Journal of Vascular Surgery
Volume 44, Issue 3 , Pages 625-632, September 2006

Treatment with an oral small molecule inhibitor of P selectin (PSI-697) decreases vein wall injury in a rat stenosis model of venous thrombosis

Presented at the Eighteenth Annual Meeting of the American Venous Forum, Miami, Fla, Feb 24, 2006.

  • Daniel D. Myers Jr, DVM, MPH

      Affiliations

    • Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan Medical Center, Ann Arbor, Mich
    • Unit for Laboratory Animal Medicine, University of Michigan Medical Center, Ann Arbor, Mich
    • Corresponding Author InformationReprint requests: Daniel D. Myers, Jr, DVM, MPH, 1150 W Medical Center Dr, Dock 6, MSRB II A570D, Ann Arbor, MI 48109-0654.
    • ,
  • Peter K. Henke, MD

      Affiliations

    • Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan Medical Center, Ann Arbor, Mich
    • ,
  • Patricia W. Bedard, BS

      Affiliations

    • Wyeth Research, Cambridge, Mass
    • ,
  • Shirley K. Wrobleski, BS

      Affiliations

    • Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan Medical Center, Ann Arbor, Mich
    • ,
  • Neelu Kaila, PhD

      Affiliations

    • Wyeth Research, Cambridge, Mass
    • ,
  • Gray Shaw, PhD

      Affiliations

    • Wyeth Research, Cambridge, Mass
    • ,
  • Thomas R.. Meier, DVM

      Affiliations

    • Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan Medical Center, Ann Arbor, Mich
    • ,
  • Angela E. Hawley, MS

      Affiliations

    • Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan Medical Center, Ann Arbor, Mich
    • ,
  • Robert G. Schaub, PhD

      Affiliations

    • Wyeth Research, Cambridge, Mass
    • ,
  • Thomas W. Wakefield, MD

      Affiliations

    • Jobst Vascular Research Laboratories, Section of Vascular Surgery, University of Michigan Medical Center, Ann Arbor, Mich

Received 24 February 2006; accepted 11 May 2006.

Article Outline

Background

Vein wall injury after thrombosis is multifactorial but seems dependent on thrombus and local thrombotic and inflammatory mechanisms. We hypothesized that inhibition of vein wall injury through reduction of thrombotic and inflammatory events with P-selectin inhibition and/or low-molecular-weight heparin (LMWH) occurs independently of thrombus resolution in a rat model of venous thrombosis.

Methods

Male rats underwent inferior vena cava (IVC) stenosis (94.4% ± 0.5% reduction in IVC diameter) to induce thrombosis. Rats were treated from 2 days after thrombosis until they were killed 7 days later. Groups consisted of (1) PSI-697, a P-selectin inhibitor (30 mg/kg; oral gavage daily); (2) LMWH-Lovenox (LOV; enoxaparin) 3 mg/kg subcutaneously daily; (3) PSI-697 (30 mg/kg; oral gavage daily) plus LOV 3 mg/kg subcutaneously daily (PSI + LOV); (4) and untreated controls. Evaluations included thrombus mass, vein wall tensiometry (stiffness [inverse of compliance]), intimal thickness scoring by light microscopy, vein wall inflammatory mediators by enzyme-linked immunosorbent assay, and vein wall inflammatory cells by histologic evaluation.

Results

Thrombus mass was not reduced by any treatment. Animals treated with PSI-697 alone, LOV alone, or PSI + LOV demonstrated significant decreases in vein wall stiffness when compared with controls. The vein wall stiffness of the PSI-697–treated groups was also significantly lower than in the LOV-only group. Animals treated with PSI-697 showed a significantly decreased intimal thickness score when compared with vehicle control IVCs. Vein wall intimal thickening was also significantly decreased in animals treated with PSI-697 vs LOV. The PSI-697 and PSI + LOV groups manifested significant decreases in the immunoregulatory and inflammatory cytokine interleukin 13 as compared with controls and LOV. Vein wall monocyte chemotactic protein 1 levels were also significantly reduced in the PSI-697 and PSI + LOV groups vs control. Only PSI-697 significantly decreased vein wall levels of platelet-derived growth factor ββ. Both the LOV and PSI + LOV groups had significant increases in vein wall monocytes and total inflammatory cells vs controls.

Conclusions

These data suggest that both LMWH and PSI-697 inhibit vein wall injury independently of thrombus mass. P-selectin inhibition seemed superior to LMWH in measured parameters of injury and mediator inhibition.

