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Isotope-assisted metabolic flux analysis (iMFA) is a powerful method to mathematically determine the metabolic fluxome from experimental isotope labeling data and a metabolic network model. While iMFA was originally developed for industrial biotechnological applications, it is increasingly used to analyze eukaryotic cell metabolism in physiological and pathological states. In this review, we explain how iMFA estimates the intracellular fluxome, including data and network model (inputs), the optimization-based data fitting (process), and the flux map (output). We then describe how iMFA enables analysis of metabolic complexities and discovery of metabolic pathways. Our goal is to expand the use of iMFA in metabolism research, which is essential to maximizing the impact of metabolic experiments and continuing to advance iMFA and biocomputational techniques.
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Análise do Fluxo Metabólico , Redes e Vias Metabólicas , Análise do Fluxo Metabólico/métodos , Isótopos , Marcação por Isótopo/métodos , Modelos BiológicosRESUMO
The human body represents a collection of interacting systems that range in scale from nanometers to meters. Investigations from a systems perspective focus on how the parts work together to enact changes across spatial scales, and further our understanding of how systems function and fail. Here, we highlight systems approaches presented at the 2022 Summer Biomechanics, Bio-engineering, and Biotransport Conference in the areas of solid mechanics; fluid mechanics; tissue and cellular engineering; biotransport; and design, dynamics, and rehabilitation; and biomechanics education. Systems approaches are yielding new insights into human biology by leveraging state-of-the-art tools, which could ultimately lead to more informed design of therapies and medical devices for preventing and treating disease as well as rehabilitating patients using strategies that are uniquely optimized for each patient. Educational approaches can also be designed to foster a foundation of systems-level thinking.
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Bioengenharia , Análise de Sistemas , Humanos , Fenômenos Biomecânicos , BiofísicaRESUMO
Endothelial cells in steady laminar flow assume a healthy, quiescent phenotype, while endothelial cells in oscillating disturbed flow become dysfunctional. Since endothelial dysfunction leads to atherosclerosis and cardiovascular disease, it is important to understand the mechanisms by which endothelial cells change their function in varied flow environments. Endothelial metabolism has recently been proven a powerful tool to regulate vascular function. Endothelial cells generate most of their energy from glycolysis, and steady laminar flow may reduce endothelial glycolytic flux. We hypothesized that steady laminar but not oscillating disturbed flow would reduce glycolytic flux and alter glycolytic side branch pathways. In this study, we exposed human umbilical vein endothelial cells to static culture, steady laminar flow (20 dynes/cm2 shear stress), or oscillating disturbed flow (4 ± 6 dynes/cm2 shear stress) for 24 h using a cone-and-plate device. We then measured glucose and lactate uptake and secretion, respectively, and glycolytic metabolites. Finally, we explored changes in the expression and protein levels of endothelial glycolytic enzymes. Our data show that endothelial cells in steady laminar flow had decreased glucose uptake and 13C labeling of glycolytic metabolites while cells in oscillating disturbed flow did not. Steady laminar flow did not significantly change glycolytic enzyme gene or protein expression, suggesting that glycolysis may be altered through enzyme activity. Flow also modulated glycolytic side branch pathways involved in proteoglycan and glycosaminoglycan synthesis, as well as oxidative stress. These flow-induced changes in endothelial glucose metabolism may impact the atheroprone endothelial phenotype in oscillating disturbed flow.
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Antioxidantes , Aterosclerose , Humanos , Antioxidantes/metabolismo , Células Cultivadas , Aterosclerose/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , GlicóliseRESUMO
[Figure: see text].
