Uptake Mechanism of ApoE-Modified Nanoparticles on Brain Capillary Endothelial Cells as a Blood-Brain Barrier Model

Background: The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. Methodology/Principal Findings: In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. Conclusions/Significance: This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier

Reconstruction and functional analysis of altered molecular pathways in human atherosclerotic arteries

<p>Abstract</p> <p>Background</p> <p>Atherosclerosis affects aorta, coronary, carotid, and iliac arteries most frequently than any other body vessel. There may be common molecular pathways sustaining this process. Plaque presence and diffusion is revealed by circulating factors that can mediate systemic reaction leading to plaque rupture and thrombosis.</p> <p>Results</p> <p>We used DNA microarrays and meta-analysis to study how the presence of calcified plaque modifies human coronary and carotid gene expression. We identified a series of potential human atherogenic genes that are integrated in functional networks involved in atherosclerosis. Caveolae and JAK/STAT pathways, and S100A9/S100A8 interacting proteins are certainly involved in the development of vascular disease. We found that the system of caveolae is directly connected with genes that respond to hormone receptors, and indirectly with the apoptosis pathway.</p> <p>Cytokines, chemokines and growth factors released in the blood flux were investigated in parallel. High levels of RANTES, IL-1ra, MIP-1alpha, MIP-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-17, PDGF-BB, VEGF and IFN-gamma were found in plasma of atherosclerotic patients and might also be integrated in the molecular networks underlying atherosclerotic modifications of these vessels.</p> <p>Conclusion</p> <p>The pattern of cytokine and S100A9/S100A8 up-regulation characterizes atherosclerosis as a proinflammatory disorder. Activation of the JAK/STAT pathway is confirmed by the up-regulation of IL-6, STAT1, ISGF3G and IL10RA genes in coronary and carotid plaques. The functional network constructed in our research is an evidence of the central role of STAT protein and the caveolae system to contribute to preserve the plaque. Moreover, Cav-1 is involved in SMC differentiation and dyslipidemia confirming the importance of lipid homeostasis in the atherosclerotic phenotype.</p

Administration of Downstream ApoE Attenuates the Adverse Effect of Brain ABCA1 Deficiency on Stroke

The ATP-binding cassette transporter member A1 (ABCA1) and apolipoprotein E (ApoE) are major cholesterol transporters that play important roles in cholesterol homeostasis in the brain. Previous research demonstrated that specific deletion of brain-ABCA1 (ABCA1-B/-B) reduced brain grey matter (GM) and white matter (WM) density in the ischemic brain and decreased functional outcomes after stroke. However, the downstream molecular mechanism underlying brain ABCA1-deficiency-induced deficits after stroke is not fully understood. Adult male ABCA1-B/-B and ABCA1-floxed control mice were subjected to distal middle-cerebral artery occlusion and were intraventricularly infused with artificial mouse cerebrospinal fluid as vehicle control or recombinant human ApoE2 into the ischemic brain starting 24 h after stroke for 14 days. The ApoE/apolipoprotein E receptor 2 (ApoER2)/high-density lipoprotein (HDL) levels and GM/WM remodeling and functional outcome were measured. Although ApoE2 increased brain ApoE/HDL levels and GM/WM density, negligible functional improvement was observed in ABCA1-floxed-stroke mice. ApoE2-administered ABCA1-B/-Bstroke mice exhibited elevated levels of brain ApoE/ApoER2/HDL, increased GM/WM density, and neurogenesis in both the ischemic ipsilateral and contralateral brain, as well as improved neurological function compared with the vehicle-control ABCA1-B/-B stroke mice 14 days after stroke. Ischemic lesion volume was not significantly different between the two groups. In vitro supplementation of ApoE2 into primary cortical neurons and primary oligodendrocyte-progenitor cells (OPCs) significantly increased ApoER2 expression and enhanced cholesterol uptake. ApoE2 promoted neurite outgrowth after oxygen-glucose deprivation and axonal outgrowth of neurons, and increased proliferation/survival of OPCs derived from ABCA1-B/-B mice. Our data indicate that administration of ApoE2 minimizes the adverse effects of ABCA1 deficiency after stroke, at least partially by promoting cholesterol traffic/redistribution and GM/WM remodeling via increasing the ApoE/HDL/ApoER2 signaling pathway

The effects of norepinephrine on the proliferation, lipid-uptake and mRNA expression of inflammatory markers in vascular smooth muscle cells

