Receptors that transport vitellogenin into oocytes are of vital importance to egg-laying species because they promote oocyte development. In this study, we describe the cloning of the first hymenopteran vitellogenin receptor (VgR) cDNA. Using reverse transcription polymerase chain reaction (RT-PCR) and both 5’- and 3’- rapid amplification of cDNA ends (RACE), cDNA fragments encompassing the entire coding region of a putative VgR from fire ant (= SiVgR) were cloned and sequenced. The complete SiVgR cDNA has a length of 5764 bp encoding a 1782-residue protein with a predicted molecular mass of 201.3 kDa. The deduced amino acid sequence of the SiVgR revealed that it encoded a protein belonging to the low-density lipoprotein receptor superfamily. The number and arrangement of modular domains of SiVgR are the same as those of mosquito and fruit fly VgRs, except there are only four Class A cysteine-rich repeats in the first ligand binding domain of SiVgR compared to five in the mosquito and fruit fly. The deduced amino acid sequence of the SiVgR exhibited 35% and 31% identity to those of the mosquito and fruit fly VgRs, respectively. Northern blot analysis demonstrated that the 7.4-kb SiVgR mRNA was present only in Northern blot analysis demonstrated that the 7.4-kb SiVgR mRNA was present only in ovaries of reproductive females − both alates (virgins) and queens (mated) and was more abundant in alates. The developmental profile of transcriptional expression was determined by semiquantitative RT-PCR. It showed that the SiVgR transcript increased 6-fold from 0- to 10-days after mating, then remained constant through 30 days. It also showed that the SiVgR transcripts increased with age in alate virgin females. The transcriptional expression of the SiVgR was up-regulated more than two-fold by methoprene, a juvenile hormone analog, as determined by using an in vitro system. This suggested the SiVgR gene is JH regulated.

Receptors that transport vitellogenin into oocytes are of vital importance to egg-laying species because they promote oocyte development. In this study, we describe the cloning of the first hymenopteran vitellogenin receptor (VgR) cDNA. Using reverse transcription polymerase chain reaction (RT-PCR) and both 5’- and 3’- rapid amplification of cDNA ends (RACE), cDNA fragments encompassing the entire coding region of a putative VgR from fire ant (= SiVgR) were cloned and sequenced. The complete SiVgR cDNA has a length of 5764 bp encoding a 1782-residue protein with a predicted molecular mass of 201.3 kDa. The deduced amino acid sequence of the SiVgR revealed that it encoded a protein belonging to the low-density lipoprotein receptor superfamily. The number and arrangement of modular domains of SiVgR are the same as those of mosquito and fruit fly VgRs, except there are only four Class A cysteine-rich repeats in the first ligand binding domain of SiVgR compared to five in the mosquito and fruit fly. The deduced amino acid sequence of the SiVgR exhibited 35% and 31% identity to those of the mosquito and fruit fly VgRs, respectively. Northern blot analysis demonstrated that the 7.4-kb SiVgR mRNA was present only in Northern blot analysis demonstrated that the 7.4-kb SiVgR mRNA was present only in ovaries of reproductive females − both alates (virgins) and queens (mated) and was more abundant in alates. The developmental profile of transcriptional expression was determined by semiquantitative RT-PCR. It showed that the SiVgR transcript increased 6-fold from 0- to 10-days after mating, then remained constant through 30 days. It also showed that the SiVgR transcripts increased with age in alate virgin females. The transcriptional expression of the SiVgR was up-regulated more than two-fold by methoprene, a juvenile hormone analog, as determined by using an in vitro system. This suggested the SiVgR gene is JH regulated.

