Fischell Department of Bioengineering
Permanent URI for this communityhttp://hdl.handle.net/1903/6626
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Item BRAIN ENDOTHELIAL BARRIER, METABOLIC, AND TRANSPORT DYSFUNCTION IN NIEMANN-PICK DISEASE TYPE C: MECHANISMS AND THERAPEUTIC STRATEGIES(2024) Moiz, Bilal; clyne, Alisa; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Brain microvascular endothelial cells (BMECs) form the blood-brain barrier, which protects the brain from neurotoxic elements and simultaneously transports glucose and other vital nutrients into the brain. Neurovascular dysfunction is implicated in pathogenesis of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease; however, little is known about how neurovascular changes contribute to rare inherited neurogenetic disorders such as Niemann-Pick Disease (NP-C). NP-C is caused by mutations in the intracellular cholesterol trafficking proteins NPC1 and NPC2, which leads to endolysosomal cholesterol accumulation and membrane cholesterol depletion. Clinical manifestations vary by age and genetic factors but include neurological symptoms such as developmental delay, cognitive impairment, ataxia, and seizures. Current clinical management strategies are challenged by diagnostic difficulties and poor therapeutic efficacy. Hydroxypropyl-beta-cyclodextrin (HPβCD), an agent believed to release accumulated cholesterol, has shown promising clinical results; however, its efficacy is limited due to poor brain penetration. The major objective of this thesis was to determine how NPC1-deficiency impacts BMEC barrier function, metabolism, and nanoparticle uptake. I found that NPC1 deficiency diminishes barrier integrity in BMECs by disrupting claudin-5 and occludin morphology. Using isotope labeling, mass spectrometry, and computational flux analysis, I also observed that NPC1 inhibition leads to systemic metabolic changes, including increased glycolytic flux, elevated activity in peripheral glycolytic pathways, and reduced mitochondrial respiration. HPβCD treatment attenuated barrier function changes and partially restored BMEC metabolic phenotype. Finally, I found that isoproprylacrylamide (NIPAA-m) nanogels loaded with HPβCD were transported across NPC1-deficient BMECs, suggesting their potential for HPβCD delivery to the brain. This thesis demonstrates a unique, integrated computational-translational approach that unveils the role of BMEC in NP-C pathology, possibly leading to improved therapeutic strategies. In addition, this thesis improves our understanding of how variants in cholesterol metabolism and trafficking, as well as in proteins such as NPC1, which has been implicated in Alzheimer’s, diabetes, obesity, and atherosclerosis, contribute to brain endothelial dysfunction.Item 13C Metabolic Flux Analysis Indicates Endothelial Cells Attenuate Metabolic Perturbations by Modulating TCA Activity(MDPI, 2021-04-07) Moiz, Bilal; Garcia, Jonathan; Basehore, Sarah; Sun, Angela; Li, Andrew; Padmanabhan, Surya; Albus, Kaitlyn; Jang, Cholsoon; Sriram, Ganesh; Clyne, Alisa MorssDisrupted endothelial metabolism is linked to endothelial dysfunction and cardiovascular disease. Targeted metabolic inhibitors are potential therapeutics; however, their systemic impact on endothelial metabolism remains unknown. In this study, we combined stable isotope labeling with 13C metabolic flux analysis (13C MFA) to determine how targeted inhibition of the polyol (fidarestat), pentose phosphate (DHEA), and hexosamine biosynthetic (azaserine) pathways alters endothelial metabolism. Glucose, glutamine, and a four-carbon input to the malate shuttle were important carbon sources in the baseline human umbilical vein endothelial cell (HUVEC) 13C MFA model. We observed two to three times higher glutamine uptake in fidarestat and azaserine-treated cells. Fidarestat and DHEA-treated HUVEC showed decreased 13C enrichment of glycolytic and TCA metabolites and amino acids. Azaserine-treated HUVEC primarily showed 13C enrichment differences in UDP-GlcNAc. 13C MFA estimated decreased pentose phosphate pathway flux and increased TCA activity with reversed malate shuttle direction in fidarestat and DHEA-treated HUVEC. In contrast, 13C MFA estimated increases in both pentose phosphate pathway and TCA activity in azaserine-treated cells. These data show the potential importance of endothelial malate shuttle activity and suggest that inhibiting glycolytic side branch pathways can change the metabolic network, highlighting the need to study systemic metabolic therapeutic effects.Item Isotope-Assisted Metabolic Flux Analysis: A Powerful Technique to Gain New Insights into the Human Metabolome in Health and Disease(MDPI, 2022-11-04) Moiz, Bilal; Li, Andrew; Padmanabhan, Surya; Sriram, Ganesh; Clyne, Alisa MorssCell metabolism represents the coordinated changes in genes, proteins, and metabolites that occur in health and disease. The metabolic fluxome, which includes both intracellular and extracellular metabolic reaction rates (fluxes), therefore provides a powerful, integrated description of cellular phenotype. However, intracellular fluxes cannot be directly measured. Instead, flux quantification requires sophisticated mathematical and computational analysis of data from isotope labeling experiments. In this review, we describe isotope-assisted metabolic flux analysis (iMFA), a rigorous computational approach to fluxome quantification that integrates metabolic network models and experimental data to generate quantitative metabolic flux maps. We highlight practical considerations for implementing iMFA in mammalian models, as well as iMFA applications in in vitro and in vivo studies of physiology and disease. Finally, we identify promising new frontiers in iMFA which may enable us to fully unlock the potential of iMFA in biomedical research.Item Induced pluripotent stem cell-derived cells model brain microvascular endothelial cell glucose metabolism(Springer Nature, 2022-12-09) Weber, Callie M.; Moiz, Bilal; Zic, Sophia M.; Vargas, Viviana Alpízar; Li, Andrew; Morss Clyne, AlisaGlucose transport from the blood into the brain is tightly regulated by brain microvascular endothelial cells (BMEC), which also use glucose as their primary energy source. To study how BMEC glucose transport contributes to cerebral glucose hypometabolism in diseases such as Alzheimer’s disease, it is essential to understand how these cells metabolize glucose. Human primary BMEC (hpBMEC) can be used for BMEC metabolism studies; however, they have poor barrier function and may not recapitulate in vivo BMEC function. iPSC-derived BMEC-like cells (hiBMEC) are readily available and have good barrier function but may have an underlying epithelial signature. In this study, we examined differences between hpBMEC and hiBMEC glucose metabolism using a combination of dynamic metabolic measurements, metabolic mass spectrometry, RNA sequencing, and Western blots. hiBMEC had decreased glycolytic flux relative to hpBMEC, and the overall metabolomes and metabolic enzyme levels were different between the two cell types. However, hpBMEC and hiBMEC had similar glucose metabolism, including nearly identical glucose labeled fractions of glycolytic and TCA cycle metabolites. Treatment with astrocyte conditioned media and high glucose increased glycolysis in both hpBMEC and hiBMEC, though hpBMEC decreased glycolysis in response to fluvastatin while hiBMEC did not. Together, these results suggest that hiBMEC can be used to model cerebral vascular glucose metabolism, which expands their use beyond barrier models.