A systems biology analysis of brain microvascular endothelial cell lipotoxicity.

BackgroundNeurovascular inflammation is associated with a number of neurological diseases including vascular dementia and Alzheimer's disease, which are increasingly important causes of morbidity and mortality around the world. Lipotoxicity is a metabolic disorder that results from accumulation of lipids, particularly fatty acids, in non-adipose tissue leading to cellular dysfunction, lipid droplet formation, and cell death.ResultsOur studies indicate for the first time that the neurovascular circulation also can manifest lipotoxicity, which could have major effects on cognitive function. The penetration of integrative systems biology approaches is limited in this area of research, which reduces our capacity to gain an objective insight into the signal transduction and regulation dynamics at a systems level. To address this question, we treated human microvascular endothelial cells with triglyceride-rich lipoprotein (TGRL) lipolysis products and then we used genome-wide transcriptional profiling to obtain transcript abundances over four conditions. We then identified regulatory genes and their targets that have been differentially expressed through analysis of the datasets with various statistical methods. We created a functional gene network by exploiting co-expression observations through a guilt-by-association assumption. Concomitantly, we used various network inference algorithms to identify putative regulatory interactions and we integrated all predictions to construct a consensus gene regulatory network that is TGRL lipolysis product specific.ConclusionSystem biology analysis has led to the validation of putative lipid-related targets and the discovery of several genes that may be implicated in lipotoxic-related brain microvascular endothelial cell responses. Here, we report that activating transcription factors 3 (ATF3) is a principal regulator of TGRL lipolysis products-induced gene expression in human brain microvascular endothelial cell

Mecanismos de protección frente a rotenona por PDGF-BB en un modelo astrocitario

Los astrocitos cumplen un importante papel neuroprotector en las diversas patologías neurodegenerativas del sistema nervioso central (SNC) ya que le brindan a las neuronas soporte trófico, metabólico y antioxidativo, debido a que poseen concentraciones elevadas de la superóxido dismutasa (SOD), la catalasa, el glutatión (GSH) y distintos factores de crecimiento como el BDNF, GDNF y PDGF. En situaciones donde los astrocitos no ejecutan tales funciones, como es el caso de la producción exacerbada de especies reactivas de oxígeno (ERO) y el daño mitocondrial, sobreviene inevitablemente la muerte neuronal. Estudios recientes sugieren que la alteración de las funciones astrocitarias se encuentra involucrada en la progresión de diversas enfermedades como Parkinson, Alzheimer, esclerosis lateral amiotrófica y otras. La producción de ERO conduce a la disfunción mitocondrial en los tejidos del sistema nervioso, acompañada por muerte neuronal excitotóxica y cambios morfológicos en las células del SNC, como son la activación astrocitaria y la inhibición de la regeneración neuronal en el sitio de la lesión. Diversos estudios han mostrado que las neuronas poseen una mayor susceptibilidad al daño oxidativo que los astrocitos, ya que estas poseen un menor número de mecanismos antioxidantes, siendo por lo tanto más propensas a la muerte celular. Por esta razón, los modelos in vitro relativos a la protección de las funciones astrocitarias antes mencionadas han sido considerados como una excelente aproximación en el estudio de las lesiones y enfermedades cerebrales. En este aspecto, se ha demostrado que el uso de factores de crecimiento como el BDNF, el FGF, el GDNF y el VEGF promueven actividades protectoras en neuronas y en células gliales en eventos como la excitotoxicidad, la producción de ERO, y la protección mitocondrial tanto en modelos in vitro como in vivo, incluyendo procesos neurodegenerativos como el Parkinson. Igualmente, el factor de crecimiento derivado de plaquetas, isoforma B (PDGF-BB), el cual expresa su receptor (PDGFR-ß), en distintos tipos celulares del SNC incluyendo los astrocitos, ha demostrado ejercer un efecto neuroprotector en demencia, modelos murinos de Parkinson, en insulto oxidativo por peróxido de hidrogeno y en protección excitotóxica de neuronas.In the present PhD Thesis, we studied the protective effects of PDGF-BB (Platelet Derived Growth Factor B), on oxidative and mitochondrial damage exerted by rotenone, in an astrocytic model (T98G cell line ). We used both experimental and computational methods for study molecular effects such as reactive oxygen species (ROS) production, changes in mitochondrial membrane potential, ultrastructural effects, changes in cell viability, and changes in the expression of proteins like GRP78 and neuroglobin (ngb). We establish the protective effects of PDGF-B in astrocytes, suggesting prospective applications for Parkinson Disease.Doctor en Ciencias BiológicasDoctorad

A systems biology analysis of brain microvascular endothelial cell lipotoxicity.

