Vein Wall Injury Research Articles

Evaluating the impact of external forces on peripheral intravenous catheter movement using ultrasound: A randomized pilot study.

A major contributor to peripheral intravenous catheter (PIVC) failure may be related to PIVC movement within the vein which is associated with vein wall damage. The magnitude of PIVC movement against the vein wall has not previously been quantified. This study aimed to examine PIVC movement within the vein when minor forces were applied to the PIVC. This was a prospective, pilot trial including healthy volunteers in an outpatient research laboratory. The primary objective was to examine the in movement (millimeters) of the PIVC using ultrasound with external pull forces (4, 5, and 6 lbs; 1.8, 2.3, and 2.7 kg, respectively) applied to the PIVC in random order. Participants (N = 11) were aged 40.36 ± 16.10 years with 54.55% being Male. Mean ± SD PIVC movement for 4, 5, and 6 lbs of force was 4.65 ± 1.88, 3.88 ± 2.28, and 5.25 ± 2.06 mm, respectively. There was substantial PIVC movement when a force was applied to the PIVC, but no statistically significant difference between 4, 5, and 6 lb forces (p > 0.05). When external pull forces were applied to the PIVC, substantial PIVC movement within the vein occurred in a healthy population. Strategies that reduce PIVC movement and/or remove or limit external pull forces from the PIVC are needed. Future studies on hospitalized patients are warranted to quantify vein wall injury and PIVC failure due to PIVC movement from various pull forces.

Research progress on prevention and treatment of deep vein thrombosis in burn patients

Deep vein thrombosis (DVT) and pulmonary embolism are common and serious complications in hospitalized patients. DVT may be induced by factors including the vein wall injury, hypercoagulability, and slow blood flow during the pathophysiological changes and treatment process of burn patients, especially severe burn patients. It is of great significance to understand the occurrence of DVT in burn patients, identify the high risk group of DVT formation in burn patients with effective evaluation methods, and carry out safe and effective prevention and treatment. This article briefly reviews research progress on the monitoring, prevention, and treatment of DVT in burn patients.

A histological study of vessel wall reaction after endovenous ultrasound ablation in an animal model

Introduction. Along with the generally accepted thermal methods of saphenous varicose veins ablation, the use of non-thermal and combined methods becomes attractive. Due to the variety of mechanisms of action, low-frequency ultrasound is a promising technology for performing a wide range of minimally invasive intravascular operations, including the treatment of varicose veins. Aim. To elucidate the mechanism of action of a novel endovenous ultrasound ablation method and evaluate its effect on vein wall injury and lumen occlusion in an animal model. Materials and methods. A total of 34 central ear vein (CEV) medial ramus were treated in 17 rabbits according to the follow-up protocol. Six femoral veins from three rabbits enrolled into the acute experiment were harvested 45 min after the procedure. All treated CEV medial ramus in the follow-up experiment were divided into four treatment groups with an observation time of: (0) 30 days, control procedure; (1) 60 days, ultrasound ablation; (2) 95 days, ultrasound ablation; (3) 138 days, ultrasound ablation. Results. Occlusion was achieved in all but one CEV medial ramus (29/30; 97%) treated with ultrasound. Histologic examination of treated veins showed heat- and mechanical-induced changes in the vein wall, such as abrasion of the endothelium and fibrotic lesions in the tunica media and tunica adventitia. The occluded segments consisted mainly of cellular fibrotic material. In the acute group, histological evidence of damage to all vessel wall layers was found. Discussion. Ultrasound ablation results in homogeneous and uniform damage to the venous wall. The results of this study suggest that the ultrasound treatment leads to venous occlusion by the additive thermomechanical mechanism of damage to the vessel wall. Conclusions. Endovenous ultrasound ablation showed a similar pattern of thermal damage as endovenous laser (EVLA) and radiofrequency ablation (RFA) and damage to the endothelium as mechanochemical ablation (MOCA). Four months after ultrasound ablation, the occlusion rate was 90%.