Background: Cardiovascular disease (CVD) is the leading, global cause of death. Elevated levels of norepinephrine (NE) are associated with CVDs such as coronary heart disease with atherosclerosis as the underlying mechanism. Oxidised low-density lipoprotein (OxLDL) has been shown to play an integral role in the formation of atherosclerosis within the vasculature. Previous studies suggest a decrease in NE by renal denervation is associated with enhanced atherosclerosis in mice and NE has anti-inflammatory effects. Therefore, we hypothesised that NE may protect against atherosclerosis. This study aimed to investigate the effects of NE on cellular proliferation, OxLDL uptake and mRNA expression of inflammatory markers in mouse aortic vascular smooth muscle cells (VSMCs), a key cell type involved in atherosclerosis. Methods: Mouse aortic VSMCs were cultured and treated with NE for three consecutive days. Cell proliferation was measured using the trypan blue exclusion and MTS proliferation assays. VSMCs were exposed to fluorescence-labelled OxLDL in the presence or absence of NE for 24 hours. Cellular uptake of fluorescence-labelled OxLDL was visualised by confocal microscopy and analysed for mean fluorescence intensity using ImageJ. To investigate the involvement of NE receptors, theMaster

Thrombosis, Atherosclerosis and Atherothrombosis – New Insights and Experimental Protocols

Previous studies have shown that TSPO as well as apolipoprotein E (Apo E) can be associated with processes such as cholesterol metabolism, oxidative stress, apoptosis, glial activation, inflammation, and immune responses. As a ligand for cell-surface lipoprotein receptors, apolipoprotein E can prevent atherosclerosis by clearing cholesterol-rich lipoproteins from plasma. Furthermore, TSPO takes part in the regulation of gene expression for proteins involved in adhesion, which potentially may play a role in platelet aggregation. There are indications that the Apo E protein is involve in platelet aggregation, while TSPO platelet levels have been found to be increased with various neurological disorders, in particular, in stress-related disorders. The role of platelets in atherogenesis and the potential therapeutic impact of TSPO ligands on disease prevention are of great interest. To determine TSPO binding characteristics in this paradigm, we applied binding assays with [3H]PK 11195 on isolated platelets and erythrocyte membranes. The in vivo findings in Apo E knockout mice revealed that TSPO levels appear to be enhanced in platelets and erythrocytes of Apo E knockout mice, and thus suggest that TSPO and Apo E expression may be interconnected in relation to some aspect of the host defense response. Other organs tested, such as liver, testis, brain, heart, aorta, lung, kidney and spleen, did not show a difference in TSPO binding levels between Apo E knockout mice and wild-type mice. This suggests that TSPO levels may be part of a feedback control system for steroid production (responding to alterations in steroid levels), rather than being regulated by a feed-forward signal provided by cholesterol (i.e. TSPO levels in relation to steroidogenesis are not being regulated by cholesterol levels in vivo)

UA12/2/1 The Xi Psi Cry, Vol. 1, Nos. 1-3

Newsletter created by and about Alpha Phi Omega

Caracterización de la respuesta inmunitaria inducida por nuevas vacunas contra la tuberculosis basadas en nanopartículas