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Acetilglucosamina/metabolismo , Circulação Sanguínea , Endotélio Vascular/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiologia , Endotélio Vascular/fisiologia , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Glicosilação , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/metabolismo , Óxido Nítrico/metabolismo , FosforilaçãoRESUMO
The endothelial cell response to glucose plays an important role in both health and disease. Endothelial glucose-induced dysfunction was first studied in diabetic animal models and in cells cultured in hyperglycemia. Four classical dysfunction pathways were identified, which were later shown to result from the common mechanism of mitochondrial superoxide overproduction. More recently, non-coding RNA, extracellular vesicles, and sodium-glucose cotransporter-2 inhibitors were shown to affect glucose-induced endothelial dysfunction. Endothelial cells also metabolize glucose for their own energetic needs. Research over the past decade highlighted how manipulation of endothelial glycolysis can be used to control angiogenesis and microvascular permeability in diseases such as cancer. Finally, endothelial cells transport glucose to the cells of the blood vessel wall and to the parenchymal tissue. Increasing evidence from the blood-brain barrier and peripheral vasculature suggests that endothelial cells regulate glucose transport through glucose transporters that move glucose from the apical to the basolateral side of the cell. Future studies of endothelial glucose response should begin to integrate dysfunction, metabolism and transport into experimental and computational approaches that also consider endothelial heterogeneity, metabolic diversity, and parenchymal tissue interactions.
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Células Endoteliais/efeitos dos fármacos , Glucose/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Diabetes Mellitus Tipo 2/fisiopatologia , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Glicólise/efeitos dos fármacos , Humanos , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Hiperglicemia/fisiopatologia , Neovascularização Fisiológica/fisiologiaRESUMO
Atherosclerosis develops at arterial sites where endothelial cells (ECs) are exposed to low time-averaged shear stress, in particular in regions of recirculating disturbed flow. To understand how hemodynamics contributes to EC dysfunction in atheroma development, an in vitro parallel plate flow chamber gasket was modified with protruding baffles to produce large recirculating flow regions. Computational fluid dynamics (CFD) predicted that more than 60% of the flow surface area was below the 12 dynes/cm2 atheroprotective threshold. Bovine aortic endothelial cells (BAECs) were then seeded in the parallel plate flow chamber with either the standard laminar or the new disturbed flow gasket (DFG) and exposed to flow for 36 h. Cell morphology, nitric oxide (NO), proliferation, permeability, and monocyte adhesion were assessed by phase contrast and confocal microscopy. BAEC exposed to 20 dynes/cm2 shear stress in the laminar flow device aligned and elongated in the flow direction while increasing nitric oxide, decreasing permeability, and maintaining low proliferation and monocyte adhesion. BAEC in the recirculating flow and low shear stress disturbed flow device regions did not elongate or align, produced less nitric oxide, and showed higher proliferation, permeability, and monocyte adhesion than cells in the laminar flow device. However, cells in disturbed flow device regions exposed to atheroprotective shear stress did not consistently align or decrease permeability, and these cells demonstrated low nitric oxide levels. The new parallel plate DFG provides a means to study recirculating flow, highlighting the complex relationship between hemodynamics and endothelial function.
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Células Endoteliais , Estresse Mecânico , Animais , Aorta , Bovinos , HemodinâmicaRESUMO
Course-based undergraduate research experiences (CURE) are a valuable tool to increase research exposure for larger undergraduate cohorts. We implemented a CURE within a senior-level biofluid mechanics course that was primarily taught using a flipped classroom approach. Due to the large class size, the students analyzed data that was publicly available and produced by one of our laboratories. Student teams then developed hypotheses based on the data analysis and designed a set of in vitro and in vivo experiments to test those hypotheses. The hypotheses and experiments that were most highly rated by the class were then tested in our laboratory. At the end of the class, student gains were assessed by self-report and compared to those self-reported by students engaging in a traditional freshman undergraduate summer research experience. While the students in the CURE reported moderate gains in self-assessment of research-based skills, their self-reported gains were statistically significantly lower than those reported by students who participated in the traditional research experience. We believe that the CURE could be improved through implementation in a lower level class, enabling students to observe laboratory experiments, and providing additional feedback throughout the hypothesis development and experimental design process. Overall, the CURE is an innovative way to expand research experiences, in particular for engineering students who often do not participate in hypothesis-driven research during their undergraduate education.