Abstract 243: Local Periadventitial Delivery Of Dexamethasone Improves Murine Deep Vein Thrombosis Resolution

Background: Post deep vein thrombosis (DVT), up to 30-50% of patients will develop post-thrombotic syndrome (PTS), despite anticoagulation. Currently no preventive therapies, including catheter-based thrombectomy/thrombolysis (CDT), have shown to reduce PTS. Local steroid therapy adjunct to CDT is being tested clinically for DVT patients in the DEXTERITY trials, but minimal mechanistic data exists on DVT resolution. Here, we investigate effects of local dexamethasone (DEX) therapy on murine DVT resolution. Method: C57/BL6 male mice (n=146) underwent inferior vena cava (IVC) complete ligation to develop stasis DVT on day 0 (D0) followed by mechanical de-ligation on D2 to spur restoration of blood flow (RBF). On D2, mice were injected with PBS/DEX (0.1, 0.2, or 0.5 mg/mouse, 60μl volume, across four zones) in the periadventitial tissue of thrombosed IVC, with or without de-ligation (Fig. A-B). Mice underwent D4&D8 serial ultrasound imaging of IVC blood flow to assess RBF-defined as IVC mean flow velocity > 0 mL/sec. D8 thrombus burden was measured followed by histological/picrosirius red assessment of vein wall fibrosis. Results: N=6-11 mice per group underwent IVC DVT creation. Low-dose (0.1 mg) but not higher doses of DEX reduced thrombus burden (p=0.02) and weight (p=0.01) on D8 (Fig. C-D). Low-dose DEX increased D4 RBF rates and mean flow velocity (MFV) (Fig. E-F). Complete IVC stasis without de-ligation but with DEX non-significantly reduced thrombus burden and weight (Fig. G-H). Low-dose DEX further decreased vein wall fibrosis (Fig. I; p=0.001). D8 thrombi exhibited reduced macrophage content with DEX (Fig. J; n=15-18 sections/group, p=0.007). Conclusion: Following stasis DVT induction, locally delivered low-dose DEX improves DVT resolution by reducing thrombus burden, vein wall injury, macrophage content, and increases RBF rate. These data provide a scientific foundation for ongoing DEXTERITY trail testing adjunct DEX for DVT patients undergoing CDT.

Endovenous radiofrequency ablation using a new bipolar electrode in a canine model: a new endovenous radiofrequency electrode.

This study aimed to determine the effectiveness and safety of a newly developed endovenous radiofrequency (RF) catheter compared with that of the existing RF catheter in a canine model. Seven dogs underwent ablation using 1 control catheter (ClosureFAST, CF; Covidien) and 1 experimental catheter (VENISTAR, VS; STARmed Co., Ltd.) in the femoral and cephalic veins. The ablated vein was evaluated macroscopically (2,3,5-triphenyltetrazolium chloride staining, TTC), microscopically (hematoxylin and eosin staining), and ultrasonographically. Vessel injury score was used to evaluate the ablating effect objectively. Veins from 1 dog were evaluated on the day of ablation, while in the remaining 6 dogs, the ablated veins were evaluated 2 weeks later. A total of 23 veins (CF, 11 veins; VS, 12 veins) were ablated in 7 dogs. Non-TTC-stained vein wall areas were identified in all ablated veins. No significant difference was observed in the mean vessel injury score (2.54 ± 1.16 vs. 2.42 ± 1.13, P = 0.656) and the mean vessel wall thickness (0.32 ± 0.03 mm vs. 0.31 ± 0.05 mm, P = 0.212) between CF and VS. There was no blood flow in all veins ablated with VS, whereas there was remaining blood flow in 1 vein ablated with CF. Perivenous complication was not observed. Endovenous RF ablation using a newly developed VS RF catheter seems to provide comparable occlusion rate and degree of vein wall injury without perivenous adverse events compared to the most commonly used RF catheter (CF).