Clinical Relevance

Deep venous thrombosis (DVT) remains a serious health care problem in this country, affecting more than 250,000 patients annually. Chronic venous insufficiency, a major complication of DVT, promotes the impairment of venous outflow, loss of vein compliance, and valvular dysfunction. The current standard of care for both treatment and prophylaxis of DVT is low-molecular-weight heparin. However, this therapy confers a bleeding risk and does not protect against postthrombotic syndrome. In previous rat studies, using a recombinant P-selectin receptor antagonist (rPSGL-Ig) 2 days after thrombosis resulted in less vein wall fibrosis without a decrease in thrombus mass. Further studies in the rat model of DVT have shown that P-selectin inhibition also inhibits profibrotic cytokine production and vein wall collagen. This study evaluated the effects of P-selectin inhibition by using a novel oral agent (PSI-697) on the postthrombotic vein wall and compared this with the current standard of care.

 

Deep venous thrombosis (DVT) remains a serious health care problem in this country, with more than 250,000 patients affected and at least 200,000 diagnosed annually.1, 2, 3 Treatment costs to the US health care system reach billions of dollars per year just for the acute treatment of venous thrombosis, without consideration of additional costs for treatment of the sequelae of DVT (chronic venous insufficiency) and pulmonary embolism (chronic pulmonary hypertension).4, 5, 6 Chronic venous insufficiency, a major complication of DVT, affects between 400,000 and 500,000 patients with skin ulcerations and 6 to 7 million patients with manifestations of chronic venous disease, including impairment of venous outflow, thickened vein walls, and valvular dysfunction.7, 8, 9, 10

Stewart et al11 originally hypothesized that procoagulant events could promote thrombosis by the activation of leukocytes and platelets. These prothrombotic events initiate the adherence and layering of leukocytes and platelets on the thrombus, thus leading to amplification and endothelial cell damage. Recent studies using multiple animal models of thrombosis have shown that the inhibition of P selectin/P-selectin ligand (PSGL-1) interactions by antibody, soluble ligand binding, or small molecule inhibition can decrease thrombosis without modulating inflammatory cell extravasation.12, 13, 14, 15, 16, 17, 18, 19 Further studies in the rat model of DVT have shown that P-selectin inhibition also inhibits profibrotic cytokine and vein wall collagen production.13, 20

The current standard of care for both treatment and prophylaxis of DVT is low-molecular-weight heparin (LMWH).21 This is effective therapy but does confer a bleeding risk, and it is unclear whether this agent decreases vein wall damage beyond preventing further thrombosis. This study evaluated the effects of P-selectin inhibition by using a novel oral agent (PSI-697; Wyeth Research, Cambridge, Mass) on the postthrombotic vein wall and compared this with the current standard of care, LMWH. It also evaluated the associated profibrotic mediators and leukocyte changes. In previous mouse19 and primate22 studies, PSI-697 was found to decrease thrombosis when present at the time thrombi were induced. However, previous rat studies with a recombinant P-selectin receptor antagonist (rPSGL-Ig) begun 2 days after thrombus induction resulted in less vein wall fibrosis without a decrease in thrombus mass.13, 20 We chose to study PSI-697 in a similar rat model starting treatment 2 days after thrombus induction. We hypothesized that inhibition of P selectin with PSI-697 would reduce vein wall injury and the sequela of venous thrombosis independently of thrombus resolution.

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Methods 

Sprague-Dawley rats (n = 79) underwent inferior vena cava (IVC) stenosis (94% ± 0.5% reduction in IVC diameter) to induce thrombosis. This model is similar to that used in studies of thrombus resolution emphasizing the importance of monocytes and tissue-type plasminogen activator.23, 24 In brief, rats are anesthetized with isoflurane gas via nose cone and undergo a midline laparotomy. The IVC is directly approached by blunt dissection. A 6-0 nonreactive ligature is placed on the IVC just below the level of the renal veins and tightened down on a 3-0 silk suture until an average 94% ± 0.5% reduction in IVC diameter is achieved (as determined by ultrasound imaging; three rats per group; unpublished observation by Wrobleski, SK; Fig 1). The 3-0 silk suture is then immediately removed once the IVC stenosis has been created.

