Martin FA, Murphy RP, Cummins PM. Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects.

Background and ObjectivesThrombomodulin (TM), an integral membrane glycoprotein expressed on the lumenal surface of vascular endothelial cells, promotes anti-coagulant and anti-inflammatory properties. Release of functional TM from the endothelium surface into plasma has also been reported. Much is still unknown however about how endothelial TM is regulated by physiologic hemodynamic forces (and particularly cyclic strain) intrinsic to endothelial-mediated vascular homeostasis.MethodsThis study employed human aortic endothelial cells (HAECs) to investigate the effects of equibiaxial cyclic strain (7.5%, 60 cycles/min, 24 hrs), and to a lesser extent, laminar shear stress (10 dynes/cm2, 24 hrs), on TM expression and release. Time-, dose- and frequency-dependency studies were performed.ResultsOur initial studies demonstrated that cyclic strain strongly downregulated TM expression in a p38- and receptor tyrosine kinase-dependent manner. This was in contrast to the upregulatory effect of shear stress. Moreover, both forces significantly upregulated TM release over a 48 hr period. With continuing focus on the cyclic strain-induced TM release, we noted both dose (0–7.5%) and frequency (0.5–2.0 Hz) dependency, with no attenuation of strain-induced TM release observed following inhibition of MAP kinases (p38, ERK-1/2), receptor tyrosine kinase, or eNOS. The concerted impact of cyclic strain and inflammatory mediators on TM release from HAECs was also investigated. In this respect, both TNFα (100 ng/ml) and ox-LDL (10–50 µg/ml) appeared to potentiate strain-induced TM release. Finally, inhibition of neither MMPs (GM6001) nor rhomboids (3,4-dichloroisocoumarin) had any effect on strain-induced TM release. However, significantly elevated levels (2.1 fold) of TM were observed in isolated microparticle fractions following 7.5% strain for 24 hrs.ConclusionsA preliminary in vitro investigation into the effects of cyclic strain on TM in HAECs is presented. Physiologic cyclic strain was observed to downregulate TM expression, whilst upregulating in a time-, dose- and frequency-dependent manner the release of TM.

Introduction: Loss of thrombomodulin (TM), a cofactor in the protein C anti-coagulant pathway, through a combination of reduced TM expression and elevated TM release, is a key feature of endothelial dysfunction leading to vascular diseases. Reduction in endothelial TM levels for example is viewed as a major cause of cyclic strain-induced early vein graft failure. In this respect, our understanding of the mechanistic relationship between vessel stretch and TM expression/release in endothelial cells is very limited, with a particular shortage of in vitro models. In this study, we investigated how cyclic circumferential strain negatively regulates thrombomodulin levels in human aortic endothelial cells (HAECs) in vitro. Methods: A Flexercell® Tension-Plus™ FX-4000T™ system (Flexcell International Corp., Hillsborough, NC) was routinely employed to subject HAECs to equibiaxial cyclic strain over a range of doses (0, 2.5 and 7.5% stretch; 60 cycles/min; cardiac waveform) and times (0-48 hours). TM expression was analyzed via Western blotting and qRT-PCR, whilst TM release was monitored by ELISA. Results: (i) Cyclic strain of HAECs led to significant force- and time-dependent reductions in TM expression (mRNA and protein) in conjunction with elevated TM release into media; (ii) Inclusion of either GM6001 (25 μM) or apocynin (10 mM) into strain experiments to block MMP-2/9 and NADPH oxidase activities, respectively, had no effect on strain-induced TM release; (iii) Control studies demonstrated that treatment of HAECs with neither TNF-α (0, 10, 100 ng/ml) nor oxLDL (10, 50, 145.7 μg/ml) increased TM release from HAECs - however, oxLDL treatment was found to dose-dependently potentiate cyclic-strain-induced TM release. Conclusions: Cyclic strain negatively regulates TM levels in endothelial cells in vitro. Moreover, release of TM from HAECs does not appear to involve the strain-dependent induction of either MMP-2/9 proteolytic activity or NADPH oxidase-dependent superoxide production. Finally, the cyclic strain-induced release of TM appears to be potentiated by inflammatory atherogenic conditions such as high oxLDL levels.

Introduction: A pivotal step in vascular calcification is the transformation of smooth muscle cells (SMCs) to osteoblast-like cells. Osteoprotegerin (OPG), a soluble protein produced by SMCs, inhibits this transformation and may represent a therapeutic target for calcification reduction. Hyperglycemia, inflammation and increased levels of cyclic strain are all important inducers of calcification in vivo. The purpose of this study was to determine the effects of these stimuli, both alone and in combination, on OPG production by human aortic SMCs (HASMCs) in vitro. Methods: HASMCs were initially exposed to a single stimulus, with OPG levels in cell-conditioned media measured at 24 and 48 hours by ELISA (n=6 for all experiments). Equibiaxial cyclic strain was produced using a Flexercell® Tension-Plus FX-4000T system (0 or 12.5%), whilst hyperglycemic and inflammatory conditions were induced using glucose (6, 15 and 30 mmol/l) and tissue necrosis factor alpha (TNFα, 10 and 100 ng/ml) respectively. HASMCs were then exposed to a combination of hyperglycemia (15 mmol/l), inflammation (10 ng/ml TNFα) and strain (10%), after which levels of OPG and runt-related transcription factor 2 (RUNX2, a marker of osteoblastic transformation) were assessed via ELISA and qRT-PCR, respectively. Results: High levels of cyclic strain (12.5%) significantly increased OPG levels in the cell-conditioned media at 24 and 48 hours. OPG levels were also dose-dependently increased by treatment with TNFα at 24 and 48 hours. At 24 hours with 15 and 30mmol/l glucose, there was no change in OPG levels, but at 48 hours increased OPG levels were observed at both concentrations. In the combination experiment, OPG levels decreased, while RUNX2 mRNA levels increased, after 48 hrs. Conclusions: Cyclic strain, hyperglycemia, and TNFα increased OPG release by HASMCs when administered in isolation. Applied in combination however, these stimuli decreased OPG levels in parallel with an increase in a marker of osteoblastic transformation. This data suggests that inhibition of vascular calcification by OPG may be actively down-regulated when multiple pro-calcification stimuli co-exist, and supports further investigation of OPG as a therapeutic target for calcification reduction.