Modulation of interleukin-6 and its effect on late vein wall injury in a stasis mouse model of deep vein thrombosis

ObjectiveDeep vein thrombosis (DVT) and its sequela, post-thrombotic syndrome (PTS), remain a clinically significant problem. Interleukin-6 (IL-6) is a proinflammatory cytokine that is elevated in patients who develop PTS. We hypothesized that genetic deletion of IL-6 and the use of anti–IL-6 pharmacologic agents would be associated with decreased late vein wall injury. MethodsWild-type C57BL/6J (WT) and IL-6−/− mice underwent induction of stasis venous thrombosis by ligation of the infrarenal IVC. Vein wall inferior vena cava and thrombus were harvested at 21 days after ligation and analyzed by Western blot and immunohistochemistry of the vein wall using monocyte markers CCR2 and arginase 1, the endothelial marker CD31, and fibroblast markers DDR2 and FSP-1. Two anti–IL-6 pharmacologic agents (gp130 [glycoprotein 130] and tocilizumab) were tested and compared with low-molecular-weight heparin (LMWH) as the reference standard in WT mice. Plasma was collected at 4 and 48 hours to confirm the pharmacologic agents’ effects. ResultsLess fibrosis but no increase in luminal endothelialization was found in IL-6−/− mice compared with WT mice at 21 days. The IL-6−/− mice had fewer DDR2- and arginase 1-positive cells in the vein wall compared with the WT mice. However, no difference was found in the CCR2+ cells. Despite documented in vivo activity, exogenous gp130 and tocilizumab were not associated with decreased vein wall fibrosis or increased endothelial luminal coverage at 21 days. LMWH therapy, both before and after treatment, was not associated with decreased vein wall fibrosis at 21 days. ConclusionsIL-6 genetic deletion was associated with less fibrotic vein wall injury at a late time point, consistent with the PTS timeframe. However, neither the standard of care LMWH nor two available anti–IL-6 agents showed antifibrotic biologic effects in this model.

Time-Restricted Salutary Effects of Blood Flow Restoration on Venous Thrombosis and Vein Wall Injury in Mouse and Human Subjects.

Up to 50% of patients with proximal deep vein thrombosis (DVT) will develop the postthrombotic syndrome characterized by limb swelling and discomfort, hyperpigmentation, skin ulcers, and impaired quality of life. Although catheter-based interventions enabling the restoration of blood flow (RBF) have demonstrated little benefit on postthrombotic syndrome, the impact on the acuity of the thrombus and mechanisms underlying this finding remain obscure. In experimental and clinical studies, we examined whether RBF has a restricted time window for improving DVT resolution. First, experimental stasis DVT was generated in C57/BL6 mice (n=291) by inferior vena cava ligation. To promote RBF, mice underwent mechanical deligation with or without intravenous recombinant tissue plasminogen activator administered 2 days after deligation. RBF was assessed over time by ultrasonography and intravital microscopy. Resected thrombosed inferior vena cava specimens underwent thrombus and vein wall histological and gene expression assays. Next, in a clinical study, we conducted a post hoc analysis of the ATTRACT (Acute Venous Thrombosis: Thrombus Removal with Adjunctive Catheter-Directed Thrombolysis) pharmacomechanical catheter-directed thrombolysis (PCDT) trial (NCT00790335) to assess the effects of PCDT on Venous Insufficiency Epidemiological and Economic Study quality-of-life and Villalta scores for specific symptom-onset-to-randomization timeframes. Mice that developed RBF by day 4, but not later, exhibited reduced day 8 thrombus burden parameters and reduced day 8 vein wall fibrosis and inflammation, compared with controls. In mice without RBF, recombinant tissue plasminogen activator administered at day 4, but not later, reduced day 8 thrombus burden and vein wall fibrosis. It is notable that, in mice already exhibiting RBF by day 4, recombinant tissue plasminogen activator administration did not further reduce thrombus burden or vein wall fibrosis. In the ATTRACT trial, patients receiving PCDT in an intermediate symptom-onset-to-randomization timeframe of 4 to 8 days demonstrated maximal benefits in Venous Insufficiency Epidemiological and Economic Study quality-of-life and Villalta scores (between-group difference=8.41 and 1.68, respectively, P<0.001 versus patients not receiving PCDT). PCDT did not improve postthrombotic syndrome scores for patients having a symptom-onset-to-randomization time of <4 days or >8 days. Taken together, these data illustrate that, within a restricted therapeutic window, RBF improves DVT resolution, and PCDT may improve clinical outcomes. Further studies are warranted to examine the value of time-restricted RBF strategies to reduce postthrombotic syndrome in patients with DVT.

Shallow-angle needle guide for ultrasound-guided internal jugular venous catheterization: A randomized controlled crossover simulation study (CONSORT).

