n = 4-8; P = .02), but no difference in neutrophils in FXIII–/– mice was observed as compared with controls. FXIII+/– mice using both VT models showed no significant differences in VT size as compared with controls. Analyses of cellular and matrix content following VT induction indicate FXIII is a critical factor for VT resolution, likely through both monocyte and matrix interactions. Whether inhibition of FXIII will accelerate fibrinolysis and enhance thrombus resolution warrants further study. Legend: Inferior vena cava histological sections of 8 days thrombus in wild-type and FXIII–/– mice at 100×. Note less matrix architecture in the FXIII–/– as compared with wild type.

Objective: Venous 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. Methods: Wild-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. Results: Optimal 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 similar to 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. Conclusions: Compared 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.

Introduction: Post-thrombotic syndrome is a chronic disabling sequelae of deep vein thrombosis (VT), for which no direct therapies beyond anticoagulation exist. Prior work has shown that monocyte/macrophages (Mo/Mø) are important for mid-term venous thrombosis resolution, in particular the pro-inflammatory Ly6C hi cells and Ly6C lo pro-healing Mo/Mø. We hypothesized that Mo/ Mø depletion would lessen vein wall fibrosis. Methods: We used CD11b-DTR mice to conditionally deplete Mo/Mø in the stasis venous thrombosis (VT) model (IVC ligation), two days after the VT was created. DTx was dosed every 48 hours to deplete >90% of circulating Mo. CCR2 -/- mice devoid of circulating Ly6C hi Mo, and Nr4a1 -/- mice devoid of circulating Ly6C lo Mo, were used to dissect the roles of the Ly6C hi pro-inflammatory and Ly6C lo pro-healing Mo/Mø, respectively. Tissue harvest at 14d assessed thrombus size, histology (trichrome, DDR2+ fibroblasts and CD68+ Mø) and immunologic assays were performed. All N > 4. Results: Conditional global Mo/Mø depletion was associated with a 37% decrease (p<.001) in vein wall thickness at 14 days, with a significant decrease in vein wall DDR2 + cells, and FSP-1, TGFb, VEGF1, and IL-6 levels. Interestingly, no difference in VT size was found. Nr4a1 -/- mice (no Ly6C lo Mo) had no difference in VT size at day 14, but exhibited a 35% increase in vein wall thickness (p<0.001) and increased DDR2 + cells (P < .001). Conversely, day 14 CCR2 -/- mice (no Ly6C hi Mo) had 40% smaller VT (p=0.007), but no change in vein wall thickness. Both Nr4a1 -/- and CCR2 -/- mice had an 85% decrease in CD68+ Mø in the vein wall at 14d (p<0.001). Conclusion: Mo/Mø drive post thrombotic vein wall fibrosis, possibly through fibroblast-mediated mechanisms. Deletion of Nr4a1, lacking Ly6C lo Mo, exaggerated vein wall fibrosis but genetic deletion of CCR2 (Ly6C hi ) cells does not significantly lessen vein wall fibrosis, suggesting that Ly6C hi are likely precursors for later Ly6C lo conversion. Further study will elucidate this lineage change as well as identify targets to enhance Ly6C lo Mo/Mø in vein wall post-thrombotic fibrosis.

Venous thrombosis (VT) damages the vein wall, both physically with prolonged distention and from inflammation. These factors underlie post-thrombotic syndrome (PTS). Interleukin 6 (IL-6) may play a role in experimental PTS and vein wall responses. Prior assessment of post-thrombotic vein wall injury has used static measures such as histology and immunologic assays. The purpose of this study was to use myography to quantify the changes in contraction and relaxation of murine vessels exposed to an acute thrombus.