Rats were treated daily, starting 2 days after thrombosis, and were killed 7 days later for tissue evaluation. Groups consisted of (1) PSI-697 inhibitor (30 mg/kg; oral gavage daily), (2) LMWH-Lovenox (LOV; enoxaparin; Aventis-Pharma, Bridgewater, NJ) 3 mg/kg subcutaneously daily; (3) PSI-697 inhibitor (30 mg/kg; oral gavage daily) plus LOV 3 mg/kg subcutaneously daily (PSI + LOV); (4), and untreated vehicle controls (Fig 2). Additionally, a group of animals that did not undergo any surgical manipulation was included (normal animals) to provide a baseline for tensiometry and chemokine/cytokine levels. The dose of PSI-697 30 mg·kg−1·d−1 was based on the effective oral dose determined in a model of rat carotid balloon injury,23 whereas the dose of enoxaparin was the amount that produced therapeutic anticoagulation in rats from previous studies.

P-selectin inhibition 

PSI-697 is an approximately 370-MW organic small molecule inhibitor of P selectin. This compound has been demonstrated in vitro to disrupt the binding of P selectin to its ligand PSGL-1 and to reduce white cell rolling in the microvasculature of mice.23 This oral formulation has been shown to significantly decrease thrombosis in a mouse and primate model of venous thrombosis.19, 22 Rats in this study were dosed with an oral suspension of PSI-697 at 30 mg/kg daily by using an 18-gauge 3-inch straight 2.25-mm-ball feeding needle (Biomedical Needles; Popper and Sons, Inc, New Hyde Park, NY). The peak circulating concentration of PSI-697 was 2400 ng/mL at 135 minutes after initial dosing in our rodent model. The serum trough concentration of PSI-697, 24 hours after the last dose, was 28.4 ± 11 ng/mL.

Thrombus mass 

The technique (weight/length measured) used is an indirect measure of thrombus dissolution over time. At death, the IVC with its associated thrombus is removed and weighed together and measured for length.27, 28, 29 In prior work, thrombus mass has been found to correlate with thrombus weight (thrombus evaluation independent of length).30

Vein wall tensiometric analysis 

Tensiometry measurements were made of individual IVC samples in this study to evaluate the mechanical properties (stiffness) of the vein wall after thrombosis.29 Harvested vein samples were divided longitudinally, placed in phosphate-buffered saline, and kept on ice until tensiometry testing (Mechanical Testing Equipment 5542 series; Instron Corp, Norwood, Mass). Each vein sample was mounted into the load frame by using pneumatic grippers, and force was applied to failure, with forces in newtons and length determined to yield a stiffness value. The results of each test were recorded via a computer program (Merlin Materials Testing Software; Instron). The force used to stretch each tissue sample was recorded in millimeters and recorded (stiffness).

Intimal thickness scoring 

Paraffin-embedded IVC tissue was sectioned (3 mm) and stained with hematoxylin and eosin. By using light microscopy in a blinded fashion, intimal thickness (IT) was scored in the following fashion, encompassing five segments around the circumference of the vein:

0.Intima appears as just a potential space with space occupied only by endothelial cells.

1.Very small spaces are present between endothelial cells and the internal elastic lamina. Intima still appears generally as thick as the nuclei of normal spindle-shaped endothelial cells.

2.Intima is at least twice as thick as an endothelial nucleus at its widest point in the high-power field.

3.Intima is at least five times the thickness of a red blood cell diameter at its widest point in the high-power field. IT tends to be highly variable and may contain cells other than endothelial cells.

4.Intima is greatly thickened and contains fibroblasts, white blood cells, and/or hemorrhage at its widest point.

Vein wall morphometrics 

The IVC samples were paraffin-embedded, stained with hematoxylin and eosin, and examined under high-power oil immersion light microscopy (×1000). Five representative high-power fields were examined around the vein wall, and the cell counts were analyzed; cells were identified as polymorphonuclear cells, monocyte/macrophages, or lymphocytes on the basis of standard histologic criteria, including nuclear size, cytoplasmic content, and total cell size, in a blinded fashion by a board-certified pathologist.26, 27, 28

Antigen determination 

Enzyme-linked immunosorbent assay was used to determine vein wall total tissue protein concentrations of interleukin (IL)-13, monocyte chemotactic protein (MCP)-1, and platelet-derived growth factor (PDGF)-ββ (R&D Systems, Minneapolis, Minn). Analysis was made with the thrombus removed. In brief, all samples were run in duplicate on freshly homogenized vein wall tissue, with homogenization performed in complete protease inhibitor cocktail (Roche, Mannheim, Germany). The suspension was sonicated on ice for 30 seconds and centrifuged at 14,000 rpm for 10 minutes, and the supernatant was used to run the tests. Samples were added and detected by a biotinylated detection antibody, followed by tetramethylbenzadine (TMB) substrate. The color reaction was stopped with 1 mol/L phosphoric acid, and the plates are read on a Plate Reader Elx808 (Biotek, Winooski, VT) at a 450-nm wavelength. Total protein was measured by using a bicinchoninic acid protein assay kit assessing colorimetric detection and quantitation of total protein, read at 590 nm (Pierce, Rockford, Ill).

