Deep vein thrombosis (DVT) is a common disease that carries serious ramifications for patients, i... more Deep vein thrombosis (DVT) is a common disease that carries serious ramifications for patients, including pulmonary embolism and post-thrombotic syndrome (PTS). Although standard treatment for DVT is anticoagulation, this carries an added risk of bleeding and increased medication monitoring. Identifying those at risk for DVT and PTS can be difficult, and current research with murine models is helping to illuminate the biologic changes associated with these two disorders. Potential novel biomarkers for improving the diagnosis of DVT and PTS include ICAM-1, P-selectin, and cell-free DNA. Inhibition of factor XI, P- and E-selectin, and neutrophil extracellular traps holds promise for novel clinical treatment of DVT. Experimental research on PTS suggests potential cellular and mediator therapy targets of TLR9, MMP-2 and-9, PAI-1, and IL-6. Although many important concepts and mechanisms have been elucidated through research on DVT and PTS, more work must be done to translate experimenta...
CCR2 is required for monocyte recruitment in many inflammatory processes, as well as conferring T... more CCR2 is required for monocyte recruitment in many inflammatory processes, as well as conferring Th1 lymphokine responses. Deep vein thrombosis (DVT) resolution represents a specific inflammatory response whereby the thrombus must be dissolved for restoration of blood flow. Using a stasis model of DVT in the mouse, we investigated the role of CCR2 on DVT resolution. Genetic deletion of CCR2 (CCR2 ؊/؊ ) was associated with larger thrombi at early and later time points, increased thrombus collagen, fewer thrombus monocytes (F4/80), and significantly impaired neovascularization. IL-2 and IFN-␥ were significantly reduced in early CCR2 ؊/؊ thrombi, whereas MCP-1 was significantly increased, and Th2 lymphokines were unaffected. Supplementation of CCR2 ؊/؊ mice with IFN-␥ normalized early thrombus resolution without increasing monocyte influx. Neither Ab depletion of IFN-␥ nor genetic deletion of IFN-␥ impaired early DVT resolution. Early fibrinolysis was not impaired in CCR2 ؊/؊ mice, but a significant reduction in both matrix metalloproteinase (MMP)-2 and MMP-9 activity was observed. However, only MMP-9 activity was restored with administration of IFN-␥. We conclude that an early CCR2-dependent Th1 lymphokine response predominates in normal DVT resolution, mediates this in part by MMP-9 activation, but is not solely dependent on IFN-␥.
Vessel wall matrix changes occur after injury, although this has not been well studied in the ven... more Vessel wall matrix changes occur after injury, although this has not been well studied in the venous system. This study tested the hypothesis that the thrombus dictates the vein wall response and vein wall damage is directly related to the duration of thrombus contact. To determine the injury response over time, rats underwent inferior vena cava (IVC) ligation to produce a stasis thrombus, with harvest at various time points to 28 days (d). Significant vein wall matrix changes occurred with biomechanical injury (stiffness) peaking at 7-14 d, with concurrent early reduction in total collagen, an increase in early matrix metalloproteinase (MMP)-9 and late MMP-2, and concomitant increase in tumor necrosis factor (TNF)alpha, monocyte chemoattractant(MCP)-1 and tumor growth factor (TGF)beta (all P<0.05). To isolate the effect of the thrombus and its mechanism of genesis, rats underwent 7 d or limited stasis (24 hours), non-stasis thrombosis, or non-thrombotic IVC occlusion (Silicone plug). Vein wall stiffness was increased seven-fold, with a five-fold reduction in collagen, and 5.5- to seven-fold increase in TNFalpha, MCP-1, and TGFbeta with 7 d stasis as compared with controls (all P<0.05). By Picosirus red staining analysis, collagenolysis was significantly greater with 7 d stasis injury (P=0.01) but neither MMP-9 nor MMP-2 activity correlated with injury mechanism. In addition, vein wall cellular proliferation and uPA gene expression paralled the stasis thrombotic injury. Limited stasis, non-stasis thrombosis and non-thrombotic IVC occlusion showed a lesser inflammatory response. These data suggest both a static component and the thrombus directs vein wall injury via multiple mechanisms.