Summary Venous thromboembolism is a major cause of death during and immediately post-sepsis. Venous thrombosis (VT) is mediated by cell adhesion molecules and leukocytes, including neutrophil extracellular traps (NETs). Sepsis, or experimentally, endotoxaemia, shares similar characteristics and is modulated via toll like receptor 4 (TLR4). This study was undertaken to determine if endotoxaemia potentiates early stasis thrombogenesis, and secondarily to determine the role of VT TLR4, ICAM-1 and neutrophils (PMNs). Wild-type (WT), ICAM-1-/- and TLR4-/- mice underwent treatment with saline or LPS (10 mg/kg i.p.) alone, or followed by inferior vena cava (IVC) ligation to generate stasis VT. In vivo microscopy of leukocyte trafficking was performed in non-thrombosed mice, and tissue and plasma were harvested during early VT formation. Pre-thrombosis, circulating ICAM-1 was elevated and increased leukocyte adhesion and rolling occurred on the IVC of LPS-treated mice. Post-thrombosis, endotoxaemic mice formed larger, platelet-poor thrombi. Endotoxaemic TLR4-/- mice did not have an augmented thrombotic response and exhibited significantly decreased circulating ICAM-1 compared to endotoxaemic WT controls. Endotoxaemic ICAM-1-/- mice had significantly smaller thrombi compared to controls. Hypothesising that PMNs localised to the inflamed endothelium were promoting thrombosis, PMN depletion using anti-Ly6G antibody was performed. Paradoxically, VT formed without PMNs was amplified, potentially related to endotoxaemia induced elevation of PAI-1 and circulating FXIII, and decreased uPA. Endotoxaemia enhanced early VT occurs in a TLR-4 and ICAM-1 dependent fashion, and is potentiated by neutropenia. ICAM-1 and/or TLR-4 inhibition may be a unique strategy to prevent sepsis-associated VT. Supplementary Material to this article is available online at www.thrombosis-online.com.

Venous thrombosis (VT) results in vein wall injury by promoting inflammation and fibrosis leading to venous reflux, swelling, pain, and potentially, recurrent thrombosis. While prior work has shown that infiltrating leukocytes are important for VT resolution, as of yet, the precise roles of different leukocyte subsets are not well understood. Monocyte/macrophages (Mo/MΦs) are essential for the repair and resolution of tissue injury in other models, and come in inflammatory (Ly6C Hi ) or pro-resolution (Ly6C Lo ) subtypes. We hypothesized that infiltrating Mo/MΦs would be critical to VT resolution. In order to study this in vivo , we utilized a conditional macrophage depletion technique, using CD11b-DTR mice, to examine the effects of Mo/MΦs in a murine model of stasis VT by inferior vena cava ligation. Administration of 10ng/g diphtheria toxoid (DTx) every 48 hours by intra-peritoneal injection in CD11b-DTR mice resulted in an 89% and 55% decrease in circulating monocytes at 24hrs and 48hrs, respectively. When compared to saline controls, DTx injection had no effects on thrombogenic response or IVC thrombus cell populations in C57BL/6 control mice. At 8 days’ post-ligation, DTx treated CD11b-DTR mice had preferentially decreased vein wall-thrombus Ly6C Lo Mo/MΦs as compared with controls. DTx treated mice had significantly larger thrombi (1.7-fold) and less TGF-β, FSP-1, and plasminogen by western immunoblotting (all P-values ≤ 0.01). Consistent with a reduction in Ly6C Lo Mo/MΦs was a significant decrease in cellular TGF-β by intra-cellular flow cytometry. These findings suggest that Ly6C Lo Mo/MΦs are essential for normal VT resolution and may promote thrombus resolution via a plasminogen-mediated mechanism.

Objective— Deep vein thrombosis (VT) can result in vein wall injury, which clinically manifests as post-thrombotic syndrome. Postinjury fibrosis may be modulated in part through cellular cysteine-cysteine receptor 7 (CCR7)–mediated events. We tested the hypothesis that late vein wall fibrotic remodeling is dependent on CCR7. Approach and Results— CCR7 −/− and C57BL/6 wild-type mice had inferior vena cava VT induced by nonstasis or stasis mechanisms. In both models, VT size was largest at day 1 and trended down by day 21, and CCR7 + cells peaked at day 8 in wild-type mice. No significant differences in VT resolution were found in CCR7 −/− as compared with wild type in either model. In the nonstasis VT model, vein wall changes consistent with fibrotic injury were evidenced by significant increases in collagen I, III, matrix metalloproteinase 2, and transforming growth factor-β gene expression, increases in α-smooth muscle actin and fibroblast specific protein-1 antigen, and total collagen at 8 days. Correspondingly, SM22α and fibroblast specific protein-1, but not DDR2 + cells, were increased at 8 days. Early wild-type thrombus exposure inhibited profibrotic gene expression in CCR7 −/− in ex vivo vein wall culture. Bone marrow chimera experiments further showed that circulating CCR7 + leukocytes partially rescued midterm profibrotic changes in CCR7 −/− mice. In human histological sections of chronic thrombosed femoral veins, CCR7 + cells were present in the fibrotic areas. Conclusions— Post-thrombotic vein wall remodeling is impaired in CCR7 −/− mice, with a profibrotic phenotype, is dependent on the thrombotic mechanism, and is mediated by circulating CCR7 + cells. Unlike other postinjury fibrotic responses, CCR7 + signaling may be important for positive vein wall remodeling after VT.