Statistical evaluation and animal use 

Statistical analysis included mean ± SEM, analysis of variance, and unpaired Student t tests for parametric data (SPSS SigmaStat 2.03, Aspire Software International, Leesburg, Va). Significance was defined as P ≤ .05. The health status of all animals was monitored, and all animals were free of pathogens. All rats were housed and cared for by the University of Michigan Unit for Laboratory Animal Medicine. The University of Michigan is an American Association for Accreditation of Laboratory Animal Care–accredited facility under the direction of a veterinarian according to the Principles of Laboratory Animal Care (formulated by the National Society for Medical Research) and the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences, 1996). The University of Michigan Committee on Use and Care of Animals approved this research protocol.

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Results 

Thrombosis is not altered with treatments 

Thrombus mass is a reliable measure of normal thrombus dissolution.11, 12, 13, 14, 16 No treatment regimen had a significant effect of decreasing thrombus mass compared with controls. Values were as follows: control, 0.051 ± 0.003 × 104 g/cm; PSI-697, 0.059 ± 0.006 × 104 g/cm; LOV, 0.057 ± 0.007 × 104 g/cm; and PSI + LOV, 0.053 ± 0.007 × 104 g/cm. This model of thrombosis produced a significant thrombosis that was five times greater than in normal animals, which did not have venous thrombi present (normal animals, 0.014 ± 0.001 × 104 g/cm).

Vein wall stiffness is decreased with P-selectin inhibition and LMWH after thrombosis 

The vein wall stiffness of animals treated with PSI-697 had measurements nearest to those of nonthrombosed normal animals, whereas animals treated with PSI-697 after thrombosis showed a significant decrease (P < .01) in vein wall stiffness compared with vehicle control by nearly fourfold. The LMWH treatment was associated with a significant reduction (P < .05) of stiffness as compared with the vehicle controls, with a nearly twofold decrease in vein wall stiffness, whereas the combination group of PSI + LOV also showed significantly decreased vein wall stiffness (P = .002). When direct comparison was made between groups treated with PSI-697 and LOV, PSI-697 treatment was associated with significantly less vein wall stiffness (P < .05; Fig 3).

Only P-selectin inhibition reduces vein wall IT after thrombosis 

Vein wall injury is accompanied by IT.22, 23, 24, 25, 26, 27, 28, 29 To assess the effects of P-selectin inhibition and LMWH on vein wall intimal response, IT was evaluated histologically. Animals treated with PSI-697 showed a significantly decreased IT score when compared with vehicle control IVCs. Vein wall IT was also significantly decreased in animals treated with PSI-697 vs LOV. No other significant differences were noted between groups, and the combination of PSI + LOV was not associated with significantly less IT (Fig 4).

Both P-selectin inhibition and the combination of PSI-697 plus LMWH decrease profibrotic vein wall mediators 

Vein wall homogenates from animals treated with PSI-697 had significantly decreased IL-13 antigen levels when compared with controls (P = .003). The combination treatment of PSI + LOV was also associated with significantly decreased IL-13 levels vs control animals (P = .007). As compared with LOV alone, PSI-697 and (P = .007) and PSI + LOV (P = .012) treatments also significantly decreased IL-13 antigen levels (Fig 5, A).

  • View full-size image.
  • Fig 5. 

    A, Vein wall interleukin 13 antigen levels. LOV, Lovenox (enoxaparin); CTR, control; NORM, normal. B, Vein wall monocyte chemotactic protein 1/JE (MCP-1/JE) antigen levels. C, Vein wall platelet-derived growth factor ββ (PDGF-BB) antigen levels.