BackgroundNeurovascular inflammation is associated with a number of neurological diseases including vascular dementia and Alzheimer's disease, which are increasingly important causes of morbidity and mortality around the world. Lipotoxicity is a metabolic disorder that results from accumulation of lipids, particularly fatty acids, in non-adipose tissue leading to cellular dysfunction, lipid droplet formation, and cell death.ResultsOur studies indicate for the first time that the neurovascular circulation also can manifest lipotoxicity, which could have major effects on cognitive function. The penetration of integrative systems biology approaches is limited in this area of research, which reduces our capacity to gain an objective insight into the signal transduction and regulation dynamics at a systems level. To address this question, we treated human microvascular endothelial cells with triglyceride-rich lipoprotein (TGRL) lipolysis products and then we used genome-wide transcriptional profiling to obtain transcript abundances over four conditions. We then identified regulatory genes and their targets that have been differentially expressed through analysis of the datasets with various statistical methods. We created a functional gene network by exploiting co-expression observations through a guilt-by-association assumption. Concomitantly, we used various network inference algorithms to identify putative regulatory interactions and we integrated all predictions to construct a consensus gene regulatory network that is TGRL lipolysis product specific.ConclusionSystem biology analysis has led to the validation of putative lipid-related targets and the discovery of several genes that may be implicated in lipotoxic-related brain microvascular endothelial cell responses. Here, we report that activating transcription factors 3 (ATF3) is a principal regulator of TGRL lipolysis products-induced gene expression in human brain microvascular endothelial cell

Role of the Swain-Langley and McCoy polymorphisms in complement receptor 1 in cerebral malaria

Malaria has been a major driving force in the evolution of the human genome. In sub-Saharan African populations, two neighbouring polymorphisms in the Complement Receptor 1 (CR1) gene, named Swain-Langley (Sl2) and McCoy (McCb), occur at high frequencies, consistent with selection by malaria. This thesis investigates the association between these two polymorphisms and severe malaria. Previous studies into this area have produced conflicting findings. Using a large case-control study of severe malaria in Kenyan children and statistical models adjusted for confounders, I found that the Sl2 polymorphism was associated with markedly reduced odds of cerebral malaria and death, while the McCb polymorphism was associated with increased odds of cerebral malaria. I also identified an interaction between Sl2 and α+thalassaemia, with the protective association of Sl2 greatest in children with normal α-globin. Following these epidemiological findings, I explored potential biological hypotheses which might explain them. The first approach examined whether the Sl2 and McCb polymorphisms affected how CR1 forms clusters on erythrocyte membranes, a process which is key in the binding and transfer of immune complexes from erythrocytes to macrophages. Using erythrocytes from Kenyan children, I performed immunofluorescence assays (IFAs) with confocal microscopy to quantify CR1 cluster number and volume. I found no association between the Sl2 and McCb polymorphisms and either the number or volume of CR1 clusters formed. The second approach investigated whether the cerebral malaria-specific associations seen with Sl2 and McCb might be due to expression of CR1 by human brain endothelial cells (HBEC). The immortalised cell line HBEC-5i was investigated for expression of CR1 using IFA, flow cytometry, western blotting, functional C3b degradation assays, mass spectrometry, immunoprecipitation and siRNA knockdown experiments. A pool of α-CR1 monoclonal antibodies recognised an intracellular antigen in permeabilised HBEC-5i cells which was a similar molecular weight to CR1 on western blotting. However, when the α-CR1 monoclonal antibodies were tested individually, only E11 recognised an HBEC-5i antigen. Further investigative approaches did not support the presence of CR1 on HBEC-5i cells, instead suggesting that E11 was not specific for CR1 and was instead recognising a protein in the Golgi apparatus. The final approach was to examine whether the Sl2 and McCb polymorphisms might influence the binding of the complement components mannose binding lectin, C1q and L-ficolin to the LHR-D region of CR1. I aimed to generate recombinant proteins of the LHR-D region which included the polymorphisms. Site-directed mutagenesis of the region was successful and subcloning and expression of the mutant amplicons will be performed at a later date. In summary, I have identified opposing associations between the Sl2 and McCb polymorphisms and cerebral malaria, which do not appear to be due to differences in CR1 clustering or expression of CR1 by human brain endothelial cells. My investigation into whether the polymorphisms might influence complement component binding is ongoing