OBJECTIVE:Deep vein thrombosis (DVT) resolution instigates an inflammatory response, resulting in vessel wall damage and scarring. Urokinase-plasminogen activator (uPA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), are integral components of the fibrinolytic system, essential for venous thrombosis (VT) resolution. This study determined the vein wall response when exposed to increased and decreased plasmin activity. METHODS:A mouse inferior vena cava (IVC) ligation model in uPA -/- or PAI-1 -/- and their genetic wild types (B6/SvEv and C57/BL6, respectively) was used to create stasis thrombi, with tissue harvest at either 8 or 21 days. Tissue analysis included gene expression of vascular smooth muscle cells (alpha smooth muscle actin [αSMA], SM22) and endothelial marker (CD31), by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, matrix metalloproteinase (MMP)-2 and -9 activity by zymography, and vein wall collagen by picro-Sirius red histologic analysis. A P < .05 was considered significant. RESULTS:Thrombi were significantly larger in both 8-day and 21-day uPA -/- as compared with wild type (WT) and were significantly smaller in both 8-day and 21-day PAI-1 -/- as compared with WT. Correspondingly, 8-day plasmin levels were reduced in half in uPA -/- and increased three-fold in PAI-1 -/- when compared with respective WT thrombi (P < .05; n = 5-6). The endothelial marker CD31 was elevated two-fold in PAI-1 -/- mice at 8 days, but reduced 2.5-fold at 21 days in uPA -/- as compared with WT (P = .02; n = 5-6), suggesting less endothelial preservation. Vein wall vascular smooth muscle cell (VSMC) gene expression showed that 8-day and 21-day PAI-1 -/- mice had 2.3- and 3.8-fold more SM22 and 1.8- and 2.3-fold more αSMA expression than respective WT (P < .05; n = 5-7), as well as 1.8-fold increased αSMA (+) cells (P ≤ .05; n = 3-5). No significant difference in MMP-2 or -9 activity was found in the PAI-1 -/- mice compared with WT, while 5.4-fold more MMP-9 was present in 21-day WT than 21-day uPA -/- (P = .03; n = 5). Lastly, collagen was ∼two-fold greater at 8 days in PAI-1 -/- IVC as compared with WT (P = .03; n = 6) with no differences observed in uPA -/- mice. CONCLUSIONS:In stasis DVT, plasmin activity is critical for thrombus resolution. Divergent vein wall responses occur with gain or loss of plasmin activity, and despite smaller VT, greater vein wall fibrosis was associated with lack of PAI-1.

BACKGROUND:Venous thrombus resolution sets up an early intense inflammatory reaction, from which vein wall damage results. Tissue response to injury includes matrix metalloproteinase (MMP) activation and extracellular matrix protein turnover. This study sought to determine the effect of exogenous MMP inhibition and its potential attenuation of early vein wall injury. METHODS:Rats received treatment beginning 24 hr after a stasis venous thrombosis by near occlusive ligation and until harvest at day 7. Three groups were evaluated: (1) vehicle saline controls (NaCl), (2) low molecular weight heparin (LMWH; Lovenox, 3 mg/kg daily SQ), and (3) doxycycline (DOXY, 30 mg/kg daily PO). Thrombus size (mg/mm), levels of tumor necrosis factor alpha (TNF alpha) and D-dimer by colorimetric assay, and monocytes counts by immunohistochemistry were assessed. Vein wall assessment included stiffness by tensiometry, interleukin 1beta (IL-1 beta protein levels by enzyme-linked immunosorbent assay, MMP2 and -9 by zymography, and histological analysis of intimal thickness (IT). Comparisons were by t-test to control. p < 0.05 was considered significant. RESULTS:Thrombus sizes were similar at days 2 and 7 for all three groups, while thrombus TNFalpha was increased in 2-day LMWH- and DOXY-treated groups (NaCl = 1.0 +/- 0.8, LWMH = 9 +/- 3, DOXY = 27 +/- 5 pg/mg protein, n = 6-8, p < 0.05) and at 7 days in the DOXY group (NaCl = 3.0 +/- 2.5, DOXY = 23 +/- 4.2 pg/mg protein, n = 5, p < 0.05). Vein wall stiffness at 7 days was less with LMWH treatment, but not with DOXY, compared to controls (NaCl = 0.33 +/- 0.05, LMWH = 0.17 +/- 0.03, DOXY = 0.43 +/- 0.09 N/mm, n = 5-7, p < 0.05). Vessel-wall IL-1 beta was reduced only in the DOXY group at 7 days (NaCl = 26 +/- 3, LMWH = 38 +/- 17, DOXY = 6 +/- 3 pg/mg protein, n = 4-6, p < 0.05), as was the IT score versus controls (NaCl = 2.2 +/- 0.6, LMWH =1.7 +/- 0.3, DOXY = 0.8 +/- 0.20, n = 4-6, p < 0.05). Zymographic MMP9 activity was significantly reduced at 2 days in the LMWH and DOXY groups (NaCl = 85 +/- 24, LMWH = 23 +/- 7( *), DOXY = 13 +/- 5 U/mg protein, n = 6-8, p < 0.05). MMP2 zymographic activity, thrombus monocyte cell counts, and D-dimer activity were not significantly different across groups. CONCLUSION:Treatment with LMWH or DOXY did not alter the size of deep vein thrombosis, mildly altered thrombus composition, and differentially affected vein wall injury, despite similar reductions in early MMP9 activity. Whether exogenous MMP inhibition affects long-term vein wall fibrosis will require further study.

