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Endothelial activation elicited by inflammatory agents is regarded as a key event in the pathogenesis of several vascular inflammatory diseases. In the present study, the inhibitory effects and underlying mechanism of C-type natriuretic peptide (CNP) on LPS-induced endothelial activation were examined in human umbilical vein endothelial cells (HUVECs). The effect of CNP on adhesion molecule expression was assessed using quantitative real-time RT-PCR and western blotting analyses. The nuclear factor-κB (NF-κB), MAPK, and PI3K/Akt signaling pathways in LPS-stimulated HUVECs were investigated using western blotting analyses, and the production of intracellular reactive oxygen species (ROS) was measured using a fluorescence method. Pretreatment with CNP inhibited LPS-induced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, and P-selectin in a concentration-dependent manner. CNP suppressed the phosphorylation of p65 and NF-κB activation in LPS-stimulated cells. Moreover, CNP reduced ERK1/2 and p38 phosphorylation induced by LPS but not JNK. Furthermore, CNP induced Akt phosphorylation and activation of hemeoxygenase-1 (HO-1) expression. CNP significantly inhibited the production of intracellular ROS. These results suggest that CNP effectively attenuated LPS-induced endothelial activation by inhibiting the NF-κB and p38 signaling pathways, eliminating LPS-induced intracellular ROS production, and activating the PI3K/Akt/HO-1 pathway in HUVECs; thereby, demonstrating that CNP may be a potential therapeutic target for the treatment of sepsis and inflammatory vascular diseases. [ABSTRACT FROM AUTHOR]

Background: The mechanical properties of cellular microenvironments play important roles in regulating cellular functions. Studies of the molecular response of endothelial cells to alterations in substrate stiffness could shed new light on the development of cardiovascular disease. Quantitative real-time PCR is a current technique that is widely used in gene expression assessment, and its accuracy is highly dependent upon the selection of appropriate reference genes for gene expression normalization. This study aimed to evaluate and identify optimal reference genes for use in studies of the response of endothelial cells to alterations in substrate stiffness. Methodology/Principal Findings: Four algorithms, GeNormPLUS, NormFinder, BestKeeper, and the Comparative ΔCt method, were employed to evaluate the expression of nine candidate genes. We observed that the stability of potential reference genes varied significantly in human umbilical vein endothelial cells on substrates with different stiffness. B2M, HPRT-1, and YWHAZ are suitable for normalization in this experimental setting. Meanwhile, we normalized the expression of YAP and CTGF using various reference genes and demonstrated that the relative quantification varied according to the reference genes. Conclusion/Significance:: Consequently, our data show for the first time that B2M, HPRT-1, and YWHAZ are a set of stably expressed reference genes for accurate gene expression normalization in studies exploring the effect of subendothelial matrix stiffening on endothelial cell function. We furthermore caution against the use of GAPDH and ACTB for gene expression normalization in this experimental setting because of the low expression stability in this study. [ABSTRACT FROM AUTHOR]

Templated assembly techniques have been extensively used to develop various types of hemoglobin (Hb) loaded particles with improved performance. However, several instability issues must still be solved, including Hb exposure, enhanced Hb auto-oxidation, and the relatively weak binding of Hb to cross-linkers. Herein, to meet the stability requirements for novel hemoglobin-based oxygen carriers (HBOCs), hemoglobin-polydopamine particles (Hb-PDA) were fabricated using a mild process that combines the co-precipitation of Hb and an inorganic template with the spontaneous adhesion of PDA. The Hb-PDA showed uniform size distribution, chemical integrity of both Hb and PDA, high biocompatibility, and robust oxygen delivery. Our results demonstrated that the use of polydopamine as a biocompatible coating material reduced Hb leakage from the particles under both static and flow conditions, thus mitigating the toxicity associated with free Hb and strengthening the stability of Hb particles. In addition, Hb-PDA reduced HUVEC (Human Umbilical Vein Cells) oxidative injury and scavenged 85% of the available hydroxyl radicals, exhibiting its potential to act as an antioxidant for encapsulated Hb. Hb-PDA therefore shows significant promise as a cell-like structurally and functionally stable HBOCs. [ABSTRACT FROM AUTHOR]