High plasma levels of lipoprotein(a) [Lp(a)] are an independent risk factor for cardiovascular disease. Currently, no drugs have proven successful at lowering Lp(a) levels. Lp(a) is comprised of a LDL particle which is covalently bound to an additional glycoprotein, apolipoprotein(a) [apo(a)]. One approach is to develop inhibitors of Lp(a) assembly. It is therefore worth investigating inhibitors for the interaction between apo(a) and apoB-100 for their therapeutic potential to reduce plasma Lp(a) levels. A synthetic α-helical peptide, apoB₄₃₇₂₋₉₂ peptide, has been shown to be an effective inhibitor of Lp(a) assembly in vitro (IC₅₀= 40 [mu]M). Complexing of the apoB₄₃₇₂₋₉₂ peptide with dimyristoylphosphatidylcholine (DMPC) in 1:45 molar ratio increased its inhibitory activity to 4 [mu]M. Since the peptide:DMPC molar ratio previously used in vitro could not be obtained for in vivo study, lower molar ratios of the apoB₄₃₇₂₋₉₂ peptide-DMPC complex were assessed for their inhibitory activities on Lp(a) assembly in vitro and the Lp(a) lowering effect in vivo. A version of the apoB peptide modified to increase stability with DMPC under physiological conditions was also assessed. The aim of this project was to investigate the Lp(a) lowering effect of this peptide-DMPC complex in vivo and to determine its possible mechanism of action.
Studies showed that a 1:20 ratio of the apoB₄₃₇₂₋₉₂ peptide-DMPC complex exhibited an effective inhibition of Lp(a) assembly in vitro (IC₅₀= 9 [mu]M). Intravenous injection of this peptide-DMPC complex (1.3 � 0.2 mg peptide/mouse) significantly decreased Lp(a) levels at 1, 2 and 4 hours after injection compared with DMPC (p< 0.01). However, a lethal effect occurred in some animals after treatment. This effect is likely associated with the aggregation of the apoB₄₃₇₂₋₉₂ peptide-DMPC complex under physiological conditions.
In order to develop a more stable peptide, a synthetic peptide, apoB[4365-4396K] based on the apoB4365-4396 sequence and substituted with extra lysines was analyzed and shown to be α-helical in the absence and presence of DMPC. Intrinsic fluorescence studies showed that the apoB[4365-96K] peptide interacted with DMPC under physiological conditions. Also, the apoB[4365-96K] peptide-DMPC complex was shown to form small stable particles by dynamic light scattering. The apoB[4365-96K] peptide proved to be an effective inhibitor of Lp(a) assembly in vitro (IC₅₀= 38 [mu]M). Complexing the apoB[4365-96K] peptide to DMPC increased its inhibitory activity to 16 [mu]M.
Intravenous injection of the apoB[4365-96K] peptide-DMPC complex decreased the Lp(a) levels at 1 hour and 2 hours after injection compared to DMPC (p< 0.05). The apoB[4365-96K] peptide was present in circulation prominently at 1 hour after intravenous injection and this coincided with a decrease in Lp(a) levels. Neither the apoB[4365-96K] peptide-DMPC complex nor DMPC had a significant effect on Lp(a) levels after intraperitoneal injection.
The mechanism by which the apoB[4365-96K] peptide-DMPC complex lowers Lp(a) levels was further investigated. The apoB[4365-96K] peptide was found to associate with plasma lipoproteins through ultrafiltration and agarose gel shift assays. The association of the peptide with lipoproteins was dependent on α-helical structure and enhanced by complexing with DMPC. The association of the apoB[4365-96K] peptide with lipoproteins was also observed in plasma samples from mice treated with the apoB[436S-96K] peptide-DMPC complex intravenously. The association of the peptide-DMPC complex with plasma lipoproteins was most prominent at 1 hour after injection and this coincided with a maximum decrease in Lp(a) levels. These results suggest that the lipoprotein association of the apoB[4365-96K] peptide-DMPC complex played a role in lowering Lp(a) levels in vivo.
The work presented in this thesis has shown that apoB peptides containing apoB4372-4392 sequence have inhibitory effect on Lp(a) assembly in vitro and lower Lp(a) levels in vivo. The α-helical structure of the peptide is important for its lipid binding ability and its inhibitory activity on Lp(a) assembly. Furthermore, the association of peptide with lipoproteins appears to play a role in its ability to inhibit Lp(a) assembly in vitro and lower Lp(a) levels in vivo. The studies undertaken in this thesis suggest that altering lipoprotein charges and conformation may reduce the efficiency of Lp(a) assembly. The knowledge gained in this study may provide a basis for future design of inhibitors of Lp(a) assembly.
| Identifer | oai:union.ndltd.org:ADTP/217869 |
| Date | January 2008 |
| Creators | Wang, Yan-Ting, n/a |
| Publisher | University of Otago. Department of Biochemistry |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Yan-Ting Wang |
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