La résolution du thrombus veineux est à la source d’une réaction inflammatoire intense précoce, provoquant les lésions de la paroi veineuse. La réponse des tissus aux lésions inclut l’activation de métalloprotéinases de la matrice (MMP) et la régénération des protéines de la matrice extracellulaire. Cette étude a cherché à déterminer l’effet de l’inhibition exogène des MMP et de son atténuation potentielle des lésions pariétales veineuses précoces.

Objective: Vein wall endothelial turnover after stasis deep vein thrombosis (DVT) has not been well characterized. The purpose of this study was to quantify re-endothelialization after DVT and determine if low-molecular-weight heparin (LMWH) therapy affects this process.Methods: Stasis DVT was generated in the rat by inferior vena cava ligation, with harvest at 1, 4, and 14 days. Immunohistologic quantification of vascular smooth muscle cells and luminal endothelialization was estimated by positive staining for a-smooth muscle actin and von Willebrand factor, respectively. In separate experiments, rats were treated either before or after DVT with subcutaneous LMWH (3 mg/kg daily) until harvesting at 4 and 14 days. The inferior vena cava was processed for histologic analysis or was processed for organ culture after the thrombus was gently removed. The vein wall was stimulated in vitro with interleukin-I beta (1 ng/mL), and the supernatant was processed at 48 hours for nitric oxide. Cells were processed by real-time polymerase chain reaction for endothelial nitric oxide synthase, inducible nitric oxide synthase, cyclooxygenase-1 and -2, and thrombomodulin at 4 and 14 days, and collagen I and III at 14 days. Comparisons were done with analysis of variance or t test. A P < .05 was significant.Results: Thrombus size peaked at 4 days, whereas luminal re-endothelialization increased over time (1 day, 11% +/- 2%; 4 days, 23% +/- 4%; 14 days, 64% +/- 7% (+) von Willebrand factor staining; P < .01, n = 3 to 4, compared with non-DVT control). Similarly, vascular smooth muscle cell staining was lowest at day I and gradually returned to baseline by 14 days. Both before and after DVT, LMWH significantly increased luminal re-endothelialization, without a difference in thrombus size at 4 days, but no significant difference was noted at 14 days despite smaller thrombi with LMWH treatment. Pretreatment with LMWH was associated with increased vascular smooth muscle cell area and recovery of certain inducible endothelial specific genes. No significant difference in nitric oxide levels in the supernatant was found at 4 days. At 14 days, type III collagen was significantly elevated with LMWH treatment.Conclusions: Venous re-endothelialization occurs progressively as the DVT resolves and can be accelerated with LMWH treatment, although this effect appears limited to the early time frame. These findings may have clinical relevance for LMWH timing and treatment compared with mechanical forms of therapy.

Sood, Vikram BS; Mitsuya, M. BS; Erin, Miller MS; Luke, Cathy LVT; Upchurch, Gilbert MD, FACS; Wakefield, Thomas MD, FACS; Henke, Peter MD, FACS Author Information