Early deep venous thrombosis (DVT) resolution is associated with neutrophil (PMN) influx. This st... more Early deep venous thrombosis (DVT) resolution is associated with neutrophil (PMN) influx. This study examined the role of PMNs in thrombus neovascularization and vein wall injury after DVT. A rat model of DVT by inferior vena cava (IVC) ligation was performed with control serum or rabbit anti-rat PMN serum administered perioperatively with sacrifice at 2 and 7 days. At 2 days, neutropenic rats had 1.6-fold larger thrombi (P = .04) and 1.4-fold higher femoral venous pressures by water manometry (P = .008) but no difference in thrombus neovascularization was observed. By 7 days, DVT sizes were similar, but vein wall injury persisted in the neutropenic rats with a 2.0-fold increase in vein wall stiffness by microtensiometry (P < .05), as well as a 1.2-fold increased thickness (P = .04). Collagen and profibrotic growth factors were significantly increased in neutropenic IVC at 7 days (all P < .05). Vein wall and intrathrombus uPA byWestern immunoblotting, and intrathrombus MMP-9 gelatinase activity were significantly less in neutropenic rats than controls (P < .001). Conversely, MMP-2 was significantly elevated in neutropenic IVC at 2 days after DVT. However, neutropenia induced 24 hours after DVT formation resulted in no significant increase in vein wall stiffness or collagen levels at 7 days, despite 1.4-fold larger thrombi (P < .05). These data suggest a critical early role for PMN in post DVT vein wall remodeling.
healthy ECs are forcibly detached by flow, or mechanical forces (shear stress and pressure) induc... more healthy ECs are forcibly detached by flow, or mechanical forces (shear stress and pressure) induce apoptosis leading to cell loss. Previously published work by others has identified that shear stress can "rescue" endothelial cells from apoptosis, utilizing a model in which apoptosis is first induced by serum starvation. We question the relevance of that model. We hypothesized that shear-induced apoptosis is a critical event leading to EC detachment and if apoptosis could be prevented, detachment from a biomaterial would be inhibited. Methods. Human Aortic ECs (passages 5-8) were established on Dacron membranes and subsequently were cultured in the presence or absence of Z-VAD-FMK (25 M), a pan-caspase inhibitor, in medium for 2 h. Membranes were placed in a parallel plate bioreactor and exposed to 0, 1, 10, and 30 dyn/cm 2 of shear stress for 24 h. Following flow, cells were stained with ethidium bromide and Hoeschst 33258. The ECs remaining on the membrane, percentage of cell retention on the membrane (normalized to no flow controls), and percentage of cells undergoing apoptosis by identification of nuclear morphology were determined by image analysis (n ϭ 7-13 per group). Results. Increasing shear stress in the absence of Z-VAD-FMK induced apoptosis and detachment as compared with the 1 dyn/cm 2 group. However, treatment with Z-VAD-FMK effectively inhibited apoptosis and resulted in significantly improved EC retention following exposure to high shear stress of 30 dyn/cm 2 . Conclusion. These data demonstrate that apoptosis induced by mechanical loading is a critical element responsible for flow-induced cell detachment from biomaterials, and can be modulated pharmacologically.
Objective: Although the treatment for acute deep vein thrombosis (DVT) is uniform, the circumstan... more Objective: Although the treatment for acute deep vein thrombosis (DVT) is uniform, the circumstances under which it develops vary widely and may impact outcomes. This study compared clinical features and outcomes in patients who developed a primary DVT associated with a defined risk to those without any proximate risk factor. Methods: Consecutive patients with a primary DVT and no past venous thromboembolism history from 2000 to 2002 were abstracted for demographics, risk factors, DVT anatomical characteristics, treatment, and outcomes of death and new pulmonary embolism. Comparison between patients with a proximate risk event within 30 days of DVT (Inpt) and those presenting with DVT with no defined proximate event (Outpt) was done by univariable and multivariable statistics. A validated survey was mailed to all living patients to assess long-term sequela. Results: A total of 293 patients with a mean age of 55 years and 49% men had confirmed DVT by objective means (92% duplex) with a mean follow-up of 25 ؎ 21 months. Inpts were more likely to have recent surgery or blunt trauma, bilateral DVT, less use of low molecular weight heparin (LMWH), and new pulmonary emboli (all P <.05). Outpts with DVT were more likely to have a history of malignancy, tibial-popliteal DVT compared with iliofemoral DVT, higher use of LMWH, and coumadin. However, there was no difference in mortality. From the patient survey (21% response),
