Blood -- Valves of the deep venous system: an overlooked risk factor

Blood, Vol. 114, Issue 6, 1276-1279, August 6, 2009

Valves of the deep venous system: an overlooked risk factor
Blood Brooks et al. 114: 1276

Supplemental materials for: Brooks et al

Methods
Confocal images were opened in MetaMorph image and analysis software as a set of four planes representing separate fluorophores. To merge all four fluorophores, the display image overlay was selected, being careful to assign different colors for each representative antibody. Blue, white, red, and green were chosen to represent nuclear staining, vWF, EPCR, and TM immunostaining, respectively. This created a reference image for accurately drawing a region of interest to be transferred to the split images for intensity measurements (see Fig. 2). Graphic outlines highlighting six 150 µm longitudinal segments of vein wall endothelium and sub-endothelium were traced on each image for quantitation (Fig. 2). The fluorescence intensity of antibody binding in each representative area (Fig. 1A) was assessed by assigning a minimum pixel intensity threshold value (based upon control incubations) for each antibody as follows: vWF (900), EPCR (500), TM (700). The mean intensity and integrated intensity were then determined automatically in each 150 µm long region of interest by measuring all pixels with intensity values greater than the set minimum threshold. The intensity values were logged into an Excel spreadsheet from which charts were created. For each factor the difference in arbitrary intensity units between the mean of areas A–C on the vein lumen and the mean of areas D–F in the valvular sinus was tested using a paired t-test.

Discussion regarding limitations of study
There are limitations to this model. One is that the proximal saphenous veins are superficial rather than deep veins. While this is mitigated to some degree by the recent observation that superficial venous thrombosis shares risk factors with deep venous thrombosis¹ and is itself a risk factor for DVT, suggesting some commonality in the causal pathway of thrombosis, saphenous veins are at best only a surrogate model for deep veins. However, at present there are no other reliable, non-invasive and readily accessible means of studying the deep veins. This may change in the near future with the advent of modern in vivo imaging technologies. A second limitation is the surgical source of the saphenous veins. After the veins are harvested, they are kept in a container on the sterile surgical tray immersed in the patient’s heparinized blood. The presence of heparin abrogates our ability to look at some coagulation proteins of interest like endothelial cell tissue factor pathway inhibitor. Finally, since the patients in this study have advanced atherosclerosis they may have endothelial dysfunction which is associated with abnormal endothelial regulation including impaired vasomotor control and a pro-thrombotic/inflammatory state.^2,3 Although impaired endothelial regulation could effect our observations, recent data suggests that endothelial dysfunction is associated with increased risk of venous thrombosis.⁴ Thus, changes associated with endothelial dysfunction may be relevant to understanding the microenvironment of the venous valvular sinus.

REFERENCES

1. Decousus H and Leizorovicz A: Superficial thrombophlebitis of the legs: still a lot to learn. J Thromb Haemost. 2005; 3:1149–1151.
2. Widlansky ME, Gokce N, Keaney JF, Jr., et al: The clinical implications of endothelial dysfunction. J Am Coll Cardiol. 2003; 42:1149–1160.
3. Bonetti PO, Lerman LO and Lerman A: Endothelial dysfunction: a marker of atherosclerotic risk. Arterioscler Thromb Vasc Biol. 2003; 23:168–175.
4. Migliacci R, Becattini C, Pesavento R, et al: Endothelial dysfunction in patients with spontaneous venous thromboembolism. Haematologica. 2007; 92:812–818.

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