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Arterial stiffness and inflammation are associated with atherosclerosis, and each have individually been shown to increase endothelial monolayer tension and permeability. The objective of this study was to determine if substrate stiffness enhanced endothelial monolayer tension and permeability in response to inflammatory cytokines. Porcine aortic endothelial cells were cultured at confluence on polyacrylamide gels of varying stiffness and treated with either tumor necrosis factor-α (TNFα) or thrombin. Monolayer tension was measured through vinculin localization at the cell membrane, traction force microscopy, and phosphorylated myosin light chain quantity and actin fiber colocalization. Cell permeability was measured by cell-cell junction confocal microscopy and a dextran permeability assay. When treated with TNFα or thrombin, endothelial monolayers on stiffer substrates showed increased traction forces, vinculin at the cell membrane, and vinculin phosphorylation, suggesting elevated monolayer tension. Interestingly, VE-cadherin shifted toward a smaller molecular weight in endothelial monolayers on softer substrates, which may relate to increased VE-cadherin endocytosis and degradation. Phosphorylated myosin light chain colocalization with actin stress fibers increased in endothelial monolayers treated with TNFα or thrombin on stiffer substrates, indicating elevated cell monolayer contractility. Endothelial monolayers also developed focal adherens intercellular junctions and became more permeable when cultured on stiffer substrates in the presence of the inflammatory cytokines. Whereas each of these effects was likely mitigated by Rho/ROCK, Rho/ROCK pathway inhibition via Y27632 disrupted cell-cell junction morphology, showing that cell contractility is required to maintain adherens junction integrity. These data suggest that stiff substrates change intercellular junction protein localization and degradation, which may counteract the inflammation-induced increase in endothelial monolayer tension and thereby moderate inflammation-induced junction loss and associated endothelial monolayer permeability on stiffer substrates.
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Células Endoteliais/metabolismo , Células Endoteliais/patologia , Estresse Mecânico , Rigidez Vascular , Animais , Fenômenos Biomecânicos , Células Endoteliais/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/patologia , Permeabilidade/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Suínos , Trombina/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Rigidez Vascular/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Quinases Associadas a rho/metabolismoRESUMO
Problem-based learning (PBL) has been shown to be effective in biomedical engineering education, particularly in motivating student learning, increasing knowledge retention, and developing problem solving, communication, and teamwork skills. However, PBL adoption remains limited by real challenges in effective implementation. In this paper, we review the literature on advantages and challenges of PBL and present our own experiences. We also provide practical guidelines for implementing PBL, including two examples of PBL modules from biomechanics courses at two different institutions. Overall, we conclude that the benefits for both professors and students support the use of PBL in biomedical engineering education.
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Engenharia Biomédica/economia , Engenharia Biomédica/educação , Educação Profissionalizante/organização & administração , Equipamentos e Provisões , Aprendizagem Baseada em Problemas/organização & administração , Ensino/organização & administração , Avaliação Educacional , Desenho de Equipamento/economia , Modelos Educacionais , Modelos Organizacionais , Estados UnidosRESUMO
Hyperglycemia is a defining characteristic of diabetes, and uncontrolled blood glucose in diabetes is associated with accelerated cardiovascular disease. Chronic hyperglycemia glycates extracellular matrix (ECM) collagen, which can lead to endothelial cell dysfunction. In healthy conditions, endothelial cells respond to mechanical stimuli such as cyclic stretch (CS) by aligning their actin cytoskeleton. Other cell types, specifically fibroblasts, align their ECM in response to CS. We previously demonstrated that glycated collagen inhibits endothelial cell actin alignment in response to CS. The aim of this study was to determine the effect of glycated collagen on ECM remodeling and protein alignment in response to stretch. Porcine aortic endothelial cells (PAEC) seeded on native or glycated collagen coated elastic substrates were exposed to 10% CS. Cells on native collagen aligned subcellular fibronectin fibers in response to stretch, whereas cells on glycated collagen did not. The loss of fibronectin alignment was due to inhibited actin alignment in response to CS, since fibronectin alignment did not occur in cells on native collagen when actin alignment was inhibited with cytochalasin. Further, while ECM protein content did not change in cells on native or glycated collagen in response to CS, degradation activity decreased in cells on glycated collagen. Matrix metalloproteinase 2 (MMP-2) and membrane-associated type 1 matrix metalloproteinase (MT1-MMP) protein levels decreased, and therefore MMP-2 activity also decreased. These MMP changes may relate to c-Jun N-terminal kinase (Jnk) phosphorylation inhibition with CS, which has previously been linked to focal adhesion kinase (FAK). These data demonstrate the importance of endothelial cell actin tension in remodeling and aligning matrix proteins in response to mechanical stimuli, which is critical to vascular remodeling in health and disease.