Any lipoproteins and cells present in the inflammatory environment of atherosclerotic plaques are likely to be exposed to high levels of oxidative stress. As 7,8-dihydroneopterin (7,8-NP) is synthesized by interferon-γ (IFN-γ)-activated macrophages, this pteridine is also thought to exist at sites of inflammation. 7,8-NP s in vivo role remains controversial, but numerous in vitro studies have identified a radical scavenging activity. The possibility of 7,8-NP protecting against oxidative damage in inflammatory environments like plaque was investigated in this thesis. Both human monocyte-derived macrophages (HMDMs) and low density lipoprotein (LDL) were used as substrates. The extent of protein hydroperoxide formation in each model, and 7,8-NP s effect on this process, were specifically studied since most previous research has focussed on lipid rather than protein peroxidation. For the first time, neopterin (including oxidized 7,8-NP) was also directly detected by high performance liquid chromatography in the inflammatory environments of 19 pus and two atherosclerotic plaque samples. Peak concentrations even reached the low micromolar range. The positive correlation identified in the pus between neopterin and a well known antioxidant, vitamin E, further hinted at a potential antioxidant function. However, no significant association was noted between neopterin and markers of protein or lipid oxidation. Exposure of HMDMs to the AAPH peroxyl radical generator resulted in significant quantities of lipid hydroperoxides but not protein hydroperoxides, as detected by the FOX assays. This is likely due to the large accumulation of polyunsaturated fatty acidrich lipid in the primary HMDMs during differentiation in 10% human serum and is of relevance to atherosclerotic plaque, where macrophages also become lipid-loaded. The addition of up to 200μM 7,8-NP failed to prevent AAPH-induced lipid peroxidation and was also unable to inhibit a loss of cellular thiols or viability. This lack of effect suggests the damaging peroxyl radicals are not being scavenged by 7,8-NP. The high lipid content of HMDM cells appears to cause the AAPH and/or 7,8-NP to localize to a cellular site, where they are unable to interact. Macrophage-mediated oxidation of LDL in iron(II)-supplemented Hams F10 was associated with the formation of 30-40 moles of protein hydroperoxides per mole of LDL. The close parallel between protein and lipid peroxidation supports the theory that lipid-derived radicals are involved in protein hydroperoxide formation on LDL and indicates that protein hydroperoxides are an early product of LDL oxidation. Their detection during exposure of LDL to both the THP-1 macrophage cell line and primary HMDM cells confirms that protein hydroperoxides are also a normal consequence of macrophage-mediated LDL oxidation. Incubation of LDL with micromolar 7,8-NP prevented macrophage-mediated protein hydroperoxide formation in a concentration-dependent manner. Lipid oxidation and vitamin E loss were similarly inhibited by 7,8-NP during the cell-mediated attack of LDL. Kinetic analysis revealed protection due to extension of the lag phase, with 7,8-NP depletion and initiation of the propagation phase coinciding. This supports a radical scavenging activity for 7,8-NP, resulting in protection of the entire LDL particle. By contrast, the release of nanomolar quantities of 7,8-NP by IFN-γ-stimulated THP-1 macrophages failed to prevent LDL oxidation. HMDMs activated by IFN-γ did significantly inhibit LDL oxidation, including protein hydroperoxide formation, for up to 48 hours but this antioxidant effect was not due to the de novo synthesis of 7,8-NP. These results indicate that both the prevalence of protein hydroperoxides, and the ability of 7,8-NP to act as an antioxidant, depend on the system under investigation. Neopterin exists in inflammatory environments but, considering the lack of protection against AAPH-mediated HMDM oxidation and the 7,8-NP concentration required to inhibit macrophage-mediated LDL oxidation, strong evidence for an antioxidant activity of 7,8-NP in atherosclerotic plaque is currently lacking.