Lung diseases are a common and diverse group of illnesses that represent a health problem affecting millions of people worldwide. The microenvironment and the immune system in the lung are very important but they are also the most challenging immunological dilemma for the host. On one hand, lung location and function are constantly exposed to pathogens; but on the other hand should be sterile and preserve lung homeostasis through the balance between tolerance and inflammation. For this reason is extremely important to characterize and understand the mechanisms and populations involved in a protective immune response, and find better targets to design vaccines. The aim of this project is to define and understand the immunological profiles that correlate with greater protection generated by preventive and therapeutic vaccines in the lung. Preventive vaccines are generally administered to healthy individuals to prevent or ameliorate infectious diseases, but therapeutic cancer vaccines are administrated once the individual has already developed the disease to strength patient's own immune responses. We will focus on two different lung diseases: an infection caused by a bacteria and lung cancer. To study a bacterial infection, we will use as a model Mycobacterium tuberculosis (Mtb), responsible for Tuberculosis (TB). TB is a serious global health problem and the only licensed vaccine against TB is "Mycobacterium bovis Bacille Calmette-Guérin" (BCG). But BCG has a poor efficacy. To achieve a better understanding of the type of immune responses that confer protection, we will use a mouse model to assess the changes in the immune system produced in the lung by vaccines and in response to Mtb infection. Lung cancer is a devastating disease and a major therapeutic burden with poor survival rates. Cancer cells have been in the focus of the research, but the importance of the immune system is becoming more important. Syngenic murine models entailing the injection of immunological compatible cancer cells in immunocompetent mice will allow us to study of the modulation of the immune system performed by cancer cells. Moreover, we will use therapeutic vaccines against tumor cells to activate anti-tumor T cells to recognize and destroy the tumor, increasing the effectiveness when used alone or in combination with conventional therapies. In both approaches, the immune cell populations present in the bronchoalverolar lavage (BAL) and in the lung parenchyma, and the ones induced by the different experimental conditions, will be analyzed and characterized by flow cytometry and ELISA. Also, cells will be obtained to perform proteomic and gene expression profiles to better define the pathways with impact in controlling infection or cancer cells. The outputs generated by our research would lead in the future to a more rational design of protective prophylactic and therapeutic vaccines against lung diseases.Las enfermedades pulmonares son un grupo diverso de enfermedades que afectan a millones de personas en todo el mundo. El microambiente y el sistema inmunitario en el pulmón son muy importantes a la vez que suponen un dilema para el huésped. Por un lado, por su ubicación y función, el pulmón está constantemente expuesto a patógenos; por otro lado debe ser estéril y preservar la homeostasis a través del equilibrio entre tolerancia e inflamación. Por ello es extremadamente importante caracterizar y comprender los mecanismos y las poblaciones involucradas en una respuesta inmunitaria protectora, y encontrar mejores dianas para diseñar vacunas. El objetivo de este proyecto es definir y comprender los perfiles inmunitarios que correlacionan con una mayor protección generada por vacunas preventivas y terapéuticas en pulmón. Las vacunas preventivas se administran a individuos sanos para prevenir o mejorar enfermedades infecciosas, mientras que las vacunas terapéuticas contra el cáncer se administran, para fortalecer la propia respuesta inmunitaria del paciente. Nos centraremos en dos enfermedades pulmonares: una infección causada por una bacteria y cáncer de pulmón. Para estudiar la infección bacteriana, utilizaremos como modelo Mycobacterium tuberculosis (Mtb), responsable de la tuberculosis. La tuberculosis es un grave problema de salud mundial. La única vacuna autorizada contra ella es "Mycobacterium bovis Bacille Calmette-Guérin" (BCG), que tiene una baja eficacia. Para comprender mejor el tipo de respuestas inmunológicas que confieren protección, utilizaremos un modelo de ratón para evaluar los cambios en el sistema inmunitario producidos por diferentes vacunas y la infección de Mtb en el pulmón. El cáncer de pulmón es una enfermedad devastadora, con una carga terapéutica importante y baja tasa de supervivencia. La investigación se ha centrado en las células tumorales, pero el sistema inmunitario se está volviendo cada vez más importante. Los modelos murinos sinogénicos permiten la inyección de células cancerosas inmunológicamente compatibles en ratones inmunocompetentes para estudiar la modulación del sistema inmunitario por las células tumorales. Además, utilizaremos vacunas terapéuticas para activar las células T a fin de que reconozcan y destruyan el tumor, aumentando su eficacia solas o en combinación con terapias convencionales. En ambos casos, las poblaciones celulares inmunitarias presentes en el lavado broncoalverolar y el parénquima pulmonar en las diferentes condiciones experimentales serán analizadas y caracterizadas por citometría de flujo y ELISA. Se obtendrán además células para estudios proteómicos y de expresión génica, para definir mejor las vías implicadas en el control de la infección o de las células cancerosas. Los resultados obtenidos podrían conducir en el futuro a un diseño más racional de vacunas profilácticas y terapéuticas contra enfermedades pulmonares.As enfermidades pulmonares son un grupo diverso de enfermidades que afectan a millóns de persoas en todo o mundo. O microambiente e o sistema inmunitario nos pulmóns son moi importantes ao mesmo tempo que representan un dilema para o hospedeiro. Por unha banda, debido á súa localización e función, o pulmón está constantemente exposto a patóxenos; por outra banda, debe ser estéril e preservar a homeostase a través do equilibrio entre tolerancia e inflamación. Por iso, é moi importante caracterizar e comprender os mecanismos e poboacións implicados nunha resposta inmune protectora e atopar mellores obxectivos para o deseño de vacinas. O obxectivo deste proxecto é definir e comprender os perfís inmunolóxicos que se correlacionan con maior protección xerada por vacinas preventivas e terapéuticas no pulmón. As vacinas preventivas son xeralmente administradas a individuos sans para previr ou mellorar doenzas infecciosas, mentres que as vacinas terapéuticas contra o cancro son administradas unha vez o individuo desenvolve a enfermidade para fortalecer a súa resposta inmunitaria. Centrarémonos en dúas enfermidades pulmonares: unha infección bacteriana e cancro de pulmón. Para estudar unha infección bacteriana, usaremos como modelo Mycobacterium tuberculosis (MTB), responsable da tuberculose (TB), un grave problema de saúde global. A única vacina con licenza actualmente é "Mycobacterium bovis Bacille Calmette-Guérin" (BCG), que ten unha baixa eficacia. Para entender mellor o tipo de respostas inmunolóxicas que confiran protección, usaremos un modelo de rato para avaliar cambios no sistema inmunitario causadas por vacinas e infección por MTB no pulmón. O cancro de pulmón é unha enfermidade devastadora, cunha importante carga terapéutica e baixa taxa de supervivencia. As células tumorais foron o foco da investigación, pero o sistema inmunolóxico é cada vez máis importante. Os modelos murinos sinxénicos, que inclúen a inxección de células de cancro imunolóxicamente compatibles en ratos inmunocompetentes, permítennos estudar a modulación do sistema inmunitario por células tumorais. Ademáis, utilizaremos vacinas terapéuticas para activar as células T que recoñezan e destrúan o tumor, aumentando a súa eficacia cando se usa só ou en combinación con terapias convencionais. En ambos casos, as poboacións de células inmunitarias presentes no lavado broncoalverolar e o parénquima pulmonar nas diferentes condicións experimentais analizaranse e caracterízanse por citometría de fluxo e ELISA. As células tamén se obterán para estudos proteómicos e de expresión xénica, para definir mellor as vías implicadas no control da infección ou as células cancerosas. Os resultados obtidos poderían levar no futuro a un deseño máis racional de vacinas profilácticas e terapéuticas contra as enfermidades pulmonares.Xunta de Galicia | Ref. ED431C 2016/041Xunta de Galicia and the European Regional Development Fund (ERDF) | Ref. ED431G2019/06Fundação para a Ciência e a Tecnologia | Ref. PTDC/SAU-INF/28463/2017Fundação para a Ciência e a Tecnologia | Ref. UIDB/50026/2020Fundação para a Ciência e a Tecnologia | Ref. UIDP/50026/2020FEDER | Ref. NORTE-01-0145-FEDER-000013FEDER | Ref. NORTE-01-0145- FEDER-00002