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Actinas/metabolismo , Colágeno/metabolismo , Células Endoteliais/metabolismo , Fibronectinas/metabolismo , Fenômenos Mecânicos , Animais , Fenômenos Biomecânicos , Citocalasinas/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Glicosilação/efeitos dos fármacos , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Suínos , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
Brain microvascular endothelial cells, which lie at the interface between blood and brain, are critical to brain energetics. These cells must precisely balance metabolizing nutrients for their own demands with transporting nutrients into the brain to sustain parenchymal cells. It is essential to understand this integrated metabolism and transport so that we can develop better diagnostics and therapeutics for neurodegenerative diseases such as Alzheimer's disease, multiple sclerosis, and traumatic brain injury. In this chapter, we first describe brain microvascular endothelial cell metabolism and how these cells regulate both blood flow and nutrient transport. We then explain the impact of brain microvascular endothelial cell metabolism on the integrity of the blood-brain barrier, as well as how metabolites produced by the endothelial cells impact other brain cells. We detail some ways that cell metabolism is typically measured experimentally and modeled computationally. Finally, we describe changes in brain microvascular endothelial cell metabolism in aging and neurodegenerative diseases. At the end of the chapter, we highlight areas for future research in brain microvascular endothelial cell metabolism. The goal of this chapter is to underscore the importance of nutrient metabolism and transport at the brain endothelium for cerebral health and neurovascular disease treatment.
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Barreira Hematoencefálica , Encéfalo , Células Endoteliais , Animais , Humanos , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Células Endoteliais/metabolismo , Microvasos/metabolismo , Doenças Neurodegenerativas/metabolismoRESUMO
Cell adhesion strength is important to cell survival, proliferation, migration, and mechanotransduction, yet changes in endothelial cell adhesion strength have not yet been examined in diseases such as diabetes with high rates of cardiovascular complications. We therefore investigated porcine aortic endothelial cell adhesion strength on native and glycated collagen-coated substrates and in low, normal, and high glucose culture using a spinning disc apparatus. Adhesion strength increased by 30 dynes/cm(2) in cells on glycated collagen as compared to native collagen. Attachment studies revealed that cells use higher adhesion strength αv ß3 integrins to bind to glycated collagen instead of the typical α2 ß1 integrins used to bind to native collagen. Similarly, endothelial cells cultured in low and high glucose had 15 dynes/cm(2) higher adhesion strength than cells in normal glucose after 2 days. Increased adhesion strength was due to elevated VEGF release and intracellular PKC in low and high glucose cells, respectively. Thus glucose increased endothelial cell adhesion strength via different underlying mechanisms. These adhesion strength changes could contribute to diabetic vascular disease, including accelerated atherosclerosis and disordered angiogenesis.