Needle guides for ultrasound-guided internal jugular venous catheterization facilitate successful cannulation. The ability of a needle guide to prevent a posterior vein wall injury which may secondarily induce lethal complications, is unknown. Previous studies showed that a shallow angle of approach may reduce the incidence of posterior wall injuries. We developed a novel needle guide with a shallow angle of approach for ultrasound-guided venous catheterization and examined whether this needle guide reduces the incidence of posterior wall injuries compared to a conventional needle guide and free-hand placement in a simulated vein. This study was a randomized crossover-controlled trial. The primary outcome was the rate of posterior vein wall injuries. Participants had a didactic lecture about three ultrasound-guided techniques using the short-axis out-of-plane approach, including free-hand (P-free), a commercial needle guide (P-com), and a novel needle guide (P-sha). The view inside a simulated vein was recorded during venipuncture. Thirty-five residents participated in this study. Posterior vein wall injuries occurred in 66% using P-free, 60% using P-com, and 0% using P-sha (p< 0.01). There was no significant difference in the incidence of posterior vein wall injuries between P-free and P-com. Use of a shallow angle of approach needle guide resulted in a lower rate of posterior vein injuries during venipuncture of a simulated vein compared with other techniques using a steeper angle techniques.

Acute experimental venous thrombosis impairs venous relaxation but not contraction

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.

Early Results of Duplex-Guided Transradial Artery Fistuloplasties

Although arteriovenous fistulae (AVFs) are the preferred mode of hemodialysis access because of their high patency rates, they are associated with an appreciable rate of nonmaturation. Balloon-assisted maturation (BAM) has been described to treat this issue. BAM is defined as repeated sequential graduated dilatation of the outflow vein. This study aims to evaluate the short-term complications of using the radial artery as an access for BAM procedures and fisutloplasties. Transradial access was used preferentially with multiple lesions in the AVF that were difficult to access with a single venous puncture. Data were collected over 3years on 44 office-based duplex-guided transradial access BAM procedures in 27 patients of whom 19 were men. BAM with ultrasound guidance was performed in 324 cases using a venous puncture during this period. The indication for the procedures was a failure of AVF maturation, and 5 cases were with short segment thrombectomy. All procedures were performed with local anesthesia only. Access site puncture, vessel cannulation, wire placement, and balloon advancement and insufflation were duplex-guided. The radial artery was punctured with ultrasound guidance and a 4-5 French low-profile sheath was placed. After crossing the lesion(s), 5,000 units of heparin was given. The radial artery was used as the access vessel for all procedures except one, in which the brachial artery was used in addition. Vascular injuries were classified based on the postprocedural duplex assessment. All patients had follow-up duplex scans within a week. The average age was 79years (±14 SD, range 39-99years). The types of AVF were 35 radio-cephalic, 1 radio-basilic, 2 brachio-brachial, 2 brachio-cephalic, and 4 brachio-basilic. The number of sites of lesions was 17 on the venous outflow, 7 perianastomotic, and 6 in the radial artery. In the remaining 14 failing AVFs, we were not able to identify any lesion. The balloon size ranged from 3-6mm (28 patients) and 7-12mm (16 patients). The most common injury was outflow vein wall injury (25), the formation of wall hematoma of the outflow vein (11), localized extravasation or rupture at the balloon site (4), spasm of the AVF (3), the formation of a puncture-site hematoma (2), and intimal flap (3). Extravasation was controlled with duplex-guided compression. There were no radial artery thromboses, and all the AVFs were patent on completion duplex and follow-up duplex. These data suggest that the radial artery could be used as a safe access route for BAM procedures with relatively low rates of complication. This approach can be considered as an adjunct in the armamentarium for angioplasty of AVF.

