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Calcific aortic valve disease affects the aortic side of the valve, exposed to low magnitude multidirectional ("disturbed) blood flow, more than it affects the ventricular side, exposed to high magnitude uniaxial flow. Overt disease is preceded by endothelial dysfunction and inflammation. Here we investigate the potential role of the transforming growth factor-β (TGF-β) receptor ALK5 in this process. Although ECs are always subject to shear stress due to blood flow, and their responses to shear stress are important in healthy valve development and homeostasis, low magnitude multidirectional flow can induce pathophysiological changes. Previous work has shown ALK5 to be an important mechanosensor. ALK5 transduces mechanically sensed signals via the activation of the SMAD2/3 transcriptional modulators. However, it is currently unclear precisely how ALK5-mediated shear stress responses translate into pathological changes under conditions of chronically disturbed flow. Here, we demonstrate that ALK5 mechanosensory signalling influences flow-induced endothelial leukocyte adhesion and paracellular permeability. Low magnitude multidirectional flow resulted in downregulation of the receptor, accompanied by increased SMAD2 phosphorylation, in human umbilical vein endothelial cell (HUVEC) monolayers. These changes correlated with elevated monocyte adhesion and significantly increased transendothelial transport of an albumin-sized tracer. These effects were abolished by inhibition of ALK5 kinase activity. Analysis of ALK5 expression patterns in porcine aortic valve tissue corroborated the findings from cell-based experiments. Together, these results suggest that ALK5 has a role in shear stress-associated cardiovascular disease pathology, emphasising the importance of further mechanistic investigations and supporting it as a potential therapeutic target. • Low magnitude multidirectional flow (LMMF) downregulate ALK5 expression in endothelial cells (EC). • LMMF increase SMAD2 phosphorylation in EC. • Inhibition of ALK5 abolishes LMMF-induced monocyte adhesion and transendothelial transport of an albumin-sized tracer. • ALK5 expression is higher in EC of the ventricularis of porcine aortic valve (compared with fibrosa). [ABSTRACT FROM AUTHOR]

Abstract: Smoking is highly correlated with enhanced likelihood of atherosclerosis by inducing endothelial dysfunction. In endothelial cells, various cell-adhesion molecules including E-selectin, are shown to be upregulated upon exposure to nicotine, the addictive component of tobacco smoke; however, the molecular mechanisms underlying this induction are poorly understood. Here we demonstrate that nicotine-induced E-selectin transcription in human aortic endothelial cells (HAECs) could be significantly blocked by α7-nAChR subunit inhibitor, α-BT, Src-kinase inhibitor, PP2, or siRNAs against Src or β-Arrestin-1 (β-Arr1). Further, chromatin immunoprecipitations show that E-selectin is an E2F1 responsive gene and nicotine stimulation results in increased recruitment of E2F1 on E-selectin promoter. Inhibiting E2F1 activity using RRD-251, a disruptor of the Rb–Raf-1 kinase interaction, could significantly inhibit the nicotine-induced recruitment of E2F1 to the E-selectin promoter as well as E-selectin expression. Interestingly, stimulation of HAECs with nicotine results in increased adhesion of U937 monocytic cells to HAECs and could be inhibited by pre-treatment with RRD-251. Similarly, depletion of E2F1 or Src using RNAi blocked the increased adhesion of monocytes to nicotine-stimulated HAECs. These results suggest that nicotine-stimulated adhesion of monocytes to endothelial cells is dependent on the activation of α7-nAChRs, β-Arr1 and cSrc regulated increase in E2F1-mediated transcription of E-selectin gene. Therefore, agents such as RRD-251 that can target activity of E2F1 may have potential therapeutic benefit against cigarette smoke induced atherosclerosis. [Copyright &y& Elsevier]

Abstract: Cellular protein delivery is an emerging technique by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an Escherichia coli expression vector including human specific gene sequences for protein cellular delivery. The plasmid was generated by ligation the nucleotides 770–817 of the homeobox A5 mRNA sequence which was matched with protein transduction domain (PTD) of homeodomain protein A5 (HOXA5) into pET expression vector. The cellular uptake of HOXA5-PTD-EGFP was detected in 1min and its transduction reached a maximum at 1h within cell lysates. The cellular uptake of HOXA5-EGFP at 37°C was greater than in 4°C. For study for the functional role of human HOXA5-PTD, we purified HOXA5-APE1/Ref-1 and applied it on monocyte adhesion. Pretreatment with HOXA5-APE1/Ref-1 (100nM) inhibited TNF-α-induced monocyte adhesion to endothelial cells, compared with HOXA5-EGFP. Taken together, our data suggested that human HOXA5-PTD vector provides a powerful research tools for uncovering cellular functions of proteins or for the generation of human PTD-containing proteins. [Copyright &y& Elsevier]

