Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA−GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED 50 ) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA−GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA−GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.
Macrophage type-I and type-II class-A scavenger receptors (MSR-A) are implicated in the pathological deposition of cholesterol during atherogenesis as a result of receptor-mediated uptake of modified low-density lipoproteins (mLDL). MSR-A can bind an extraordinarily wide range of ligands, including bacterial pathogens, and also mediates cation-independent macrophage adhesion in vitro. Here we show that targeted disruption of the MSR-A gene in mice results in a reduction in the size of atherosclerotic lesions in an animal deficient in apolipoprotein E. Macrophages from MSR-A-deficient mice show a marked decrease in mLDL uptake in vitro, whereas mLDL clearance from plasma occurs at a normal rate, indicating that there may be alternative mechanisms for removing mLDL from the circulation. In addition, MSR-A-knockout mice show an increased susceptibility to infection with Listeria monocytogenes or herpes simplex virus type-1, indicating that MSR-A may play a part in host defence against pathogens.
Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins
Lung Krüppel-like factor (LKLF/KLF2) is an endothelial transcription factor that is crucially involved in murine vasculogenesis and is specifically regulated by flow in vitro. We now show a relation to local flow variations in the adult human vasculature: decreased LKLF expression was noted at the aorta bifurcations to the iliac and carotid arteries, coinciding with neointima formation. The direct involvement of shear stress in the in vivo expression of LKLF was determined independently by in situ hybridization and laser microbeam microdissection/reverse transcriptase-polymerase chain reaction in a murine carotid artery collar model, in which a 4-to 30-fold induction of LKLF occurred at the high-shear sites. Dissection of the biomechanics of LKLF regulation in vitro demonstrated that steady flow and pulsatile flow induced basal LKLF expression 15-and 36-fold at shear stresses greater than ϳ5 dyne/cm 2 , whereas cyclic stretch had no effect. The focal development of atherosclerosis has been linked to the local variations in blood flow that are observed near the irregular blood vessel geometries of bifurcations and bends. 1,2 Continuous exposure of endothelial cells to flow in vivo generates a tangential force, shear stress, across their apical surfaces. A large number of studies support the hypothesized antiatherosclerotic effect of shear stress on the endothelium, and are mainly based on the ability of shear stress to modulate endothelial gene expression. 3 Throughout the recent years, a collection of shear stress-responsive endothelial genes has been established. 4 -8 Usually no clear distinction is made between genes induced by prolonged shear and those induced by short-term shear (Ͻ24 hours), although the latter class typically represents a general stress response also observed with turbulent flow types and seems more related to endothelial dysfunction. Based on the rationale that only genes induced by prolonged shear would represent the healthy transcriptome, we previously identified a limited number of genes that are still highly induced after exposing human umbilical vein endothelial cells (HUVECs) to flow for 7 days, but which are not induced by various other (inflammatory) stimuli. 6 The expression of one of those genes, the transcription factor lung Krü ppel-like factor (LKLF/
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