Abstract 354: Early Intentional Restoration of Blood Flow Reduces Thrombus Burden and Vein Wall Scarring Following Dvt: Implications for Preventing the Post-thrombotic Syndrome

Background: Despite anticoagulation therapy, up to 50% of deep vein thrombosis (DVT) patients still develop the post-thrombotic syndrome (PTS), a condition that arises from thrombus obstruction and vein wall damage, leading to venous hypertension. While catheter-based intervention may reduce PTS, the CaVenT and ATTRACT trials demonstrated little clinical benefit of intentional restoration of blood flow (RBF) for reducing PTS. However, these trials have not explored the time-dependence of RBF, a key factor as aging VT are associated with greater vein wall injury. Methods: To investigate the temporal effects of intentional RBF following VT, we modified a classic complete ligation inferior vena cava (IVC) stasis VT protocol in C57BL/6 mice by de-ligating the day 0 ligature at day 2 to allow RBF. Compared to intravital microscopy, the sensitivity and specificity of noninvasive ultrasound (US) to detect RBF was 82.1% and 94.4%, respectively. Serial US was then performed on mice. Mice were classified as early RBF (RBF by day 4), late RBF (RBF after day 4), no RBF, or sham de-ligation groups. Follow day 8 sacrifice, thrombus burden (mg/cm) and vein wall scarring (VWS) were measured. Kruskal-Wallis followed by Dunn’s test was used to compare statistical significance between groups. Results: After de-ligation, RBF through VT increased gradually over time (Fig. A). The early RBF group exhibited significant reductions in VT burden (N=9) and VWS (N=5) at day 8 ( p &lt;0.05 vs. comparator groups, Fig B-C). In contrast, the late RBF group did not experience significant reductions in thrombus burden (N=6) nor VWS (N=4), with measures similar to mice with no RBF (N=3-4), or sham de-ligation (N=8-9). Conclusions: Intentional early RBF reduces venous thrombus burden and VWS, factors that drive PTS. Mechanistic studies to address these findings are going. The overall results suggest that anti-PTS benefits of catheter-based intervention may be enhanced in subjects receiving early RBF.

Impaired Inferior Vena Cava Relaxation but not Contraction in Two Models of Murine Acute Deep Venous Thrombosis

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. Wild-type (WT) C57BL/6 mice were used to determine baseline vein wall passive tension on a DMT 610M myograph (DMT, Ann Arbor, Mich), including dosing concentrations of phenylephrine (Phe) and acetylcholine (Ach). WT and IL-6 knockout mice underwent VT using inferior vena cava (IVC) ligation (complete stasis) and stenosis (partial stasis), and no-surgery mice were used as controls. Mice were harvested at 2 days (2D) and analyzed using a myograph. Vessels were stimulated with Phe and Ach to stimulate a contraction and relaxation response. Immunohistochemistry for CD31 was used to identify endothelial cells on WT no-surgery and WT 2D ligation vessels. Optimal passive tension was determined to be 2 mN, with the optimal concentrations of Phe and Ach being 7E-3M and 1E-5M, respectively. There was no significant difference in contraction on exposure to Phe between the WT no-thrombus mice, WT 2D ligation, and WT 2D stenosis mice and the IL-6 knockout mice with or without thrombus (all P > .05). When mice were treated with Ach, there was significantly more relaxation in the nonthrombosed control IVC segments than in those mice IVC segments that had a 2D thrombus of either ligation- or stenosis-derived mechanisms (Fig). The CD31 staining confirmed the presence of endothelium in the WT 2D IVC thrombus mice, ruling out tissue lost during harvesting. Compared with controls, acute thrombosis with total or partial stasis did not impair IVC contractile responses, suggesting no effect on medial vascular smooth muscle response. The relaxation response, however, was significantly reduced in the post-thrombotic groups. These findings suggest that at acute time points, VT impairs the endothelial function of a vein wall while retaining the vascular smooth muscle cell function and may be a mechanism that promotes PTS.

Abstract 32: Ly6C Lo Monocyte/Macrophages Are Essential for Thrombus Resolution in a Murine Model of Venous Thrombosis

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.

Abstract 530: In vivo DVT Inflammation Presages Vein Wall Injury, and is Ameliorated by Statin Therapy

