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.

Apolipoprotein C-1 (apo C-1) enriched HDL has been described as an atherogenic form of HDL associated with an increased risk for cardiovascular disease (CVD). The objective of the present study was to develop a rapid method for the separation, purification, and characterization of Apo C-1 from serum. We isolated and characterize HDL subclasses from individuals with and without angiographically-proven CVD who have elevated and normal-to-low HDL-C levels. Ultracentrifugation was linked with immunoaffinity separations for the specific separation of Apo C-1 enriched HDL from other lipoproteins. A 50 μL sample of serum is diluted in TRIS HCl buffer (pH 7.5) and incubated with CNBr-activated Sepharose (Amersham) containing antibodies to apo C-1 (Academy Bio-medical Company). The apo C-1-depleted serum is removed by centrifugation and all apo C-1-containing lipoproteins are released from the Sepharose beads at pH 2. The apo C-1-depleted sample and the apo C-1-containing sample were ultracentrifuged to obtain a lipoprotein density profile in the absence and presence of apo C-1. Density Lipoprotein Profiling (DLP) gives relevant information of lipoproteins, such as density and subclass characterization, and is a novel approach to purify apo C-1-enriched HDL. An additional advantage of this approach is that lipoprotein-a (Lp(a)), which is often an interfering component in the HDL density region, is eliminated. Results show feasibility that these methods could be used in a clinical setting, was achieved. This measurement may yield a precise and quantitative profile of the distribution of apo C-1 for all lipoprotein particles including HDL.

Since cardiovascular disease is one of the leading causes of mortality all over the world, it is becoming increasingly important and relevant to develop new analytical techniques for the analysis of the mechanisms of this complex disease as well as for clinical applications. The overall objective of this research was to develop an array of methods for the analysis of triglyceride rich lipoproteins (TRL) in human serum and to apply these methods to clinical samples. TRL particles are mainly derived from dietary fats, which are positively correlated with cardiovascular disease. The mechanism behind which triglycerides cause cardiovascular disease is not well understood. The analysis of TRL by novel methods including density gradient ultracentrifugation and density profiling, gel electrophoresis, in vitro enzymatic assays, and capillary zone electrophoresis are presented here. The development of a novel density profiling method for the remnant lipoproteins class of TRL and its application to clinical samples was successful. In addition, TRL were successfully evaluated for their composition by gel electrophoresis, in vitro enzymatic assays and capillary zone electrophoresis. The results of these analyses demonstrate great potential for the use of these new methods as analytical tools for researchers in understanding the mechanism behind the onset of cardiovascular disease by TRL and their triglycerides as well as diagnostic tools for clinicians.

Lipoprotein(a) (Lp(a)) has been described as an emerging risk factor for cardiovascular disease. The complexity of the Lp(a) molecule sets a challenge for the determination of the risk it represents for the cardiovascular system. The objective of the present study was to develop a rapid method for the separation, purification, density measurement, and characterization of Lp(a) from serum using a procedure that is isoform independent. The objective was met by linking ultracentrifugation with affinity separations for the specific separation of Lp(a) from other lipoproteins. The mean density distribution of Lp(a) was determined by a differential density lipoprotein profile (DDLP). For DDLP, the lipoprotein density distribution of a serum sample with elevated Lp(a) levels was determined by ultracentrifugation using NaBiEDTA complex as a density gradient. Lp(a) was removed from a second aliquot of the same serum sample by carbohydrate affinity using wheat germ agglutinin (WGA). WGA was demonstrated to have high specificity for Lp(a) in serum. The Lp(a)-depleted sample was ultracentrifuged to obtain a lipoprotein density distribution in the absence of Lp(a). A DDLP was obtained after subtracting the Lp(a)-depleted lipoprotein density profile from the untreated lipoprotein density profile. DDLP gives relevant information of the lipoproteins in serum such as density, Lp(a) isoform, and subclass characteristics. Lp(a) was quantitatively removed from serum with a recovery efficiency of more than 80%. Lp(a) was purified by ultracentrifugation. Lp(a) obtained in this way retained its inherent density and immunoreactivity. Lp(a) was further characterized by gel electrophoresis and Western blot as well as by capillary electrophoresis. Capillary electrophoresis demonstrated to be a powerful analytical technique for the characterization of Lp(a) and apoprotein(a) isoforms. The major outcome of this research was the effectiveness of using affinity separations coupled with density ultracentrifugation for the isolation of pure Lp(a) from serum and its isoform characterization based on density and electromobility. The methodology developed and described here are relevant in a clinical setting for the analysis of Lp(a).

