There is much interest in the significance of apolipoproteins and proteins that are noncovalently associated with lipoproteins. It is possible that the high ionic strength used for isolation of lipoproteins with KBr and NaI could alter the pattern of associated exchangeable proteins. Here we describe lipoprotein classes fractionation from up to 0.5 ml of serum or plasma with buffers of physiological ionic strength and pH prepared with deuterium oxide (D 2 O) and sucrose. An advantage of the D 2 O/sucrose procedure was that the lipoproteins could be directly analyzed by the techniques described without need for desalting. We compared the isolated lipoproteins with those obtained using ultracentrifugation in KBr from the same plasma pool. Electrophoretic homogeneity of the lipoproteins was very similar using the two methods, as well as their lipid composition evaluated by HPLC. Two-dimensional electrophoresis and surface-enhanced laser adsorption/ionization time-offlight mass spectrometry indicated that the patterns of exchangeable proteins of VLDL isolated using with the two procedures were very similar. However, significant differences were found in the profiles of LDL and HDL, indicating that the D 2 O/sucrose method allowed a more complete characterization of its exchangeable apolipoproteins and proteins.-Ståhlman, M., P. Davidsson, I. Kanmert, B. Rosengren, J. Borén, B. Fagerberg, and G. Camejo. Proteomics and lipids of lipoproteins isolated at low salt concentrations in D 2 O/sucrose or in KBr. J. Lipid Res. 2008. 49: 481-490.
The reversible interaction of low density lipoprotein (LDL) with arterial chondroitin sulfate proteoglycans (CSPGs) or glycosaminoglycans (GAGs) selects LDL particles with a high affinity for sulfated GAGs and also induces modifications in apolipoprotein B (apo B) and the lipid organization of the lipoprotein. In the present work we studied the effect that the reversible interaction with sulfated polysaccharides has on the susceptibility of LDL to in vitro oxidation. For this purpose soluble, nonaggregated CSPG-or GAG-treated LDL was subjected to oxidation in the presence of 5 fiM CuSO 4 for as long as 48 hours. The rate of formation of thiobarbituric acid-reactive substances, the decrease in isoelectric point, the increase in relative electrophoretic mobility of LDL, the higher degradation rate by human macrophages, and the lower degradation rate by human arterial smooth muscle cells showed that LDLs exposed to CSPGs and GAGs were significantly more susceptible to oxidation than native LDL. Results from competition experiments indicate that C6S-treated LDL after 4 hours of oxidation is taken up via the acetylated LDL receptor in human macrophages. Coincubation of lipoproteins with human macrophages or human arterial smooth muscle cells for 24 hours also indicated that C6S-treated LDL was more susceptible to cell-induced modifications than native LDL. The occurrence in vivo of similar processes may contribute to focal retention, increased rate oxidation of LDL in the arterial intima, and foam cell formation during atherogenesis. ( 11 The interaction of low density lipoprotein (LDL) with human arterial chondroitin sulfate proteoglycans (CSPGs) or glycosaminoglycans (GAGs) induces changes in the apo B lipoprotein and lipid organization that can be observed by
Objective-We investigated the potential role of ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motif type I) in atherogenesis. Methods and Results-ADAMTS-1 is expressed at the highest levels in the aorta when compared with other human tissues examined. Immunolocalization studies in human aorta and coronary artery indicate that ADAMTS-1 expression is mainly seen at low levels in the medial layer, but upregulated in the intima when plaque is present. We found that ADAMTS-1 mRNA levels are significantly higher in proliferating/migrating cultured primary aortic vascular smooth muscle cells (VSMCs) compared with resting/confluent cells. Using the mouse carotid artery flow cessation model, we show that there are differences in vessel remodeling in ADAMTS-1 transgenic/apoE-deficient mice compared with apoE deficiency alone, particularly a significant increase in intimal hyperplasia. We show that ADAMTS-1 can cleave the large versican containing proteoglycan population purified from cultured human aortic VSMCs. Finally, using versican peptide substrates, we show data suggesting that ADAMTS-1 cleaves versican at multiple sites.
Conclusion-We
See page 12Early in atherogenesis, VSMCs from the media are thought to migrate into the intima and contribute to the development of atherosclerotic lesions. Although what initially triggers these events is not known, it is thought that proteases released by VSMCs degrade the matrix proteins in the intima, particularly the main proteoglycan of the arterial intima versican, making the intima more permissive for invasion by VSMCs. One recently discovered family of metalloproteases, the ADAMTS family, might play a key role in atherogenesis by modulating the degradation of versican and possibly other proteoglycans.The first member of this family to be identified is ADAMTS-1. 3,4 It has been observed that ADAMTS-1 mRNA is upregulated substantially in human umbilical vein endothelial cells and cardiac microvascular endothelial cells under shear stress, suggesting regulation during flow-dependent vascular remodeling. 5 ADAMTS-1 has been shown to cleave the proteoglycan versican, which is expressed by VSMCs. 6 -8 Versican can exist in 4 isoforms (V0, V1, V2, and V3), depending on alternative splicing of the chondroitin sulfate containing glycosaminoglycan domains. V0 versican contains all possible domains, whereas the glycosaminoglycan-alpha and glycosaminoglycan-beta domains are spliced out in the V1 and V2 versican isoforms, respectively. 7 ADAMTS-1 and ADAMTS-4 have been shown to cleave V1/V0 versican at the Glu 441 -Ala 442 /Glu 1428 -Ala 1429 bond and the product of this cleavage was shown to be present in human atherosclerotic plaques by immunohistochemistry using neoepitope antibodies. 8 ADAMTS-1 has also been shown to have a role in matrix remodeling during ovulation in mice, which involves dissolution of connective matrix and cellular layers. A. ADAMTS-1-deficient mice displayed impaired ovulation, and the authors proposed that this was at least partially caused by...
Inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) reduce low-density lipoprotein (LDL) cholesterol and are used for treatment of dyslipidemia. Current PCSK9 inhibitors are administered via subcutaneous injection. We present a highly potent, chemically modified PCSK9 antisense oligonucleotide (ASO) with potential for oral delivery. Past attempts at oral delivery using earlier-generation ASO chemistries and transient permeation enhancers provided encouraging data, suggesting that improving potency of the ASO could make oral delivery a reality. The constrained ethyl chemistry and liver targeting enabled by N-acetylgalactosamine conjugation make this ASO highly potent. A single subcutaneous dose of 90 mg reduced PCSK9 by >90% in humans with elevated LDL cholesterol and a monthly subcutaneous dose of around 25 mg is predicted to reduce PCSK9 by 80% at steady state. To investigate the feasibility of oral administration, the ASO was coformulated in a tablet with sodium caprate as permeation enhancer. Repeated oral daily dosing in dogs resulted in a bioavailability of 7% in the liver (target organ), about fivefold greater than the plasma bioavailability. Target engagement after oral administration was confirmed by intrajejunal administration of a rat-specific surrogate ASO in solution with the enhancer to rats and by plasma PCSK9 and LDL cholesterol lowering in cynomolgus monkey after tablet administration. On the basis of an assumption of 5% liver bioavailability after oral administration in humans, a daily dose of 15 mg is predicted to reduce circulating PCSK9 by 80% at steady state, supporting the development of the compound for oral administration to treat dyslipidemia.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.