Objective: Inflammation mediates early venous thrombosis (VT) resolution and can induce vein wall scarring (VWS), a key driver of the morbid post-thrombotic syndrome (PTS). Statins exhibit anti-inflammatory properties, and may positively impact VWS after VT. However, whether early inflammation contributes to this process and can be detected is not known. In this study, we hypothesized that early VT inflammation detected by 18F-fludeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) could predict subsequent VWS and that both VT inflammation and VWS would be attenuated by statin therapy. Methods: Stasis VT was induced by complete ligation in male C57BL/6J mice (n=55) in either the infrarenal inferior vena cava (IVC, n=42) or right jugular vein (n=13). IVC VT mice were randomized to statin or control groups. Statin (rosuvastatin 5mg/kg) was given by oral gavage starting one day prior to VT induction; control mice received PBS. All mice underwent survival FDG-PET/CT venography imaging on day 2. FDG-PET inflammation signals (standard uptake value (SUV), SUVmax, target-to-background ratios (TBR)) were measured. Picrosirius red staining of day 14 VT sections measured vein wall collagen/thickness. Ex vivo VT tissue gamma counting of a subgroup was performed at day 2. Whole-thrombus protein/mRNA and VT tissue sections assessed neutrophil content. Results: FDG-PET/CT at day 2 revealed increased FDG uptake in jugular VT over the contralateral sham surgery vein (p&lt;0.001). Statin-treated mice showed a decrease in FDG-PET SUV, SUVmax and TBR (p&lt;0.05 for all). Whole-thrombus analyses and tissue section immunostaining showed reduced thrombus neutrophil content at day 2, without reducing GLUT1 or MPO expression (p&gt;0.05). At day 14, statin therapy significantly reduced VWS (p=0.02). In mice undergoing survival imaging, the day 2 FDG-PET VT inflammation signal correlated significantly with the magnitude of day 14 VWS (IVC VT r=0.74, p&lt;0.001) and jugular models of VT (r=0.62, p=0.02). Conclusions: Quantitative FDG inflammation imaging demonstrates that early VT inflammation presages subsequent VWS, and is ameliorated by prophylactic statin therapy. The overall findings support the concept that statins and could reduce VWS and PTS.

Macroscopic and Histologic Analysis of Vessel Wall Reaction After Mechanochemical Endovenous Ablation Using the ClariVein OC Device in an Animal Model

Mechanochemical endovenous ablation (MOCA) has been developed as a tumescentless technique to ablate saphenous veins and to avoid heat induced complications and post-procedural pain. The mechanism of action of MOCA is poorly understood. The present experiments were conducted to determine the effect of MOCA on vein wall injury and sclerosis in an animal model. A total of 36 lateral saphenous veins (LSVs) were treated in 18 goats according to the human protocol. Veins from nine goats were evaluated 45min after the procedure, while in the remaining nine, the treated veins were evaluated 6 weeks later. All treated veins were divided equally over three treatment groups: (i) MOCA, (ii) mechanical ablation without the sclerosant, and (iii) liquid sclerotherapy alone. The histological effects of treatment on the vein wall were systematically evaluated. The average diameter of the LSV was 4.0±0.5mm. Technical success was achieved in all but one LSV (35/36; 97%), with a median procedure time of 14min (range 9-22min). In the acute group, histological examination showed that mechanical ablation (alone or MOCA) induced severe injury to the endothelium in 82% but no damage to other layers of the vein wall. Mechanical ablation led to vasoconstriction. After 6 weeks follow-up, four of six MOCA treated veins were occluded. The occluded segments consisted mainly of fibrotic lesions probably evolved from organised thrombus. No occlusions were observed after sclerotherapy or mechanical treatment alone. No major complications occurred during procedures or follow-up. MOCA is associated with an increased occlusion rate compared with its separated components of mechanical ablation or sclerotherapy. The occlusion consists of cellular fibrotic material likely to be evolved from organised thrombus with fibrotic alterations to the surrounding media and adventitia. This study underlines the hypothesis that the additive use of MOCA increases the effectiveness of sclerosants alone by inducing endothelial damage and probably vasoconstriction.

