Abstract 161: Cardiomyocytes Release Tissue Factor-positive Microparticle In Response To TNF-α: Increase Of The Extracellular Thrombogenicity By Microparticle Diffusion Through Endothelium.
Tissue Factor (TF) is constitutively expressed within the myocardium especially by cardiomyocytes (CM). Myocardial inflammation is associated with an altered TF expression and increased local and systemic thrombogenicity. We investigated the transcriptional regulation of CM TF expression under inflammatory conditions. In a second step, TF-bearing microparticle (MP) generation from CM and diffusion through an endothelial monolayer was evaluated.
Methods: Murine CM (HL-1) were stimulated with TNF-α (10ng/mL) and/or pre-incubated with specific inhibitor for JNK, p38, ERK1/2 and NFκB. The TF expression was quantified by real-time PCR, western blot and TF activity assay. The release of MP from CM was assessed by FACS. A trans-well chamber system for co-cultivation of endothelial cells and CM was used to determine whether CM-derived thrombogenic MP would diffuse through an endothelial monolayer upon stimulation with TNF-α.
Results: TNF-α induced the TF expression in CM (mRNA: 3.45 ± 0.65-fold; protein: 3.26 ± 0.65-fold; TF activity: 2.40 ± 0.30-fold; P<0.005 vs. control). The inhibition of JNK (10μM SP600125) reduced the constitutive TF expression to 64.69 ± 0.07% (mRNA) and to 36.17 ± 0.05% (protein) of the control levels (P<0.001). JNK inhibition also reduced the TF induction after TNF-α stimulation to 18.75 ± 0.65% (mRNA) and 10.78 ± 0.66% (protein) of TNF-α stimulated controls (P<0.001). Increased amounts of thrombogenic MP were released in response to TNF-α (2.47 ± 0.15-fold; P<0.01). The inhibition of JNK led to an increase in the release of MP from CM (4.88 ± 0.63-fold; P<0.01), but not of MP-associated TF activity. TF-positive MP from CM diffused upon stimulation with TNF-α through an endothelial monolayer into the extracellular endothelial compartment, (MP diffusion 18.15 ± 1.67% vs. 44.36 ± 5.45%; P<0.05 control vs. TNF-α) thereby increased its thrombogenicity.
Conclusion: Cardiomyocytic TF expression and MP release was regulated through JNK. The permeability of an endothelial monolayer for MP of cardiomyocytic origin was increased after TNF-α stimulation. Thus, myocardial inflammation is associated with TF upregulation and release of TF-bearing MP from cardiomyocytes, contributing to an increased thrombogenicity in the heart.