Callie Weber

All Publications

• Instationary metabolic flux analysis reveals that NPC1 inhibition increases glycolysis and decreases mitochondrial metabolism in brain microvascular endothelial cells. Neurobiology of disease Moiz, B., Walls, M., Alpizar, V., Addepall, A., Weber, C., Li, A., Sriram, G., Clyne, A. M. 2024: 106769

  Abstract

  Niemann Pick Disease Type C (NP-C), a rare neurogenetic disease with no known cure, is caused by mutations in the cholesterol trafficking protein NPC1. Brain microvascular endothelial cells (BMEC) are thought to play a critical role in the pathogenesis of several neurodegenerative diseases; however, little is known about how these cells are altered in NP-C. In this study, we investigated how NPC1 inhibition perturbs BMEC metabolism in human induced pluripotent stem cell-derived BMEC (hiBMEC). We incorporated extracellular metabolite and isotope labeling data into an instationary metabolic flux analysis (INST-MFA) model to estimate intracellular metabolic fluxes. We found that NPC1 inhibition significantly increased glycolysis and pentose phosphate pathway flux while decreasing mitochondrial metabolism. These changes may have been driven by gene expression changes due to increased cholesterol biosynthesis, in addition to mitochondrial cholesterol accumulation. We corroborated these findings in primary BMEC, an alternative in vitro human brain endothelial model. Finally, we found that co-treatment with hydroxypropyl-β cyclodextrin (HPβCD) partially restored metabolic phenotype in U18666A-treated BMECs, suggesting that this drug may have therapeutic effects on the brain endothelium in NP-C. Together, our data highlight the importance of NPC1 in BMEC metabolism and implicate brain endothelial dysfunction in NP-C pathogenesis.

  View details for DOI 10.1016/j.nbd.2024.106769

  View details for PubMedID 39706535

• Impacts of APOE-ε4 and exercise training on brain microvascular endothelial cell barrier function and metabolism. EBioMedicine Weber, C. M., Moiz, B., Pena, G. S., Kheradmand, M., Wunderler, B., Kettula, C., Sangha, G. S., Smith, J. C., Clyne, A. M. 2024; 111: 105487

  Abstract

  The APOE-ε4 genotype is the highest genetic risk factor for Alzheimer's disease (AD), and exercise training can reduce the risk of AD. Two early pathologies of AD are degradation of tight junctions between brain microvascular endothelial cells (BMEC) and brain glucose hypometabolism. Therefore, the objective of this work was to determine how the APOE-ε4 genotype and serum from exercise trained individuals impacts BMEC barrier function and metabolism.iPSC homozygous for the APOE-ε3 and APOE-ε4 alleles were differentiated to BMEC-like cells and used to measure barrier function and metabolism. To investigate exercise effects, serum was collected from older adults pre- and post- 6 months of exercise training (n = 9 participants per genotype). APOE-ε3 and APOE-ε4 BMEC were treated with genotype-matched serum, and then barrier function and metabolism were measured.APOE-ε4 genotype impaired BMEC barrier function and metabolism by reducing sirtuin 1 (SIRT1) levels by 27% (p = 0.0188) and baseline insulin signalling by 37% (p = 0.0186) compared to APOE-ε3 BMEC. Exercise-trained serum increased SIRT1 by 33% (p = 0.0043) in APOE-ε3 BMEC but decreased SIRT1 by 22% (p = 0.0004) in APOE ε4 BMEC.APOE-ε4 directly impairs glucose metabolism and barrier function. Serum from exercise trained individuals alters SIRT1 in a genotype-dependent manner but may require additional cues from exercise to decrease AD pathologies.Brain and Behaviour Initiative at the University of Maryland through the Seed Grant Program, NSF-GRFP DGE 1840340, Fischell Fellowship in Biomedical Engineering, NSF CBET-2211966 and DGE-1632976, National Niemann-Pick Disease Foundation, University of Maryland ASPIRE Program, NIH R01HL165193, R01HL140239-01, and R01AG057552.