The immunoregulatory cytokine MCP-1 is responsible for the recruitment of monocytes to sites of injury and inflammation and was significantly decreased, by 52% (P = .011), in vein wall homogenates from animals treated with PSI-697 when compared with thrombosed controls. The combination therapy of PSI + LOV was also associated with significantly decreased MCP-1 antigen levels (P = .02). LOV treatment itself was not associated with decreased MCP-1 in the vein wall after thrombosis (Fig 5, B).

PDGF is produced by a number of cell types and elements, including platelets that promote the migration of monocytes, fibroblasts, and smooth muscle cells to areas of injury. Only animals treated with PSI-697 had significantly decreased vein wall levels of PDGF-ββ when compared with controls (P = .041), and neither LOV alone nor in combination with PSI-697 was associated with a significant decrease (Fig 5, C).

Vein wall leukocytes are increased with LMWH but not P-selectin inhibition by visual cell counting 

P-selectin inhibition by PSI-697 did not decrease the number of vein wall inflammatory cells compared with thrombosed controls. LOV treatment was associated with significantly increased numbers of vein wall monocyte/macrophages by nearly 2-fold (P = .007) and with total inflammatory cell populations by 1.5-fold (P = .026) when compared with thrombosed controls. Animals treated with the combination PSI + LOV also had significantly increased monocyte/macrophages (P = .003), lymphocytes (P = .003), and total inflammatory cells (P = .001) in the vein wall compared with thrombosed controls (Fig 6).

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  • Fig 6. 

    Vein wall morphometrics. LOV, Lovenox (enoxaparin); CTR, control; TC, total inflammatory cell; MONO, monocytes; LYMPH, lymphocytes; PMN, polymorphonuclear cells; HPF, high-power field.

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Discussion 

The occurrence of DVT and the sequela of chronic venous insufficiency remains a significant and underappreciated heath care problem. New and safer therapies are needed to treat DVT. In this study, the novel findings include the fact that vein wall injury was most significantly attenuated by the small molecule inhibitor of P selectin (PSI-697) in a rodent stenosis model of venous thrombosis. The data suggest that this effect is independent of thrombus size, with P-selectin inhibition having direct anti-inflammatory effects on the vein wall. Support for this notion derives from the observation that profibrotic mediators such as IL-13, MCP-1, and PDGF-ββ were significantly decreased with PSI-697 treatment. Lesser, but still significant, reductions in vein wall injury were also observed with LMWH, and this serves to confirm our prior work (although at a lower dose and a different LMWH preparation) showing that LMWH decreased perithrombotic vein wall inflammation.31

On venous endothelial cell stimulation, PSGL-1 ligand initiates the adhesion interactions responsible for the initial rolling of neutrophils along stimulated vascular endothelium, thus creating platelet-leukocyte complexes on the vascular surface and in the circulation.25, 26, 27, 28, 29, 30, 31, 32, 33 Targeted P-selectin inhibition has been shown to modulate thrombogenesis by counteracting platelet-endothelial, platelet-platelet, and platelet-leukocyte interactions in several animal models of venous thrombosis. Although in this study, treatment after thrombosis did not decrease overall thrombus mass, we have noted that P-selectin inhibition in the primate after the thrombus has been present for 48 hours still results in less thrombus burden over time.18 The difference may relate to the type of animal species studied (rat vs baboon) or to the model used. The primate model involves temporary balloon occlusion to form thrombosis without vessel ligation and stenosis, whereas the current rat model involved a significant 94% stenosis to produce thrombosis. We chose the stenosis model in the rat to align with models in the literature that have been used to evaluate thrombus resolution.25, 26 The advantages of this model include but are not limited to the fact that clinical DVT often begins as a nonocclusive event that then progresses to occlusion (as in the current rat protocol).

It has been suggested that the inhibition of platelet-leukocyte interactions modulates the release of PDGFs and proteases, with promotion of vein remodeling and fibrosis.34, 35, 36 Previous work in our laboratory showed that low-dose LMWH demonstrated anti-inflammatory properties during stasis-induced thrombosis.31 However, LMWHs have decreased inhibitory effects on both P- and L-selectin ligand binding as compared with standard unfractionated heparin,21, 37, 38 and this may be why no difference in profibrotic mediators was observed with LMWH. Additionally, in our previous study, there was no inflammatory protection at the anticoagulant dose of LMWH, which is closer to the dose used in this study.

Vein wall injury was primarily reflected by measuring vein wall stiffness, a biomechanical measure. Our findings showed that daily treatment with PSI-697, LMWH, or PSI-697 in combination with LMWH was associated with decreased vein wall stiffness, independently of thrombus size (Fig 3).