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Colágeno/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Glucose/metabolismo , Glicoproteínas/metabolismo , Animais , Fenômenos Biomecânicos , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Células Cultivadas , Materiais Revestidos Biocompatíveis , Angiopatias Diabéticas/etiologia , Angiopatias Diabéticas/patologia , Angiopatias Diabéticas/fisiopatologia , Endotélio Vascular/patologia , Glucose/química , Especificidade por Substrato/fisiologia , Suínos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Introduction: Women are at elevated risk for certain cardiovascular diseases, including pulmonary arterial hypertension, Alzheimer's disease, and vascular complications of diabetes. Angiotensin II (AngII), a circulating stress hormone, is elevated in cardiovascular disease; however, our knowledge of sex differences in the vascular effects of AngII are limited. We therefore analyzed sex differences in human endothelial cell response to AngII treatment. Methods: Male and female endothelial cells were treated with AngII for 24 h and analyzed by RNA sequencing. We then used endothelial and mesenchymal markers, inflammation assays, and oxidative stress indicators to measure female and male endothelial cell functional changes in response to AngII. Results: Our data show that female and male endothelial cells are transcriptomically distinct. Female endothelial cells treated with AngII had widespread gene expression changes related to inflammatory and oxidative stress pathways, while male endothelial cells had few gene expression changes. While both female and male endothelial cells maintained their endothelial phenotype with AngII treatment, female endothelial cells showed increased release of the inflammatory cytokine interleukin-6 and increased white blood cell adhesion following AngII treatment concurrent with a second inflammatory cytokine. Additionally, female endothelial cells had elevated reactive oxygen species production compared to male endothelial cells after AngII treatment, which may be partially due to nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX2) escape from X-chromosome inactivation. Conclusions: These data suggest that endothelial cells have sexually dimorphic responses to AngII, which could contribute to increased prevalence of some cardiovascular diseases in women. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00762-2.
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Within the biopharmaceutical sector, there exists the need for a contactless multiplex sensor, which can accurately detect metabolite levels in real time for precise feedback control of a bioreactor environment. Reported spectral sensors in the literature only work when fully submerged in the bioreactor and are subject to probe fouling due to a cell debris buildup. The use of a short-wave infrared (SWIR) hyperspectral (HS) cam era allows for efficient, fully contactless collection of large spectral datasets for metabolite quantification. Here, we report the development of an interpretable deep learning system, a convolution metabolite regression (CMR) approach that detects glucose and lactate concentrations using label-free contactless HS images of cell-free spent media samples from Chinese hamster ovary (CHO) cell growth flasks. Using a dataset of <500 HS images, these CMR algorithms achieved a competitive test root-mean-square error (RMSE) performance of glucose quantification within 27 mg/dL and lactate quantification within 20 mg/dL. Conventional Raman spectroscopy probes report a validation performance of 26 and 18 mg/dL for glucose and lactate, respectively. The CMR system trains within 10 epochs and uses a convolution encoder with a sparse bottleneck regression layer to pick the best-performing filters learned by CMR. Each of these filters is combined with existing interpretable models to produce a metabolite sensing system that automatically removes spurious predictions. Collectively, this work will advance the safe and efficient adoption of contactless deep learning sensing systems for fine control of a variety of bioreactor environments.
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Superparamagnetic iron oxide nanoparticles are widely used in biomedical applications, yet questions remain regarding the effect of nanoparticle size and coating on nanoparticle cytotoxicity. In this study, porcine aortic endothelial cells were exposed to 5 and 30 nm diameter iron oxide nanoparticles coated with either the polysaccharide, dextran, or the polymer polyethylene glycol (PEG). Nanoparticle uptake, cytotoxicity, reactive oxygen species (ROS) formation, and cell morphology changes were measured. Endothelial cells took up nanoparticles of all sizes and coatings in a dose dependent manner, and intracellular nanoparticles remained clustered in cytoplasmic vacuoles. Bare nanoparticles in both sizes induced a more than 6 fold increase in cell death at the highest concentration (0.5 mg/mL) and led to significant cell elongation, whereas cell viability and morphology remained constant with coated nanoparticles. While bare 30 nm nanoparticles induced significant ROS formation, neither 5 nm nanoparticles (bare or coated) nor 30 nm coated nanoparticles changed ROS levels. Furthermore, nanoparticles were more toxic at lower concentrations when cells were cultured within 3D gels. These results indicate that both dextran and PEG coatings reduce nanoparticle cytotoxicity, however different mechanisms may be important for different size nanoparticles.