Abstract 35: DVT Inflammation Assessed by 18F-FDG-PET/CT Predicts Subsequent Vein Wall Scarring

Objective: While venous thrombosis (VT)-induced inflammation facilitates thrombus resolution, inflammation causes vein wall scarring (VWS). Recently, statins have shown to improve VT resolution and reduce VT inflammatory components. In this study, we hypothesized that early VT inflammation detected by 18F-FDG positron emission tomography/computed tomography (PET/CT) could predict subsequent late stage VWS, and would be attenuated by statin therapy. Methods: Stasis VT was induced in 8-12 week old male C57BL/6 mice (n=31) in either the right jugular vein (n=13) or inferior vena cava (IVC,n=18). Animals in the IVC VT cohort were randomized to statin (n=8) or control (n=10) treatment. Statin, rosuvastatin (5mg/kg), was administered by oral gavage, daily starting 24 hours prior to VT induction; control mice received saline. All mice underwent survival FDG-PET/CT venography imaging on day 2. FDG inflammation signals (standard uptake value=SUV) were measured in the thrombosed vein and compared to the sham-operated venous segments or treatment control. On day 14, mice were sacrificed and VT tissue was resected. Picrosirius red staining allowed measurement of collagen and vein wall thickness in VT sections. Results: FDG-PET/CT at day 2 revealed increased inflammation signal activity in jugular VT (SUV 1.43 ± 0.3 VT vs. 0.81 ± 0.3 contralateral vein, p&lt;0.0001). Statin-treated mice showed a trend of decreased inflammation signal at day 2 in the IVC VT models (SUV 1.02 ± 0.1 statin VT vs. 1.42 ± 0.2 control VT, p=0.07). Day 14 histological analysis revealed significantly reduced vein wall injury in statin-treated animals (thickness, 32±9.4 μm statin; vs. 56.2±14.7 μm control, p=0.02). Day 2 FDG-PET inflammation in VT correlated positively with the magnitude of day 14 VWS (jugular VT, Spearman r=0.62, p=0.02; IVC VT r=0.74, p&lt;0.001, respectively). Conclusions: Quantitative FDG-PET/CT imaging demonstrates that early in vivo VT inflammation predicts subsequent VWS, a driver of post-thrombotic syndrome (PTS). The overall findings strengthen: (i) the link between inflammation and PTS; (ii) the translational potential of FDG-PET inflammation to predict VWS and PTS; and (iii) the concept that statins and other anti-inflammatory therapies could reduce VWS and PTS.

Statins improve the resolution of established murine venous thrombosis: reductions in thrombus burden and vein wall scarring.

Despite anticoagulation therapy, up to one-half of patients with deep vein thrombosis (DVT) will develop the post-thrombotic syndrome (PTS). Improving the long-term outcome of DVT patients at risk for PTS will therefore require new approaches. Here we investigate the effects of statins—lipid-lowering agents with anti-thrombotic and anti-inflammatory properties—in decreasing thrombus burden and decreasing vein wall injury, mediators of PTS, in established murine stasis and non-stasis chemical-induced venous thrombosis (N = 282 mice). Treatment of mice with daily atorvastatin or rosuvastatin significantly reduced stasis venous thrombus burden by 25% without affecting lipid levels, blood coagulation parameters, or blood cell counts. Statin-driven reductions in VT burden (thrombus mass for stasis thrombi, intravital microscopy thrombus area for non-stasis thrombi) compared similarly to the therapeutic anticoagulant effects of low molecular weight heparin. Blood from statin-treated mice showed significant reductions in platelet aggregation and clot stability. Statins additionally reduced thrombus plasminogen activator inhibitor-1 (PAI-1), tissue factor, neutrophils, myeloperoxidase, neutrophil extracellular traps (NETs), and macrophages, and these effects were most notable in the earlier timepoints after DVT formation. In addition, statins reduced DVT-induced vein wall scarring by 50% durably up to day 21 in stasis VT, as shown by polarized light microscopy of picrosirius red-stained vein wall collagen. The overall results demonstrate that statins improve VT resolution via profibrinolytic, anticoagulant, antiplatelet, and anti-vein wall scarring effects. Statins may therefore offer a new pharmacotherapeutic approach to improve DVT resolution and to reduce the post-thrombotic syndrome, particularly in subjects who are ineligible for anticoagulation therapy.

Altered elastase-alpha1-antitrypsin balance in the blood of patients with chronic venous disease.