A aterosclerose é uma doença crônico-inflamatória multifatorial com o envolvimento do sistema imunológico, sendo o resultado da interação de diferentes elementos celulares. A lipoproteína de baixa densidade eletronegativa [LDL(-)], capaz de induzir o acúmulo de ésteres de colesterol em macrófagos e a subsequente formação de células espumosas, desempenha um papel-chave na doença. Anticorpos recombinantes têm sido gerados nas últimas décadas, como o scFv (single chain fragment variable), com o intuito de serem utilizados como uma novas alternativas de prevenção para o surgimento da lesão. Diante do papel da LDL(-) na aterosclerose, este projeto avaliou o efeito da imunização passiva de camundongos LDLr-/-- com scFv anti-LDL(-) em solução e scFv anti-LDL(-) conjugado a nanocápsulas, em relação ao desenvolvimento e progressão da aterosclerose. Após obtenção do scFv e sua conjugação à nanocápsulas (NC-scFv), ensaios in vitro determinaram a diminuição da captação de LDL(-) em macrófagos tratados com o scFv 2C7 anti-LDL(-) em solução. No entanto, o tratamento com NC-scFv promoveu o aumento da internalização de LDL(-) em relação ao controle, possivelmente por um mecanismo de endocitose mediada por receptor específico. Estudos in vivo determinaram que camundongos LDLr-/- com idade entre 2 e 3 meses tratados com o scFv em solução apresentaram menor área de lesão aterosclerótica (p<0,05) quando comparados ao controle e que animais com 3 a 4 meses de idade tratados com NC-scFv demonstraram uma tendência à diminuição do mesmo parâmetro. Na análise da expressão de proteínas por imunohistoquímica, ambos os grupos tratados com scFv 2C7 anti-LDL(-) em solução e NC-scFv demostraram redução significativa da expressão dos receptores CD14 e TLR-4 no local da lesão. Esse achado tem grande importância, uma vez que dados da literatura apresentam ambos os receptores como possíveis candidatos ao reconhecimento da LDL(-). Diante dos dados obtidos, o estudo evidenciou a eficácia do scFv 2C7 anti-LDL(-) em solução e da formulação NC-scFv no contexto da aterosclerose, possibilitando a sua utilização como estratégias terapêuticas na intervenção precoce para prevenir o desenvolvimento e a progressão da doença. / Atherosclerosis is a chronic inflammatory multifactorial disease related to the immune system and being the result of interaction of different cellular elements. The electronegative LDL, since the changes undergone by this particle are able to induce the accumulation of cholesterol esters in macrophages and the subsequent formation of foam cells, plays a key role in atherosclerosis. Recombinant antibodies have been generated in recent decades, such as scFv, (single chain fragment variable), and they may be used as a new alternative treatment for atherosclerosis treatment or prevention. Considering the role of LDL(-) in atherosclerosis, this project evaluated the effects of the treatment with anti-LDL(-) scFv 2C7 solution and anti-LDL(-) scFv conjugated to nanocapsules as a passive immunization strategy on atherosclerosis induced in LDL receptor knockout mice. After obtaining the anti-LDL(-) scFv 2C7 solution and its conjugation to nanocapsules (NC-scFv), in vitro tests led to the decrease in LDL(-) uptake in macrophages treated with anti-LDL(-) scFv 2C7. However, the treatment of macrophages with NC-scFv promoted increased internalization of LDL(-) as compared to control, possibly due to a mechanism of specific receptor-mediated endocytosis. In vivo studies have determined that LDLR-/- mice aged 2 and 3 months treated with anti-LDL(-) scFv 2C7 solution showed less atherosclerotic lesion area (p <0.05) compared to control and animals aged 3 to 4 months treated with NC-scFv showed a decreasing tendency of the same parameter. In the analysis of protein expression by immunohistochemistry, both groups treated with anti-LDL(-) scFv 2C7 solution and NC-scFv showed significant reduction of CD14 receptor expression and TLR-4 at the lesion site. This finding is of great importance, since the literature has both receptors as candidates for recognition of the LDL(-). From the data obtained, the study demonstrated the efficacy of treatments anti-LDL(-) scFv 2C7 in solution and NC-scFv in the context of atherosclerosis, enabling their use as therapeutic strategies in the early intervention to prevent the development and progression of the disease.

Low-density lipoproteins (LDL) are heterogeneous nanoparticles containing one copy of apolipoprotein B (~550 kDa) and thousands of lipids. LDL are the main plasma carriers of cholesterol and the major risk factor for atherosclerosis, the number one cause of death in the developed world. In atherosclerosis, LDL lipids are deposited in the arterial intima. Fusion of modified LDL in the arterial wall is an important underexplored triggering event in early atherosclerosis. Previous studies from our laboratory showed that thermal denaturation mimics LDL remodeling and fusion, and revealed the kinetic origin of LDL stability. Here, we report the first quantitative kinetic analysis of LDL stability. We show that LDL denaturation monitored by turbidity follows a sigmoidal time course that is unique among lipoproteins, suggesting that slow conformational changes in apoB precede lipoprotein fusion. High activation energy of LDL denaturation, Ea~100 kcal/mol, indicates disruption of extensive protein-protein and protein-lipid interactions involving large apoB domains. Next, we combined size-exclusion chromatography, gel electrophoresis and electron microscopy to show that dimerization is a common early step preceding LDL fusion. Monoclonal antibody binding studies indicated that α-helices in the N-terminal βα1 domain of apoB undergo conformational changes at early stages of LDL aggregation and fusion. Better understanding of these structural changes that prime LDL for fusion is important, as it may help control this pathogenic process before it occurs. We applied the kinetic approach to test how selected factors that are expected to contribute to LDL fusion in vivo affect the rate of LDL fusion and rupture in vitro. The results show that LDL fusion accelerates at pH<7, which may contribute to LDL retention in acidic atherosclerotic lesions. Fusion also accelerates upon increasing LDL concentration in near-physiologic range, which likely contributes to atherogenesis. Further, we showed that thermal stability of LDL decreases with increasing particle size, indicating that the pro-atherogenic properties of small dense LDL do not result from their enhanced fusion. Our work provides the first kinetic approach to measuring LDL stability and suggests that lipid-lowering therapies that reduce LDL concentration but increase the particle size may have opposite effects on LDL fusion.

