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Background and objectives: Vascular calcification (VC) is a major risk factor for elevated cardiovascular morbidity/mortality. Underlying this process is osteoblastic signalling within the vessel wall involving complex and interlinked roles for receptor-activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). RANKL promotes vascular cell osteoblastic differentiation, whilst OPG acts as a neutralizing decoy receptor for RANKL (and TRAIL). With respect to TRAIL, much recent evidence points to a vasoprotective role for this ligand, albeit via unknown mechanisms. In order to shed more light on TRAILs vasoprotective role therefore, we employed in vitro cell models to test the hypothesis that TRAIL can counteract the RANKL-mediated signalling that occurs between the vascular cells that comprise the vessel wall. Methods and results: Human aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0–25 ng/mL ± 5 ng/mL TRAIL) for 72 hr. Furthermore, to better recapitulate the paracrine signalling that exists between endothelial and smooth muscle cells within the vessel wall, non-contact transwell HAEC:HASMC co-cultures were also employed and involved RANKL treatment of HAECs (±TRAIL), subsequently followed by analysis of pro-calcific markers in the underlying subluminal HASMCs. RANKL elicited robust osteoblastic signalling across both mono- and co-culture models (e.g. increased BMP-2, alkaline phosphatase/ALP, Runx2, and Sox9, in conjunction with decreased OPG). Importantly, several RANKL actions (e.g. increased BMP-2 release from mono-cultured HAECs or increased ALP/Sox9 levels in co-cultured HASMCs) could be strongly blocked by co-incubation with TRAIL. In summary, this paper clearly demonstrates that RANKL can elicit pro-osteoblastic signalling in HAECs and HASMCs both directly and across paracrine signalling axes. Moreover, within these contexts we present clear evidence that TRAIL can block several key signalling actions of RANKL in vascular cells, providing further evidence of its vasoprotective potential. [ABSTRACT FROM AUTHOR]

Background and Objectives: Thrombomodulin (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. Methods: This 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. Results: Our 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. Conclusions: A 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. [ABSTRACT FROM AUTHOR]

Background and Objectives: Blood-brain barrier (BBB) dysfunction is an integral feature of neurological disorders and involves the action of multiple proinflammatory cytokines on the microvascular endothelial cells lining cerebral capillaries. There is still however, considerable ambiguity throughout the scientific literature regarding the mechanistic role(s) of cytokines in this context, thereby warranting a comprehensive in vitro investigation into how different cytokines may cause dysregulation of adherens and tight junctions leading to BBB permeabilization. Methods: The present study employs human brain microvascular endothelial cells (HBMvECs) to compare/contrast the effects of TNF-α and IL-6 on BBB characteristics ranging from the expression of interendothelial junction proteins (VE-cadherin, occludin and claudin-5) to endothelial monolayer permeability. The contribution of cytokine-induced NADPH oxidase activation to altered barrier phenotype was also investigated. Results: In response to treatment with either TNF-α or IL-6 (0–100 ng/ml, 0–24 hrs), our studies consistently demonstrated significant dose- and time-dependent decreases in the expression of all interendothelial junction proteins examined, in parallel with dose- and time-dependent increases in ROS generation and HBMvEC permeability. Increased expression and co-association of gp91 and p47, pivotal NADPH oxidase subunits, was also observed in response to either cytokine. Finally, cytokine-dependent effects on junctional protein expression, ROS generation and endothelial permeability could all be attenuated to a comparable extent using a range of antioxidant strategies, which included ROS depleting agents (superoxide dismutase, catalase, N-acetylcysteine, apocynin) and targeted NADPH oxidase blockade (gp91 and p47 siRNA, NSC23766). Conclusion: A timely and wide-ranging investigation comparing the permeabilizing actions of TNF-α and IL-6 in HBMvECs is presented, in which we demonstrate how either cytokine can similarly downregulate the expression of interendothelial adherens and tight junction proteins leading to elevation of paracellular permeability. The cytokine-dependent activation of NADPH oxidase leading to ROS generation was also confirmed to be responsible in-part for these events. [ABSTRACT FROM AUTHOR]

Background and Objectives: Vascular calcification (VC) is a major risk factor for elevated cardiovascular morbidity/mortality. Underlying this process is osteoblastic signalling within the vessel wall involving complex and interlinked roles for receptor-activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). RANKL promotes vascular cell osteoblastic differentiation, whilst OPG acts as a neutralizing decoy receptor for RANKL (and TRAIL). With respect to TRAIL, much recent evidence points to a vasoprotective role for this ligand, albeit via unknown mechanisms. In order to shed more light on TRAILs vasoprotective role therefore, we employed in vitro cell models to test the hypothesis that TRAIL can counteract the RANKL-mediated signalling that occurs between the vascular cells that comprise the vessel wall., Methods and Results: Human aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0-25 ng/mL ± 5 ng/mL TRAIL) for 72 hr. Furthermore, to better recapitulate the paracrine signalling that exists between endothelial and smooth muscle cells within the vessel wall, non-contact transwell HAEC:HASMC co-cultures were also employed and involved RANKL treatment of HAECs (±TRAIL), subsequently followed by analysis of pro-calcific markers in the underlying subluminal HASMCs. RANKL elicited robust osteoblastic signalling across both mono- and co-culture models (e.g. increased BMP-2, alkaline phosphatase/ALP, Runx2, and Sox9, in conjunction with decreased OPG). Importantly, several RANKL actions (e.g. increased BMP-2 release from mono-cultured HAECs or increased ALP/Sox9 levels in co-cultured HASMCs) could be strongly blocked by co-incubation with TRAIL. In summary, this paper clearly demonstrates that RANKL can elicit pro-osteoblastic signalling in HAECs and HASMCs both directly and across paracrine signalling axes. Moreover, within these contexts we present clear evidence that TRAIL can block several key signalling actions of RANKL in vascular cells, providing further evidence of its vasoprotective potential.