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Materiais Revestidos Biocompatíveis/química , Dextranos/química , Compostos Férricos/toxicidade , Nanopartículas/química , Nanopartículas/toxicidade , Polietilenoglicóis/química , Citoesqueleto de Actina/metabolismo , Animais , Aorta/citologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Compostos Férricos/química , Nanopartículas/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , SuínosRESUMO
Cell 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.
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Glucose 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.
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Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Células Endoteliais/metabolismo , Barreira Hematoencefálica/metabolismo , Glucose , Diferenciação Celular/fisiologia , Encéfalo/irrigação sanguínea , Células CultivadasRESUMO
Fiber alignment plays a critical role in the structure and function of cells and tissues. While fiber alignment quantification is important to experimental analysis and several different methods for quantifying fiber alignment exist, many studies focus on qualitative rather than quantitative analysis perhaps due to the complexity of current fiber alignment methods. Speed and sensitivity were compared in edge detection and fast Fourier transform (FFT) for measuring actin fiber alignment in cells exposed to shear stress. While edge detection using matrix multiplication was consistently more sensitive than FFT, image processing time was significantly longer. However, when MATLAB functions were used to implement edge detection, MATLAB's efficient element-by-element calculations and fast filtering techniques reduced computation cost 100 times compared to the matrix multiplication edge detection method. The new computation time was comparable to the FFT method, and MATLAB edge detection produced well-distributed fiber angle distributions that statistically distinguished aligned and unaligned fibers in half as many sample images. When the FFT sensitivity was improved by dividing images into smaller subsections, processing time grew larger than the time required for MATLAB edge detection. Implementation of edge detection in MATLAB is simpler, faster, and more sensitive than FFT for fiber alignment quantification.
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Actinas/química , Análise de Fourier , Processamento de Imagem Assistida por Computador/métodos , Actinas/metabolismo , Animais , Células Cultivadas , Células Endoteliais/química , Células Endoteliais/metabolismo , Sensibilidade e Especificidade , Software , SuínosRESUMO
The number of people diagnosed with neurodegenerative diseases is on the rise. Many of these diseases, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and motor neuron disease, demonstrate clear sexual dimorphisms. While sex as a biological variable must now be included in animal studies, sex is rarely included in in vitro models of human neurodegenerative disease. In this Review, we describe these sex-related differences in neurodegenerative diseases and the blood-brain barrier (BBB), whose dysfunction is linked to neurodegenerative disease development and progression. We explain potential mechanisms by which sex and sex hormones affect BBB integrity. Finally, we summarize current in vitro BBB bioengineered models and highlight their potential to study sex differences in BBB integrity and neurodegenerative disease.
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Vascular smooth muscle cells align circumferentially around the vessel lumen, which allows these cells to control vascular tone by contracting and relaxing. It is essential that this circumferential alignment is recapitulated in tissue engineered blood vessels. While many methods have been reported to align cells on 2D polymeric substrates, few techniques enable cell alignment on a 3D physiologically relevant hydrogel substrate. We hypothesized that the ridges inherent to the sides of fused deposition modeling 3D printed molds could be used to topographically pattern both stiff and soft substrates and thereby align cells on flat and curved surfaces. Flat and curved molds with 150, 250, and 350 µm ridges were 3D printed and used to topographically pattern polydimethylsiloxane and gelatin-methacryloyl. The ridges transferred to both substrates with less than 10% change in ridge size. Vascular smooth muscle cells were then seeded on each substrate, and nuclear and actin alignment were quantified. Cells were highly aligned with the molded ridges to a similar extent on both the stiffer polydimethylsiloxane and the softer gelatin-methacryloyl substrates. These data confirm that fused deposition modeling 3D printed molds are a rapid, cost-effective way to topographically pattern stiff and soft substrates in varied 3D shapes. This method will enable investigators to align cells on 3D polymeric and hydrogel structures for tissue engineering and other applications.