Development of atherosclerosis, the hardening of the arteries, is dependent on levels of serum cholesterol, which is regulated by the liver via LDL receptors (LDLR). The expression and internalization of LDL receptors depend on several proteins including PCSK9. In fact, previous studies in our laboratory have shown that NEU1 down regulation leads to LDLR hypersialylation which results in its stabilization via reduced interactions with PCSK9. New evidence suggests that NEU1 which de-sialylates LDLR, may affect the ability of another hepatic receptor, the asialoglycoprotein receptor (ASGR), which is comprised of ASGR1 and ASGR2, to interact with LDLR potentially causing its internalization and therefore reduced ability to take up LDL. We investigated how sialidase plays a role in the interaction of ASGR with LDLR. Knockdown and overexpression experiments suggest that NEU1 allows stabilization of LDLR at the cell membrane via ASGR interactions. Treatment of HepG2 cells with monensin which inhibits recycling from the early endosome, unveiled a new truncated ASGR1 isoform potentially lacking its lectin motif. This may be a novel regulatory step in ASGR biosynthesis that warrants further studies. Lysosomal inhibition with chloroquine resulted in concurrent accumulations of NEU1, LDLR and ASGR1, further suggesting these proteins are biosynthetically connected. Our studies revealed a novel isoform of ASGR1 in membrane fractions of HepG2 cell lysates that can associate with NEU1 and LDLR. The impact of NEU1 and ASGR1 on the function and stability of LDLR might lead to new clues for lowering serum cholesterol and reducing atherosclerosis. / Thesis / Master of Science (MSc)

AIMS: Macrophages (MPs) and vascular smooth muscle cells (VSMCs) closely interact within the growing atherosclerotic plaque. An in vitro co-culture model was established to study how MPs modulate VSMC behaviour. METHODS AND RESULTS: MPs were exposed to fluorescence-labelled-acetylated LDL (FL-acLDL) prior to co-culture with VSMCs. Fluorescence microscopy visualized first transport of FL-acLDL within 6 h after co-culture implementation. When MPs had been fed with FL-acLDL in complex with fluorescence-labelled cholesterol (FL-Chol), these complexes were also transferred during co-culture and resulted in cholesterol positive lipid droplet formation in VSMCs. When infected with a virus coding for a fusion protein of Rab5a and fluorescent protein reporter (FP) to mark early endosomes, no co-localization between Rab5a-FP and the transported FL-acLDL within VSMCs was detected implying a mechanism independent of phagocytosis. Next, expression of lysosome-associated membrane glycoprotein 1 (LAMP1)-FP, marking all lysosomes in VSMCs, revealed that the FL-acLDL was located in non-acidic lysosomes. MPs infected with virus encoding for LAMP1-FP prior to co-culture demonstrated that intact fluorescence-marked lysosomes were transported into the VSMC, instead. Xenogenic cell composition (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat-specific primers rendered induction of genes typical for MPs and down-regulation of the cholesterol sensitive HMG-CoA reductase. CONCLUSION: Our results demonstrate that acLDL/cholesterol-loaded lysosomes are transported from MPs into VSMCs in vitro. Lysosomal transfer results in a phenotypic alteration of the VSMC towards a foam cell-like cell. This way VSMCs may lose their plaque stabilizing properties and rather contribute to plaque destabilization and rupture.

C-reactive protein (CRP) is secreted by hepatocytes as a pentameric molecule made up of identical monomers, circulates in the plasma as pentamers, and localizes in atherosclerotic lesions. In some cases, localized CRP was detected by using monoclonal antibodies that did not react with native pentameric CRP but were specific for isolated monomeric CRP. It has been reported that, once CRP is bound to certain ligands, the pentameric structure of CRP is altered so that it can dissociate into monomers. Accordingly, the monomeric CRP found in atherosclerotic lesions may be a stationary, ligand-bound, by-product of a ligand-binding function of CRP. CRP binds to modified forms of low-density lipoprotein (LDL). The binding of CRP to oxidized LDL requires acidic pH conditions; the binding at physiological pH is controversial. The binding of CRP to enzymatically-modified LDL occurs at physiological pH; however, the binding is enhanced at acidic pH. Using enzymatically-modified LDL, CRP has been shown to prevent the formation of enzymatically-modified LDL-loaded macrophage foam cells. CRP is neither pro-atherogenic nor atheroprotective in ApoE-/-and ApoB100/100Ldlr-/-murine models of atherosclerosis, except in one study where CRP was found to be slightly atheroprotective in ApoB100/100Ldlr-/-mice. The reasons for the ineffectiveness of human CRP in murine models of atherosclerosis are not defined. It is possible that an inflammatory environment, such as those characterized by acidic pH, is needed for efficient interaction between CRP and atherogenic LDL during the development of atherosclerosis and to observe the possible atheroprotective function of CRP in animal models.