Background. Deep venous thrombosis (DVT) confers vein wall damage associated with fibrosis and ex... more Background. Deep venous thrombosis (DVT) confers vein wall damage associated with fibrosis and extracellular matrix turnover, mediated by proteases. This study investigated the molecular expression of proteases and collagen involved in early vein wall remodeling. Methods. In the mouse, DVT was produced by ligation of the infrarenal inferior vena cava (IVC), or sham operation, and tissue harvested at 4, 8, and 12 days. The vein wall tissue was processed for real-time PCR (ratio of gene to b-actin), Western immunoblotting, and gelatin zymography. ANOVA was used for multiple comparisons and a P < 0.05 was significant. All N = 6–8. Results. Thrombus resolution was documented by a 1.6-fold decrease in the thrombosed IVC weight:length ratio over 12 days (P = 0.007). MMP-2 gene expression was 23-fold greater at 12 days as compared to sham or the 4-day time point (P < 0.05). Total MMP-2 activity by zymography was significantly elevated at 12 days as compared with sham (P = 0.047). MMP-7 expression peaked at 4 days (76-fold; P = 0.003) but approached baseline thereafter. MMP-9 expression was 19–27 higher at days 4 and 8, respectively, both relative to shams (P < 0.05), but no difference in activity was found. MMP-14 expression was 2- to 3.6-fold greater at day 12 compared to earlier time points and shams (P < 0.001), but no difference in protein levels was found. Plasminogen activator inhibitor was increased at 4 days relative to sham, but not after 8 days. Procollagen I and III increased over time and peaked at 12 days (24-fold, 6.1-fold, respectively, P < 0.02). Conclusions. Vein wall remodeling after DVT is associated with persistent procollagen up regulation and primarily MMP-2 expression and activity, whereas other proteases may be less important.
Methods: A rat model of stasis DVT was produced with proximal ligation of the inferior vena cava ... more Methods: A rat model of stasis DVT was produced with proximal ligation of the inferior vena cava (IVC) and all visible tributaries. One g 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. Results: Peripheral 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. Conclusion: Pro-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. (J Vasc Surg 2004;40:536-42.)
and SMCs (p Ͻ 0.05). However 6M R136K-CBD had equivalent activity to R136K on EC and SMCs (p Ͼ 0.... more and SMCs (p Ͻ 0.05). However 6M R136K-CBD had equivalent activity to R136K on EC and SMCs (p Ͼ 0.05).
(1 g) or vehicle control was administered, with sacrifice at 4 and 8 days. Prior to sacrifice and... more (1 g) or vehicle control was administered, with sacrifice at 4 and 8 days. Prior to sacrifice and at harvest, duplex ultrasound of the DVT and femoral venous pressure measurements were performed. Thrombi were analyzed by immunohistochemical techniques for PMN, monocytes, and neovascularization; for chemokines, by enzyme-linked immunoassay; and fibrosis, by hydroxyproline assay and trichrome staining.
Objectives: Neutrophil influx is one of the first events in a formed deep venous thrombosis (DVT)... more Objectives: Neutrophil 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. Methods: Thrombosis 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), ␥ interferon inducible protein-10 (IP-10), macrophage inflammatory protein-1 ␣ (MIP-1␣), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor (TNF)-␣ were quantified by enzyme immunoassay at each time point and normalized to total protein. Total collagen was determined at day 7. Results: Peripheral blood smears showed a 94% PMN reduction at 2 days (P <.05), recovering to 44% of control at 7 days. Intrathrombus PMNs were significantly lower in neutropenic rats at 2 and 4 days, but there were no differences in intrathrombus monocytes. The MPO assay confirmed reduced neutrophil activity at 4 days. Thrombi from neutropenic rats were larger at 2 and 7 days compared with controls. In vivo thrombus area at 7 days as assessed by venography was also greater in neutropenic rats as compared with controls. The intrathrombus KC concentration was increased more than 20-fold in the neutropenic rats at 2 days, but there were no significant differences in other intrathrombus chemokines. Finally, intrathrombus collagen was increased over threefold in neutropenic rats as compared with controls. Conclusion: Neutropenia impairs DVT resolution by several measures, most likely by altering normal fibrinolytic activity and thrombus collagen turnover. (J Vasc Surg 2003;38:1090-8.)
