An in situ inferior vena cava ligation-stenosis model to study thrombin generation rates with flow.

BACKGROUND: Blood flow-induced shear stress affects platelet participation in coagulation and thrombin generation. We aimed to develop an in vivo model to characterize thrombin generation rates under flow. METHODS: An in situ inferior vena cava (IVC) ligation-stenosis model was established using C57BL/6 mice. Wild type C57BL/6 mice were fed normal chow diet for two weeks before experiments. On the day of experiments, mice were anesthetized, followed by an incision through the abdominal skin to expose the IVC, which was then ligated (followed by reperfusion through a stenosis for up to 2 h). IVC blood flow rate was monitored using a Transonic ultrasound flow meter. In sham animals, the IVC was exposed following the same procedure, but no ligation was applied. Thrombin generation following IVC ligation was estimated by measuring mouse plasma prothrombin fragment 1-2 concentration. Mouse plasma factor Va concentration was measured using phospholipids and a modified prothrombinase assay. Blood vessel histomorphology, vascular wall ICAM-1, von Willebrand Factor, tissue factor, and PECAM-1 expression were measured using immunofluorescence microscopy. RESULTS: IVC blood flow rate increased immediately following ligation and stenosis formation. Sizable clots formed in mouse IVC following ligation and stenosis formation. Both plasma factor Va and prothrombin fragment 1-2 concentration reduced significantly following IVC ligation/stenosis, while no changes were observed with ICAM-1, von Willebrand Factor, tissue factor and PECAM-1 expression. CONCLUSION: Clot formation was successful. However, the prothrombin-thrombin conversion rate constant in vivo cannot be determined as local thrombin and FVa concentration (at the injury site) cannot be accurately measured. Modification to the animal model is needed to further the investigation.

Endothelial Cell Biomechanical Responses are Dependent on Both Fluid Shear Stress and Tensile Strain.

INTRODUCTION: The goal of this study was to investigate how concurrent shear stress and tensile strain affect endothelial cell biomechanical responses. METHODS: Human coronary artery endothelial cells were exposed to concurrent pulsatile shear stress and cyclic tensile strain in a programmable shearing and stretching device. Three shear stress-tensile strain conditions were used: (1) pulsatile shear stress at 1 Pa and cyclic tensile strain at 7%, simulating normal stress/strain conditions in a healthy coronary artery; (2) shear stress at 3.7 Pa and tensile strain at 3%, simulating pathological stress/strain conditions near a stenosis; (3) shear stress at 0.7 Pa and tensile strain at 5%, simulating pathological stress/strain conditions in a recirculation zone. Cell morphology was quantified using immunofluorescence microscopy. Cell surface PECAM-1 phosphorylation, ICAM-1 expression, ERK1/2 and NF-κB activation were measured using ELISA or Western blot. RESULTS: Simultaneous stimulation from pulsatile shear stress and cyclic tensile strain induced a significant increase in cell area, compared to that induced by shear stress or tensile strain alone. The combined stimulation caused significant increases in PECAM-1 phosphorylation. The combined stimulation also significantly enhanced EC surface ICAM-1 expression (compared to that under shear stress alone) and transcriptional factor NF-κB activation (compared to that under control conditions). CONCLUSION: Pulsatile shear stress and cyclic tensile strain could induce increased but not synergistic effect on endothelial cell morphology or activation. The combined mechanical stimulation can be relayed from cell membrane to nucleus. Therefore, to better understand how mechanical conditions affect endothelial cell mechanotransduction and cardiovascular disease development, both shear stress and tensile strain need to be considered.