Prior work has suggested that less vein wall collagen occurs with P-selectin inhibition,20 and in this study a complementary parameter of intimal injury (IT) was determined. However, only PSI-697 had associated decreased IT scores when compared with thrombosed controls. IT is well established to occur after arterial injury and is related to selectin interactions.36 Stiffness is related to vein wall injury conferred by proteinases,29 whereas vein IT may be more related to levels of profibrotic mediators, such as IL-13.

This study did not confirm the specific downstream signaling mechanisms associated with the antifibrotic properties of PSI-697. However, the evaluation of proinflammatory mediators from vein wall homogenates showed that P-selectin inhibition significantly decreased vein wall antigen levels of IL-13, MCP-1, and PDGF-ββ. Treatment with LMWH alone was not associated with reduced levels. The relationship between IL-13 and fibrosis has been shown in animal models of pulmonary fibrosis and visceral parasitic migrations.39 IL-13 is released from activated T-helper lymphocytes and promotes the expression of MCP-1, a chemokine that directs monocytes to areas of inflammation.29, 40 It is interesting to note that IL-13 and MCP-1 elevations were found in pulmonary tissue after pulmonary embolism in a rat model and were associated with significant intimal hyperplasia.41 Although MCP-1 may confer organ fibrosis, it may also hasten DVT resolution when infused into the thrombus.24

Similarly, activated platelets have been shown to release PDGF which was increased in thrombosed control vein walls.42 PDGF is active in signaling that occurs in wound healing and is implicated in atherosclerosis, fibrotic disease, and cancer.43, 44, 45 It is likely that the decreased IT in rats treated with PSI-697 was related to significantly less PDGF-ββ present.

Of interest, animals treated with LMWH alone and in combination with PSI-697 had a significant increase in monocyte/macrophage populations and overall total numbers of inflammatory cells present in the vein wall (Fig 6). Conversely, animals treated with PSI-697 had inflammatory cell populations similar to those of untreated controls by visual counting. It is not clear from this study whether the decreased MCP-1 is causative of fewer monocytes or is a secondary marker of fewer leukocytes in the vein wall. These observations support similar findings in which P-selectin inhibition did not decrease the vein wall extravasation of inflammatory cells in the presence of thrombosis.16 A recent study by Henke et al29 suggests a temporally important relationship between neutrophil migration and vein wall injury response—namely, that the early cellular composition affects how the vein wall remodels, independently of thrombus size. The presence of neutrophils in the vein wall seems to be important to limit vein wall fibrosis.

Taken together, these data suggest that both LMWH and PSI-697 inhibit vein wall injury independently of thrombus size in a rodent model of DVT. P-selectin inhibition with PSI-697 seems superior to LMWH or the combination regarding vein wall stiffness, the production of proinflammatory cytokines, and IT scoring. PSI-697 with LOV did not have a greater response than PSI-697 administered alone and in some instances (IT, PDGF-ββ levels, and cell infiltrates) did not exhibit a beneficial response. This raises a question about the combination of these treatments and should be examined in future studies. In addition, the mechanisms by which P-selectin inhibition decreases the severity of postthrombotic changes to the vein wall need to be further elucidated. These results show the potential benefits of P-selectin inhibition in modulating the pathophysiological changes in the vein wall that promote chronic venous insufficiency.

Author contributions 

Conception and design: DDM, PKH, RGS, TWW

Analysis and interpretation: DDM, PKH, SKW, RGS, TWW

Data collection: DDM, SKW, AEH, TRM

Writing the article: DDM, PKH, TWW

Critical revision of the article: DDM, PKH, SKW, RGS, TWW

Final approval of the article: DDM, PKH, SKW, RGS, TRM, TWW

Statistical analysis: DDM, PKH, TWW

Obtained funding: PWB, NK, GS, TWW

Overall responsibility: DDM

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We thank Derek A. Dubay, MD, for his assistance with vein wall tensiometric measurements. We also thank Robert E. Sigler, DVM, PhD, for his assistance with vein wall IT scoring and morphometrics.

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 Supported by NIH grant RO1 HL 70766 (T.W.W.) and Wyeth Research.Competition of interest: none.

PII: S0741-5214(06)00929-3

doi:10.1016/j.jvs.2006.05.021

Journal of Vascular Surgery
Volume 44, Issue 3 , Pages 625-632, September 2006

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