Deep vein thrombosis (DVT) is a common disease that carries serious ramifications for patients, i... more Deep vein thrombosis (DVT) is a common disease that carries serious ramifications for patients, including pulmonary embolism and post-thrombotic syndrome (PTS). Although standard treatment for DVT is anticoagulation, this carries an added risk of bleeding and increased medication monitoring. Identifying those at risk for DVT and PTS can be difficult, and current research with murine models is helping to illuminate the biologic changes associated with these two disorders. Potential novel biomarkers for improving the diagnosis of DVT and PTS include ICAM-1, P-selectin, and cell-free DNA. Inhibition of factor XI, P- and E-selectin, and neutrophil extracellular traps holds promise for novel clinical treatment of DVT. Experimental research on PTS suggests potential cellular and mediator therapy targets of TLR9, MMP-2 and-9, PAI-1, and IL-6. Although many important concepts and mechanisms have been elucidated through research on DVT and PTS, more work must be done to translate experimenta...
CCR2 is required for monocyte recruitment in many inflammatory processes, as well as conferring T... more CCR2 is required for monocyte recruitment in many inflammatory processes, as well as conferring Th1 lymphokine responses. Deep vein thrombosis (DVT) resolution represents a specific inflammatory response whereby the thrombus must be dissolved for restoration of blood flow. Using a stasis model of DVT in the mouse, we investigated the role of CCR2 on DVT resolution. Genetic deletion of CCR2 (CCR2 ؊/؊ ) was associated with larger thrombi at early and later time points, increased thrombus collagen, fewer thrombus monocytes (F4/80), and significantly impaired neovascularization. IL-2 and IFN-␥ were significantly reduced in early CCR2 ؊/؊ thrombi, whereas MCP-1 was significantly increased, and Th2 lymphokines were unaffected. Supplementation of CCR2 ؊/؊ mice with IFN-␥ normalized early thrombus resolution without increasing monocyte influx. Neither Ab depletion of IFN-␥ nor genetic deletion of IFN-␥ impaired early DVT resolution. Early fibrinolysis was not impaired in CCR2 ؊/؊ mice, but a significant reduction in both matrix metalloproteinase (MMP)-2 and MMP-9 activity was observed. However, only MMP-9 activity was restored with administration of IFN-␥. We conclude that an early CCR2-dependent Th1 lymphokine response predominates in normal DVT resolution, mediates this in part by MMP-9 activation, but is not solely dependent on IFN-␥.
Vessel wall matrix changes occur after injury, although this has not been well studied in the ven... more Vessel wall matrix changes occur after injury, although this has not been well studied in the venous system. This study tested the hypothesis that the thrombus dictates the vein wall response and vein wall damage is directly related to the duration of thrombus contact. To determine the injury response over time, rats underwent inferior vena cava (IVC) ligation to produce a stasis thrombus, with harvest at various time points to 28 days (d). Significant vein wall matrix changes occurred with biomechanical injury (stiffness) peaking at 7-14 d, with concurrent early reduction in total collagen, an increase in early matrix metalloproteinase (MMP)-9 and late MMP-2, and concomitant increase in tumor necrosis factor (TNF)alpha, monocyte chemoattractant(MCP)-1 and tumor growth factor (TGF)beta (all P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05). To isolate the effect of the thrombus and its mechanism of genesis, rats underwent 7 d or limited stasis (24 hours), non-stasis thrombosis, or non-thrombotic IVC occlusion (Silicone plug). Vein wall stiffness was increased seven-fold, with a five-fold reduction in collagen, and 5.5- to seven-fold increase in TNFalpha, MCP-1, and TGFbeta with 7 d stasis as compared with controls (all P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.05). By Picosirus red staining analysis, collagenolysis was significantly greater with 7 d stasis injury (P=0.01) but neither MMP-9 nor MMP-2 activity correlated with injury mechanism. In addition, vein wall cellular proliferation and uPA gene expression paralled the stasis thrombotic injury. Limited stasis, non-stasis thrombosis and non-thrombotic IVC occlusion showed a lesser inflammatory response. These data suggest both a static component and the thrombus directs vein wall injury via multiple mechanisms.
