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BackgroundPulmonary embolism (PE) is a life-threatening condition that is associated with the long-term sequelae of chronic pulmonary hypertension. Prior experimental work has suggested that post-PE inflammation is accompanied by pulmonary artery intimal hyperplasia. This study evaluated the effect of the thrombus and tested the hypothesis that thrombolytic, antiplatelet, and anticoagulant agents would decrease pulmonary injury.MethodsMale Sprague-Dawley rats (n = 267) underwent laparotomy and temporary clip occlusion of the infrarenal inferior vena cava for the formation of endogenous thrombus or placement of an inert silicone “thrombus.” Two days later, repeat laparotomy was performed, the clip removed, and the thrombus or silicone plug was embolized to the lungs. The endogenous thrombus group received normal saline, low-molecular-weight heparin (LMWH), tissue plasminogen activator (tPA), or a gIIB/IIIA antagonist (abciximab). Lung tissue was harvested at various times over 21 days and assayed for total collagen, monocyte chemoattractant protein-1 (MCP-1), interleukin-13 (IL-13), and transforming growth factor-β (TGF-β). Fixed sections were stained with trichrome for intimal hyperplasia determination and ED-1 monocytes and α-actin-positive staining.ResultsThe overall survival for rats undergoing PE was 90%, was not affected by treatment, and 84% of all PE localized to the right pulmonary artery. The PE significantly reduced Pao2 in all groups. Compared with controls, the silicone emboli group had an increased level of IL-13 on day 1, an increased level of MCP-1 on day 4, and an increase in the levels of all inflammatory mediators on day 14 (P < .05). Accompanying these differences were greater pulmonary artery intimal hyperplasia at days 4 and 21 in the silicone group compared with controls (P < .05). LMWH treatment in the thrombus of PE rats significantly decreased IL-13 levels at all time points, whereas treatment with abciximab or tPA significantly increased IL-13 levels compared with controls. TGF-β levels were significantly increased by LMWH at day 4 and 14, and abciximab was associated with lower TGF-β at day 14. Only LMWH was associated with less pulmonary artery intimal hyperplasia at day 14 compared with controls and the other treatment groups.ConclusionsPersistent pulmonary artery distention by an inert material is sufficient to invoke significant inflammation and intimal hyperplasia independent of the thrombus itself. Compared with nontreated PE, LMWH is the only therapy associated with a significant reduction in late intimal hyperplasia and, with the exception of TGF-β, lower profibrotic growth-factor production.Clinical RelevancePulmonary embolism is a highly fatal disease and may be associated with long-term pulmonary hypertension. Few good animal models exist for this condition, and the aim of this report was to evaluate the role of the thrombus itself on the pulmonary artery injury and to assess the effect of currently available therapies. Our data suggest that rapid treatment of pulmonary embolism with low-molecular-weight heparin is associated with least injury response, and that persistent occlusion of the pulmonary artery is associated with significant injury.

IntroductionDeep venous thrombosis (DVT) resolution involves fibrinolysis, neovascularization, and fibrosis. We hypothesized that promoting neovascularization would accelerate DVT resolution.MethodsA rat model of stasis DVT was produced with proximal ligation of the inferior vena cava (IVC) and all visible tributaries. One μg of interferon inducible protein (IP-10; angiostatic chemokine), basic fibroblast growth factor (bFGF; pro-angiogenic cytokine), epithelial neutrophil activating protein (ENA-78; pro-angiogenic chemokine), or saline solution control was injected into the IVC after ligation, and then via tail vein injection daily until sacrifice at either 4 or 8 days. Peripheral blood counts were measured, and thrombus weight was recorded at sacrifice. Laser Doppler in vivo imaging was used to estimate post-thrombotic IVC blood flow. Immunohistologic assessment of the thrombosed IVC for polymorphonuclear neutrophils (PMNs), monocytes (ED-1), and laminin (neovascular channels) was performed or the thrombus was separated from the IVC and assayed for keratinocyte cytokine (KC), monocyte chemotactic protein-1 (MCP-1), bFGF with enzyme-linked immunosorbent assay (ELISA), and total collagen with a direct colorimetric assay.ResultsPeripheral blood and intrathrombus PMNs and monocytes were not significantly different in the treated or control rats. There were no differences in any measure at 4 days. At 8 days, thrombus neovascularity, but not weight or collagen content, was increased in rats treated with bFGF or ENA-78 compared with control rats (17.6 ± 0.93, 16.2 ± 0.97 vs 13.2 ± 0.79; channels/5 high-power fields (hpf; n = 6-10; P < .05). Post DVT IVC blood flow was significantly increased in bFGF-treated rats but not in rats treated with IP-10 or ENA-78, as compared with control rats. Rats treated with ENA-78 had increased intrathrombus bFGF compared with control rats (85 ± 27 pg/mg protein vs 20 ± 6 pg/mg protein; n = 6; P < .05), but other mediators were not significantly different in treated rats compared with control rats.ConclusionPro-angiogenic compounds increase thrombus neovascularization, but this does not correlate with smaller or less fibrotic DVT. Mechanisms other than neovascularization may be more important to hasten DVT dissolution.AbstractClinical relevanceImproved therapy for deep venous thrombosis (DVT) will ideally increase the rate of thrombus dissolution and eliminate the bleeding risks of anticoagulation. This study evaluated promoting DVT neovascularization with angiogenic chemokines, and, while successful by experimental measures, this did not translate into smaller DVT. Solely promoting thrombus neovascularization will not likely speed resolution.

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 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 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.

ObjectivesNeutrophil influx is one of the first events in a formed deep venous thrombosis (DVT), but whether these cells are active participants in the resolution process is not clear. This study tests the hypothesis that neutrophils (PMN) are active participants in DVT resolution.MethodsThrombosis was induced by inferior vena caval (IVC) ligation in male Sprague-Dawley rats, and rats were sacrificed at 2, 4, or 7 days for evaluation of the thrombus. Neutropenia was induced by rabbit anti-rat PMN serum, and controls received rabbit serum. Venography was performed at the 7-day time point. Immunohistochemical staining was performed to quantify intrathrombus PMNs and monocytes, and the myeloperoxidase (MPO) assay was performed to assess intrathrombus neutrophil activity. Intrathrombus concentrations of kerotinocyte cytokine (KC), macrophage inflammatory protein-2 (MIP-2), γ interferon inducible protein-10 (IP-10), macrophage inflammatory protein-1 α (MIP-1α), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor (TNF)–α were quantified by enzyme immunoassay at each time point and normalized to total protein. Total collagen was determined at day 7.ResultsPeripheral blood smears showed a 94% PMN reduction at 2 days (P