  View details for DOI 10.1016/j.ebiom.2024.105487

  View details for PubMedID 39647262

• Fabrication of endothelialized capillary-like microchannel networks using sacrificial thermoresponsive microfibers. Biofabrication Rector, J. A., McBride, L., Weber, C., Grossman, K., Sorets, A., Ventura-Antunes, L., Holtz, I. K., Young, K., Schrag, M., Lippmann, E. S., Bellan, L. M. 2024

  Abstract

  In the body, capillary beds fulfill the metabolic needs of cells by acting as the sites of diffusive transport for vital gasses and nutrients. In artificial tissues, replicating the scale and complexity of these capillary vessels has proved challenging, especially in a three-dimensional context. In order to better develop thick artificial tissues, it will be necessary to recreate both the form and function of capillaries. Here we demonstrate a top-down method of patterning hydrogels using sacrificial templates formed from thermoresponsive microfibers whose size and architecture approach those of natural capillaries. Within the resulting microchannels, we cultured endothelial monolayers that remain viable for over three weeks and exhibited functional barrier properties. Additionally, we cultured endothelialized microchannels within hydrogels containing fibroblasts and characterized the viability of the co-cultures to demonstrate this approach's potential to when applied to cell-laden hydrogels. This method represents a step forward in the evolution of artificial tissues and a path towards producing viable capillary-scale microvasculature for engineered organs.

  View details for DOI 10.1088/1758-5090/ad867d

  View details for PubMedID 39401530

• Brain microvascular endothelial cell metabolism and its ties to barrier function. Vitamins and hormones Weber, C. M., Moiz, B., Clyne, A. M. 2024; 126: 25-75

  Abstract

  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.

  View details for DOI 10.1016/bs.vh.2024.05.002

  View details for PubMedID 39029976

• Mechanical stimuli such as shear stress and piezo1 stimulation generate red blood cell extracellular vesicles. Frontiers in physiology Sangha, G. S., Weber, C. M., Sapp, R. M., Setua, S., Thangaraju, K., Pettebone, M., Rogers, S. C., Doctor, A., Buehler, P. W., Clyne, A. M. 2023; 14: 1246910

  Abstract

  Introduction: Generating physiologically relevant red blood cell extracellular vesicles (RBC-EVs) for mechanistic studies is challenging. Herein, we investigated how to generate and isolate high concentrations of RBC-EVs in vitro via shear stress and mechanosensitive piezo1 ion channel stimulation. Methods: RBC-EVs were generated by applying shear stress or the piezo1-agonist yoda1 to RBCs. We then investigated how piezo1 RBC-EV generation parameters (hematocrit, treatment time, treatment dose), isolation methods (membrane-based affinity, ultrafiltration, ultracentrifugation with and without size exclusion chromatography), and storage conditions impacted RBC-EV yield and purity. Lastly, we used pressure myography to determine how RBC-EVs isolated using different methods affected mouse carotid artery vasodilation. Results: Our results showed that treating RBCs at 6% hematocrit with 10 µM yoda1 for 30 min and isolating RBC-EVs via ultracentrifugation minimized hemolysis, maximized yield and purity, and produced the most consistent RBC-EV preparations. Co-isolated contaminants in impure samples, but not piezo1 RBC-EVs, induced mouse carotid artery vasodilation. Conclusion: This work shows that RBC-EVs can be generated through piezo1 stimulation and may be generated in vivo under physiologic flow conditions. Our studies further emphasize the importance of characterizing EV generation and isolation parameters before using EVs for mechanistic analysis since RBC-EV purity can impact functional outcomes.