Associação entre variações alélicas clássicas do gene da ApoE com fatores de risco cardiovascular em indivíduos muito idosos

Monografia (graduação)—Universidade de Brasília, Faculdade de Ceilândia, Curso de Farmácia, 2013.Introdução: Levantamentos epidemiológicos indicam a influência de polimorfismos da ApoE sobre níveis plasmáticos de lipídeos e lipoproteínas ricas em triglicerídeos, com impactos sobre fenótipos ateroscleróticos. Objetivo: Estudar a associação dos genótipos clássicos do gene da ApoE com fatores de risco clínicos e bioquímicos para a aterosclerose em um segmento muito idoso da população brasileira, com destaque para o perfil lipêmico. Métodos: Foram realizadas avaliações transversais de parâmetros clínicos e laboratoriais, incluindo tomografia cardíaca computadorizada, dos 208 participantes com base nos alelos ε2, ε3 e ε4 do gene da ApoE. Resultados: Quando foram comparados os não-carreadores do alelo ε4 com os carreadores, níveis plasmáticos mais baixos de ApoB, assim como da razão ApoB/ApoA foram observados no primeiro grupo. A avaliação do polimorfismo da ApoE com outras variáveis e com a calcificação arterial não apresentaram diferenças significativas entre os grupos. Conclusão: O estudo sugere que o papel aterogênico do alelo ε4 pode, pelo menos em parte, ser atribuído a um aumento na razão ApoB/ApoA, devido aos altos níveis de ApoB em carreadores ε4 em comparação aos não-portadores. _________________________________________________________________________ ABSTRACTBackground: Epidemiological surveys indicate the influence of polymorphisms of the Apolipoprotein (Apo) E on plasma lipids and triglyceride-rich lipoprotein levels, with impact on atherosclerotic phenotypes. Objective: To study the association of classic genotypes of the ApoE gene with clinical and biochemical risk factors for atherosclerosis in a segment of the very old Brazilian individuals, with emphasis to the lipemic profile. Methods: We performed cross-sectional analyses of clinical and laboratory assessments, including cardiac computed tomography, across ε2, ε3 and ε4 carriers of the ApoE gene in 208 participants eligible for primary prevention. Results: When non-ε4 carries were compared with ε4 carrying subjects, lower levels of ApoB as well as on the ApoB/ApoA ratio were observed in the former group. Tests between ApoE polymorphisms with other variables and with arterial calcification showed no significant differences between groups. Conclusion: The study suggests that the atherogenic role of ε4 allele can at least in part be attributed to an increased ApoB/ApoA ratio due to higher levels of ApoB in ε4 carriers compared to non-carriers