Early deep venous thrombosis (DVT) resolution is associated with neutrophil (PMN) influx. This st... more Early deep venous thrombosis (DVT) resolution is associated with neutrophil (PMN) influx. This study examined the role of PMNs in thrombus neovascularization and vein wall injury after DVT. A rat model of DVT by inferior vena cava (IVC) ligation was performed with control serum or rabbit anti-rat PMN serum administered perioperatively with sacrifice at 2 and 7 days. At 2 days, neutropenic rats had 1.6-fold larger thrombi (P = .04) and 1.4-fold higher femoral venous pressures by water manometry (P = .008) but no difference in thrombus neovascularization was observed. By 7 days, DVT sizes were similar, but vein wall injury persisted in the neutropenic rats with a 2.0-fold increase in vein wall stiffness by microtensiometry (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .05), as well as a 1.2-fold increased thickness (P = .04). Collagen and profibrotic growth factors were significantly increased in neutropenic IVC at 7 days (all P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .05). Vein wall and intrathrombus uPA byWestern immunoblotting, and intrathrombus MMP-9 gelatinase activity were significantly less in neutropenic rats than controls (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .001). Conversely, MMP-2 was significantly elevated in neutropenic IVC at 2 days after DVT. However, neutropenia induced 24 hours after DVT formation resulted in no significant increase in vein wall stiffness or collagen levels at 7 days, despite 1.4-fold larger thrombi (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .05). These data suggest a critical early role for PMN in post DVT vein wall remodeling.
healthy ECs are forcibly detached by flow, or mechanical forces (shear stress and pressure) induc... more healthy ECs are forcibly detached by flow, or mechanical forces (shear stress and pressure) induce apoptosis leading to cell loss. Previously published work by others has identified that shear stress can "rescue" endothelial cells from apoptosis, utilizing a model in which apoptosis is first induced by serum starvation. We question the relevance of that model. We hypothesized that shear-induced apoptosis is a critical event leading to EC detachment and if apoptosis could be prevented, detachment from a biomaterial would be inhibited. Methods. Human Aortic ECs (passages 5-8) were established on Dacron membranes and subsequently were cultured in the presence or absence of Z-VAD-FMK (25 M), a pan-caspase inhibitor, in medium for 2 h. Membranes were placed in a parallel plate bioreactor and exposed to 0, 1, 10, and 30 dyn/cm 2 of shear stress for 24 h. Following flow, cells were stained with ethidium bromide and Hoeschst 33258. The ECs remaining on the membrane, percentage of cell retention on the membrane (normalized to no flow controls), and percentage of cells undergoing apoptosis by identification of nuclear morphology were determined by image analysis (n ϭ 7-13 per group). Results. Increasing shear stress in the absence of Z-VAD-FMK induced apoptosis and detachment as compared with the 1 dyn/cm 2 group. However, treatment with Z-VAD-FMK effectively inhibited apoptosis and resulted in significantly improved EC retention following exposure to high shear stress of 30 dyn/cm 2 . Conclusion. These data demonstrate that apoptosis induced by mechanical loading is a critical element responsible for flow-induced cell detachment from biomaterials, and can be modulated pharmacologically.
Objective: Although the treatment for acute deep vein thrombosis (DVT) is uniform, the circumstan... more Objective: Although the treatment for acute deep vein thrombosis (DVT) is uniform, the circumstances under which it develops vary widely and may impact outcomes. This study compared clinical features and outcomes in patients who developed a primary DVT associated with a defined risk to those without any proximate risk factor. Methods: Consecutive patients with a primary DVT and no past venous thromboembolism history from 2000 to 2002 were abstracted for demographics, risk factors, DVT anatomical characteristics, treatment, and outcomes of death and new pulmonary embolism. Comparison between patients with a proximate risk event within 30 days of DVT (Inpt) and those presenting with DVT with no defined proximate event (Outpt) was done by univariable and multivariable statistics. A validated survey was mailed to all living patients to assess long-term sequela. Results: A total of 293 patients with a mean age of 55 years and 49% men had confirmed DVT by objective means (92% duplex) with a mean follow-up of 25 ؎ 21 months. Inpts were more likely to have recent surgery or blunt trauma, bilateral DVT, less use of low molecular weight heparin (LMWH), and new pulmonary emboli (all P <.05). Outpts with DVT were more likely to have a history of malignancy, tibial-popliteal DVT compared with iliofemoral DVT, higher use of LMWH, and coumadin. However, there was no difference in mortality. From the patient survey (21% response),