Thesis (doctoral)--University of Helsinki, 1986. / Includes bibliographical references (p. 31-38).

To overcome the membrane permeability barrier and low nutrient availability in the environment, Gram-negative organisms have evolved many mechanisms dedicated to the intake of nutrients. One such mechanism is the long-chain fatty acid uptake pathway. This pathway involves machinery that transports fatty acids across the outer membrane and into the cell, where the lipid can be used for either nutrition or for remodeling the structure of the membrane. Interestingly, the fatty acid species that can be recognized by this machinery differ between organisms; the aquatic pathogen Vibrio cholerae demonstrates a much wider substrate recognition profile than other Gram-negative species. In this work we elaborate on the lipid nutrients accessible to V. cholerae, demonstrating that it can use lysophosphatidylcholine as both a carbon source as well as a source of fatty acids for remodeling its outer membrane. We identify the enzyme responsible for the breakdown of lysophosphatidylcholine, VolA (Vibrio outer membrane lysophospholipase A). VolA is conserved in many Vibrio species as well as other aquatic Gram-negatives, annotated as a putative lipase. We show VolA is co-expressed with the outer membrane fatty acid transporter FadL; FadL serves to transport across the outer membrane those long-chain fatty acids freed from lysophosphatidylcholine by VolA. VolA is expressed on the surface of the cell as a membrane anchored lipoprotein; this is novel as VolA is the first identified example of a surface-localized lipoprotein phospholipase. Biochemical characterization of VolA shows that it acts as a canonical lysophospholipase in vitro, suggesting that it works in tandem with the FadL transporter, freeing fatty acids from lysophosphatidylcholine at the surface of the cell to be brought in via the fatty acid uptake pathway. This work expands on the currently understood lipid uptake abilities of Vibrio cholerae, demonstrating a novel mechanism for utilizing a nutrient not previously thought to metabolized. VolA is an important to our understanding of the larger picture of lipid uptake and how it contributes to the survival of Gram-negative organisms. / text

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are a risk factor for a variety of atherosclerotic disorders including coronary heart disease. In the current study, the investigators report that incubation of cultured human umbilical vein endothelial cells (HUVECs) with high concentrations of apolipoprotein(a)(apo(a)/Lp(a)) induces apoptosis and endothelial dysfunction in a dose dependent manner. Apo(a), the component of Lp(a) mediates these effects by inducing externalization of Annexin V, DNA condensation and fragmentation which are the hallmarks of death by apoptosis. The pathway of apo(a)-induced apoptosis is associated with overexpression of Bax, caspase-9, p53 phosphorylation, decreased in Bcl-2 expression and activation of caspase-3. Taken together, the data suggest that elevated concentration of apo(a) induces apoptosis in endothelial cells probably by activating the intrinsic pathway. The data also showed that apo(a) induces increased expression of the growth arrest protein (Gas1), which has been known to induce apoptosis and growth arrest in vitro. In addition the data showed that elevated apo(a)/Lp(a) attenuates endothelial nitric oxide (eNOS) activity and endothelin-1 (ET-1) in a dose and time-dependent manner, particularly with small apo(a) isoforms. In summary, the authors proposed a new signaling pathway by which apo(a)/Lp(a) induce apoptosis and this finding could help explain how apo(a)/Lp(a) mediate atherosclerosis related diseases. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2011-06-20 13:59:48.473

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.

Regensburg, Univ., Diss., 2008

Thesis (Ph. D.)--University of Hong Kong, 1997. / Includes bibliographical references (leaf 155-192).