8. Thrombomodulin Demonstrates Critical Beneficial Direct Effects On Smooth Muscle Cell Physiology

Abstract

Introduction and Objectives: Following vascular injury, critical endothelial cell (EC) function is lost. Thrombomodulin (TM), an endothelial anticoagulant, is one lost regulator. TM has known effects through inhibition of thrombin, but here we sought to examine direct, independent effects of TM on SMC physiology. We hypothesized that exogenous TM would induce a favorable SMC phenotype and would inhibit SMC migration. Methods: Primary human saphenous vein SMC were explanted and used in early passage (1-4). SMCmigration was measured using: (1) a 4-hour Boyden-chamber chemotaxis assay, and (2) a 24-hour ElectricCell-substrate Impedance Sensing (ECIS) wound assay.Migration experiments were conducted with serum-starved SMC exposed to increasing doses of soluble human TM and results quantified by cell counts. Thrombin served as positive control and serum-free media as negative control. Dose-dose comparisons were performed using a t-test (a< .05). Multiphoton laser scanning microscopy was used to assess the effect of exogenous TM on SMC phenotype. Results: SMC demonstrated low, baseline migration in serum-free conditions. Thrombin significantly stimulated SMCmigration as expected. TM, independent of thrombin, significantly inhibited SMC migration in a dose-dependent fashion (Figure 1). 70% reduction was observed at 5mg/mL (56±1.7 vs. 18±3.5 cells/5hpf, P1⁄4 .0005). SMC exposed to TM demonstrated a spindle-shapedmorphology with organized stress fibers consistent with a differentiated, contractile phenotype, whereas, control thrombin stimulation lead to a dedifferentiated, synthetic phenotype (Figure 2). Conclusions: Thrombomodulin demonstrated direct effects on SMC physiology, independent from thrombin, includingmaintenance of a differentiated, contractile phenotype and inhibition of migration. These findings provide new knowledge in understanding the biology of vascular injury, and supports a strategy focusedon restoring key endothelial function to prevent intimal hyperplasia.