Of the challenges facing the improvement of human health, none has taken the forefront quite like the endeavor to discover novel treatments for heart disease. As heart disease has now become the leading cause of death throughout the world [1], the medical community has made incredible strides in the mission to treat atherosclerosis which is the major contributor to heart disease. Very Low Density Lipoproteins (VLDL) are secreted by the liver and subsequently converted to Low Density Lipoproteins (LDL). Many factors contribute to the narrowing of the arterial walls, however oxidized LDL is the main factor that leads to the deposition of plaque, leading to atherosclerosis pathologies. Recently, a main focus of research into atherosclerotic processes has been the synthesis and trafficking of VLDL in hepatocytes. The rate-limiting step for the secretion of VLDL from the liver has been determined to be the transport of VLDL from the endoplasmic reticulum (ER) to the Golgi apparatus. VLDL molecules are transported in a specialized transport vesicle the Very Low Density Lipoprotein Transport Vesicle (VTV) [2]. VLDL’s core protein, apolipoproteinB-100 (apoB100), is initially lipidated in the ER, and then subsequently delivered to the Golgi apparatus where the VLDL molecule undergoes maturation involving further lipidation and glycosylation of apoB100. Oxysterol Binding Proteins (OSBP) and the sub family OSBP Related Proteins (ORP) have been implicated in many different trafficking processes, mainly the trafficking of sterols, cholesterol, and lipids. Recently, ORP 10 was shown to be a negative regulator of apoB100 secretion in growth medium [3]. Using co-immunoprecipitation, the current study shows that ORP 10 interacts with VLDL’s core protein apoB100 directly. Employing an in vitro budding assay, we show that the blocking of ORP 10 with a specific antibody against ORP10 increases VTV formation from the ER. Given that the ER to Golgi pathway is the rate-limiting step in overall VLDL secretion, these findings support the conclusion that ORP 10 is a negative regulator of VLDL trafficking between the ER and Golgi, and that this process is mediated by the ORP 10 protein binding with apoB100.

AIMS: Macrophages (MPs) and vascular smooth muscle cells (VSMCs) closely interact within the growing atherosclerotic plaque. An in vitro co-culture model was established to study how MPs modulate VSMC behaviour. METHODS AND RESULTS: MPs were exposed to fluorescence-labelled-acetylated LDL (FL-acLDL) prior to co-culture with VSMCs. Fluorescence microscopy visualized first transport of FL-acLDL within 6 h after co-culture implementation. When MPs had been fed with FL-acLDL in complex with fluorescence-labelled cholesterol (FL-Chol), these complexes were also transferred during co-culture and resulted in cholesterol positive lipid droplet formation in VSMCs. When infected with a virus coding for a fusion protein of Rab5a and fluorescent protein reporter (FP) to mark early endosomes, no co-localization between Rab5a-FP and the transported FL-acLDL within VSMCs was detected implying a mechanism independent of phagocytosis. Next, expression of lysosome-associated membrane glycoprotein 1 (LAMP1)-FP, marking all lysosomes in VSMCs, revealed that the FL-acLDL was located in non-acidic lysosomes. MPs infected with virus encoding for LAMP1-FP prior to co-culture demonstrated that intact fluorescence-marked lysosomes were transported into the VSMC, instead. Xenogenic cell composition (rat VSMC, human MP) and subsequent quantitative RT-PCR with rat-specific primers rendered induction of genes typical for MPs and down-regulation of the cholesterol sensitive HMG-CoA reductase. CONCLUSION: Our results demonstrate that acLDL/cholesterol-loaded lysosomes are transported from MPs into VSMCs in vitro. Lysosomal transfer results in a phenotypic alteration of the VSMC towards a foam cell-like cell. This way VSMCs may lose their plaque stabilizing properties and rather contribute to plaque destabilization and rupture.