Cathy Luke

Urokinase plasminogen activator independent early experimental thrombus resolution: MMP2 as an alternative mechanism

Deep-vein thrombosis (DVT) resolution is thought to be primarily a urokinase plasminogen activator (uPA) -dependent mechanism, although observations suggest other non-fibrinolytic mechanisms may exist. We explored the role of matrix metalloproteinase (MMP) -2 and -9 in early DVT resolution in uPA-deficient mice. Male B6/SVEV (WT) and genetically matched uPA -/- mice underwent inferior vena cava (IVC) ligation to create stasis venous thrombi, with IVC and thrombus harvest. Thrombus size was similar between WT and uPA -/- mice at day 4, suggesting early non uPA-dependent resolution. Intrathrombus neutrophils and monocytes were reduced 3- and 3.5-fold in uPA -/- mice as compared with WT. By ELISA, tumour necrosis factor α and interleukin 1β were not altered, while interferon (IFN)γ was significantly elevated in uPA -/- mice. A compensatory increase in thrombus tPA was not observed, plasmin activity was reduced and PAI-1 was elevated 2.5-fold in uPA -/- mice. Active MMP2, but not MMP9, was elevated 3-fold in uPA -/- mice as compared with WT as well as MMP-14, an MMP2 activator. Collagen type IV and fibrinogen were reduced in uPA -/- mice thrombi as compared with WT. IFNγ induces MMP2, and blockade of IFNγ was associated with larger venous thrombi and reduced active MMP2, as compared with WT. Consistently, MMP2 -/- mice had larger VT as compared with WT controls, despite normal thrombus plasmin levels. Taken together, early experimental venous thrombus resolution is independent of uPA, and, in part, inflammatory cell influx. MMP2-dependent thrombolysis is an important compensatory mechanism of venous thrombus resolution, possibly by collagen type IV metabolism, and may represent an exploitable therapeutic avenue.

The role of urokinase plasminogen activator and plasmin activator inhibitor-1 on vein wall remodeling in experimental deep vein thrombosis

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.

Vein wall re-endothelialization after deep vein thrombosis is improved with low-molecular-weight heparin.

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 alpha-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-1beta (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 1 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. CLINICAL RELEVANCE How the vein wall endothelium responds after deep vein thrombosis (DVT) has not been well documented owing to limited human specimens. This report shows that low-molecular-weight heparin accelerates or protects the endothelium and preserves medial smooth muscle cell integrity after DVT, but that this effect is limited to a relatively early time period. Although most DVT prophylaxis is pharmacologic (a heparin agent), use of nonpharmacologic measures is also common. The use of heparin prophylaxis, compared with after DVT treatment, and the acceleration of post-DVT re-endothelialization require clinical correlation.

Vein wall remodeling after deep vein thrombosis: differential effects of low molecular weight heparin and doxycycline.

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.

Deletion of Cysteine-Cysteine Receptor 7 Promotes Fibrotic Injury in Experimental Post-Thrombotic Vein Wall Remodeling

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-&bgr; gene expression, increases in &agr;-smooth muscle actin and fibroblast specific protein-1 antigen, and total collagen at 8 days. Correspondingly, SM22&agr; 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.

Endotoxaemia-augmented murine venous thrombosis is dependent on TLR-4 and ICAM-1, and potentiated by neutropenia

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.

Human Chronic Post-Thrombotic Intraluminal Venous Obstruction Involves Neovascularization

or venography. When findings are “normal,” the physician is left with no explanation for recurrent venous hypertension. The purpose of this study is to show the value of looking inside the stent, with intravenous ultrasound (IVUS), to identify treatable hemodynamically significant lesions that cause symptomatic venous hypertension. Methods: Patients underwent standard measurements of limbs, duplex of lower extremities and iliac veins with attention to waveform analysis and velocities. Symptomatic patients were imaged from a jugular or popliteal approach with multi-plane venography and IVUS, to evaluate the stents and veins. In the event of in-stent stenosis, measurements of cross-sectional area were obtained before and after intervention. Treatment included angioplasty, atherectomy, or strategic placement of a new stent. Procedures were performed on an outpatient basis. No change of therapeutic international normalized ratio for patients on warfarin. A postprocedure duplex was performed and compared to the preoperative study. Results: Between 2002 and 2013, we evaluated 37 patients (17 M, 20 F) with 47 limbs (17 R, 30 L; 9 bilateral + 13 inferior vena cava) with previously placed iliocaval venous stents. Each person presented with new onset pain and/or edema that had been absent after initial stenting. The onset of recurrent venous hypertension ranged between 6 months and 10 years. Twenty-seven of 47 limbs had occluded stents, while 20 of 47 had one or more stenoses. The mean age was 41.4 years (range, 21-70 years). Lumen-compromising stenosis was identified within stents, with IVUS, in 90% of symptomatic patients, all of whom had “normal” findings on duplex. Tissue specimens were obtained in three cases, and evaluation showed fibrosis, not thrombus. Balloon dilatation effectively increased luminal area. Recalcitrant lesions (4) were successfully enlarged with balloonexpandable nitinol stents. Sustained patency of the revisions has been excellent. Mean follow-up was 38 months. All patients reported clinical improvement consistent with decreased venous pressure. Conclusions: Patients with failing venous stents present with recurrent limb edema or pain, heaviness, tightness, etc. Hyperplasia can narrow the diameter up to 30% and not be appreciated with standard duplex or venography. Restenosis typically occurs, circumferentially, where stents overlap and at the leading edges. IVUS is an important tool for investigating stent integrity in symptomatic patients. Timely endovenous evaluation and treatment of failing stents is clinically indicated to preserve patency.