Background. Deep venous thrombosis (DVT) confers vein wall damage associated with fibrosis and ex... more Background. Deep venous thrombosis (DVT) confers vein wall damage associated with fibrosis and extracellular matrix turnover, mediated by proteases. This study investigated the molecular expression of proteases and collagen involved in early vein wall remodeling. Methods. In the mouse, DVT was produced by ligation of the infrarenal inferior vena cava (IVC), or sham operation, and tissue harvested at 4, 8, and 12 days. The vein wall tissue was processed for real-time PCR (ratio of gene to b-actin), Western immunoblotting, and gelatin zymography. ANOVA was used for multiple comparisons and a P < 0.05 was significant. All N = 6–8. Results. Thrombus resolution was documented by a 1.6-fold decrease in the thrombosed IVC weight:length ratio over 12 days (P = 0.007). MMP-2 gene expression was 23-fold greater at 12 days as compared to sham or the 4-day time point (P < 0.05). Total MMP-2 activity by zymography was significantly elevated at 12 days as compared with sham (P = 0.047). MMP-7 expression peaked at 4 days (76-fold; P = 0.003) but approached baseline thereafter. MMP-9 expression was 19–27 higher at days 4 and 8, respectively, both relative to shams (P < 0.05), but no difference in activity was found. MMP-14 expression was 2- to 3.6-fold greater at day 12 compared to earlier time points and shams (P < 0.001), but no difference in protein levels was found. Plasminogen activator inhibitor was increased at 4 days relative to sham, but not after 8 days. Procollagen I and III increased over time and peaked at 12 days (24-fold, 6.1-fold, respectively, P < 0.02). Conclusions. Vein wall remodeling after DVT is associated with persistent procollagen up regulation and primarily MMP-2 expression and activity, whereas other proteases may be less important.
Methods: A rat model of stasis DVT was produced with proximal ligation of the inferior vena cava ... more Methods: A rat model of stasis DVT was produced with proximal ligation of the inferior vena cava (IVC) and all visible tributaries. One g 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. Results: Peripheral 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. Conclusion: Pro-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. (J Vasc Surg 2004;40:536-42.)
and SMCs (p Ͻ 0.05). However 6M R136K-CBD had equivalent activity to R136K on EC and SMCs (p Ͼ 0.... more and SMCs (p Ͻ 0.05). However 6M R136K-CBD had equivalent activity to R136K on EC and SMCs (p Ͼ 0.05).
(1 g) or vehicle control was administered, with sacrifice at 4 and 8 days. Prior to sacrifice and... more (1 g) or vehicle control was administered, with sacrifice at 4 and 8 days. Prior to sacrifice and at harvest, duplex ultrasound of the DVT and femoral venous pressure measurements were performed. Thrombi were analyzed by immunohistochemical techniques for PMN, monocytes, and neovascularization; for chemokines, by enzyme-linked immunoassay; and fibrosis, by hydroxyproline assay and trichrome staining.
Objectives: Neutrophil influx is one of the first events in a formed deep venous thrombosis (DVT)... more Objectives: Neutrophil 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. Methods: Thrombosis 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), ␥ interferon inducible protein-10 (IP-10), macrophage inflammatory protein-1 ␣ (MIP-1␣), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor (TNF)-␣ were quantified by enzyme immunoassay at each time point and normalized to total protein. Total collagen was determined at day 7. Results: Peripheral blood smears showed a 94% PMN reduction at 2 days (P <.05), recovering to 44% of control at 7 days. Intrathrombus PMNs were significantly lower in neutropenic rats at 2 and 4 days, but there were no differences in intrathrombus monocytes. The MPO assay confirmed reduced neutrophil activity at 4 days. Thrombi from neutropenic rats were larger at 2 and 7 days compared with controls. In vivo thrombus area at 7 days as assessed by venography was also greater in neutropenic rats as compared with controls. The intrathrombus KC concentration was increased more than 20-fold in the neutropenic rats at 2 days, but there were no significant differences in other intrathrombus chemokines. Finally, intrathrombus collagen was increased over threefold in neutropenic rats as compared with controls. Conclusion: Neutropenia impairs DVT resolution by several measures, most likely by altering normal fibrinolytic activity and thrombus collagen turnover. (J Vasc Surg 2003;38:1090-8.)
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