Although leukocyte elastase is suspected to be involved in the damage of vein wall during chronic venous disease, the equilibrium between this protease and its inhibitor, alpha1-antitrypsin, has not yet been evaluated. The aim of the present study was to determine the relationship between leukocyte elastase and alpha1-antitrypsin, in the blood of patients with chronic venous disease. The concentration and the activity of leukocyte elastase along with the activity of alpha1-antitrypsin were evaluated in the blood of 55 chronic venous disease patients. The results were compared with those obtained in 33 healthy age and sex-matched volunteers. A significant decrease in the leukocyte elastase activity that correlated with an increased alpha1-antitrypsin activity was observed in the serum of patients with mild clinical symptoms of chronic venous disease. The results of the study did not confirm a hypothesis about an important role of proteolytic activity of leukocyte elastase in the vein wall injury mechanism. They show that the leukocyte elastase-alpha1-antitrypsin balance is rather shifted toward antiprotease activity, especially in an early stage of chronic venous disease.

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

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.

Neutrophils in Deep Venous Thrombosis

Neutrophils are involved in venous thrombogenesis, through P-selectin and neutrophil endothelial traps (NETS). In order to determine the importance of neutrophils to thrombogenesis, neutrophil depletion was performed in our rat model of stasis-induced deep venous thrombosis (DVT). Animals were treated with control serum or rabbit anti-rat PMN serum administered perioperatively and sacrificed at two and seven days. At two days, neutropenic rats had 1.6-fold larger thrombi (P = 0.04) and 1.4-fold higher femoral venous pressures by manometry (P = 0.008) without a difference in thrombus neovascularization. By seven days, DVT sizes were similar, but vein wall injury persisted in neutropenic rats with a two-fold increase in vein wall stiffness by microtensiometry (P < 0.05), as well as a 1.2-fold increased thickness (P = 0.04). Vein wall and intrathrombus uPA by Western immunoblotting, as well as intrathrombus MMP-9 gelatinase activity was significantly less in neutropenic rats than controls (P < 0.001). Conversely, MMP-2 was significantly elevated in neutropenic inferior vena cava (IVC) at two days after DVT.1 P-selectin inhibition has been found to limit venous thrombosis in mice. Animals with high circulating levels of sP-selectin (^CT) were compared to selectin gene-deleted animals (PKO, EPKO) and wild-type (WT) mice. ^CT mice showed a significant 50% increase in thrombosis in our IVC ligation model while EPKO mice had the smallest thrombi. A significant difference was noted between ^CT and EPKO for neutrophils, monocytes, and total inflammatory cells at day two. Microparticle (MP) analysis revealed that in the ^CT, WT and PKO mice, a mixed leukocyte (MAC-1) and platelet (CD41) MP population was present. EPKO mice (with the smallest thrombi) revealed primarily a platelet-derived MP population, suggesting the importance of leukocyte-derived MPs in venous thrombogenesis. Of interest, the ^CT mice with the highest TM showed an elevated level of mean channel fluorescence for MAC-1 antibody, indicative of leukocyte derived MPs.2 Through processes that also involve the initial activation of leukocytes and platelets, neutrophils initiate and amplify thrombosis through the formation of NETS, which are extracellular fragments of DNA containing histones and antimicrobial proteins.3,4In vitro and in vivo, NETs provide a scaffold and stimulus for thrombus formation.5 In order to investigate if plasma DNA (surrogate for NETS) is elevated in patients with DVT and to determine correlations with other biomarkers of DVT, we studied patients presenting to our diagnostic vascular laboratory. From December 2008 to August 2010, patients were divided into three distinct groups: (1) DVT positive, patients symptomatic for DVT confirmed by ultrasound (n = 47); (2) DVT negative, patients with leg pain but negative by ultrasound (n = 28); and (3) control healthy non-pregnant volunteers without signs or symptoms of active or previous DVT (n = 19). Blood was collected for biomarkers and the Wells score risk of DVT was assessed. Results showed that circulating DNA was significantly elevated in DVT patients, compared with both DVT-negative patients (57.7 vs. 17.9 ng/mL; P < 0.01) and controls (57.7 vs. 23.9 ng/mL; P < 0.01). There was a strong positive correlation with C-reactive protein (P < 0.01), D-dimer (P < 0.01), VWF (P < 0.01), Wells score (P < 0.01) and MPO (P < 0.01), along with a strong negative correlation with ADAMTS13 (P < 0.01) and the ADAMTS13/VWF ratio. The logistic regression model showed a strong association between plasma DNA and the presence of DVT (ROC curve 0.814) suggesting a role for DNA in venous thrombogenesis.6