Abstract: Resistin and high glucose (HG) are concomitantly present at elevated concentration in diabetic’s plasma; both are pro-inflammatory agents acting on vascular cells by mechanisms that are not fully understood. We questioned whether resistin and HG affect the expression of major adhesion molecules, P-selectin and fractalkine in human endothelial cells (HEC). The results showed that in HEC (i) resistin increased P-selectin expression; (ii) HG up-regulated Fk expression; (iii) P-selectin and fractalkine were functional increasing monocyte adhesion to activated cells. Co-stimulation with resistin and HG increased P-selectin and fractalkine mRNA and protein and induced monocyte adhesion, generated an increase in NADPH oxidase activity and of the intracellular reactive oxygen species and activated the NF-kB and AP-1 transcription factors at similar values as those of each activator. In conclusion in HEC, resistin and HG induce the up-regulation of P-selectin and fractalkine and the ensuing increased monocyte adhesion by a mechanism involving oxidative stress and NF-kB and AP-1 activation. [Copyright &y& Elsevier]

Abstract: Nitration products (nitroalkenes) of linoleic acid (LNO2) and oleic acid (OA-NO2) can act as endogenous PPARγ ligands with electrophilic properties to exert anti-inflammatory effects on atherosclerotic plaques in the vasculature. Here, we show that OA-NO2 and LNO2 prevent tumor necrosis factor α (TNFα)-stimulated inflammatory and atherogenic responses in human umbilical vein endothelial cells (HUVECs). Both OA-NO2 and LNO2 prevented TNFα-stimulated release of the cytokines, IL-6, IL-8, IL-12/p40, IFNγ, MCP-1, and IP-10, and inhibited NF-κB activation. OA-NO2 and LNO2 also blocked TNFα-induced expression of the adhesion molecules, ICAM-1, VCAM-1, and E-selectin, and suppressed monocyte adhesion to HUVECs. In each case, OA-NO2 was more potent and efficacious than was LNO2, possibly due to increased stability in aqueous media. Collectively, these results substantiate a new functional role for nitrated fatty acids, demonstrating that OA-NO2 and LNO2 exert an anti-inflammatory function against the inflammatory cascade initiated by the representative pro-inflammatory cytokine, TNFα. [Copyright &y& Elsevier]

Abstract: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. In this study we evaluated the protective role of Tat-mediated APE1/ref-1 transduction on the tumor necrosis factor (TNF)-α-activated endothelial activation in cultured human umbilical vein endothelial cells. To construct Tat-APE1/ref-1 fusion protein, human full length of APE1/ref-1 was fused with Tat-protein transduction domain. Purified Tat-APE1/ref-1 fusion protein efficiently transduced cultured endothelial cells in a dose-dependent manner and reached maximum expression at 1h after incubation. Transduced Tat-APE1/ref-1 showed inhibitory activity on the TNF-α-induced monocyte adhesion and vascular cell adhesion molecule-1 expression in cultured endothelial cells. These results suggest Tat-APE1/ref-1 might be useful to reduce vascular endothelial activation or vascular inflammatory disorders. [Copyright &y& Elsevier]

Abstract: The aim of this study was to investigate the effect of fluctuations in blood glucose levels on atherogenesis. Apolipoprotein (apo) E-deficient mice fed maltose twice daily were used as a model of repetitive postprandial glucose spikes. We investigated the number of macrophages adherent to the endothelium and the area of fibrotic arteriosclerotic lesions, with and without administration of miglitol, an α-glucosidase inhibitor. Macrophage adhesion to endothelial cells in thoracic aorta was quantitated by the en face method for optimal observation of endothelial surface after immunohistochemical staining for Mac-2. The area of arteriosclerotic lesions was measured in elastica van Giesson-stained proximal aorta. The number of adherent macrophages increased at 1 week after commencement of maltose feeding and the size of arteriosclerotic lesion increased at 5 weeks after such feeding. These increases were prevented by simultaneous use of miglitol. Our data demonstrated that glucose fluctuations accelerate atherogenesis. This was independent of changes in serum cholesterol level in vivo. Reduction of glucose fluctuation by α-glucosidase inhibitor efficiently controlled the progression of atherosclerosis. [Copyright &y& Elsevier]