  View details for DOI 10.3389/fphys.2023.1246910

  View details for PubMedID 37719461

  View details for PubMedCentralID PMC10502313

• Fast-Training Deep Learning Algorithm for Multiplex Quantification of Mammalian Bioproduction Metabolites via Contactless Short-Wave Infrared Hyperspectral Sensing. ACS omega Hevaganinge, A., Weber, C. M., Filatova, A., Musser, A., Neri, A., Conway, J., Yuan, Y., Cattaneo, M., Clyne, A. M., Tao, Y. 2023; 8 (16): 14774-14783

  Abstract

  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.

  View details for DOI 10.1021/acsomega.3c00861

  View details for PubMedID 37125125

  View details for PubMedCentralID PMC10134457

• Angiotensin II Increases Oxidative Stress and Inflammation in Female, But Not Male, Endothelial Cells. Cellular and molecular bioengineering Weber, C. M., Harris, M. N., Zic, S. M., Sangha, G. S., Arnold, N. S., Dluzen, D. F., Clyne, A. M. 2023; 16 (2): 127-141

  Abstract

  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.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.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.These data suggest that endothelial cells have sexually dimorphic responses to AngII, which could contribute to increased prevalence of some cardiovascular diseases in women.The online version contains supplementary material available at 10.1007/s12195-023-00762-2.

  View details for DOI 10.1007/s12195-023-00762-2

  View details for PubMedID 37096068

  View details for PubMedCentralID PMC10121986

• Induced pluripotent stem cell-derived cells model brain microvascular endothelial cell glucose metabolism. Fluids and barriers of the CNS Weber, C. M., Moiz, B., Zic, S. M., Alpízar Vargas, V., Li, A., Clyne, A. M. 2022; 19 (1): 98

  Abstract

  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.

  View details for DOI 10.1186/s12987-022-00395-z

  View details for PubMedID 36494870

  View details for PubMedCentralID PMC9733016

• Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity. Circulation research Basehore, S. E., Bohlman, S., Weber, C., Swaminathan, S., Zhang, Y., Jang, C., Arany, Z., Clyne, A. M. 2021; 129 (11): 1054-1066

  Abstract

  [Figure: see text].

  View details for DOI 10.1161/CIRCRESAHA.121.318982

  View details for PubMedID 34605247

  View details for PubMedCentralID PMC8653916

• Sex differences in the blood-brain barrier and neurodegenerative diseases. APL bioengineering Weber, C. M., Clyne, A. M. 2021; 5 (1): 011509

  Abstract

  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.

  View details for DOI 10.1063/5.0035610

  View details for PubMedID 33758788

  View details for PubMedCentralID PMC7968933

• Thermoresponsive Transient Radio Frequency Antennas: Toward Triggered Wireless Transient Circuits ADVANCED MATERIALS TECHNOLOGIES Zhang, X., Weber, C. M., Bellan, L. M. 2019; 4 (11)

  View details for DOI 10.1002/admt.201900528

  View details for Web of Science ID 000486529200001

• iPSC-Derived Brain Endothelium Exhibits Stable, Long-Term Barrier Function in Perfused Hydrogel Scaffolds. Stem cell reports Faley, S. L., Neal, E. H., Wang, J. X., Bosworth, A. M., Weber, C. M., Balotin, K. M., Lippmann, E. S., Bellan, L. M. 2019; 12 (3): 474-487

  Abstract

  There is a profound need for functional, biomimetic in vitro tissue constructs of the human blood-brain barrier and neurovascular unit (NVU) to model diseases and identify therapeutic interventions. Here, we show that induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (BMECs) exhibit robust barrier functionality when cultured in 3D channels within gelatin hydrogels. We determined that BMECs cultured in 3D under perfusion conditions were 10-100 times less permeable to sodium fluorescein, 3 kDa dextran, and albumin relative to human umbilical vein endothelial cell and human dermal microvascular endothelial cell controls, and the BMECs maintained barrier function for up to 21 days. Analysis of cell-cell junctions revealed expression patterns supporting barrier formation. Finally, efflux transporter activity was maintained over 3 weeks of perfused culture. Taken together, this work lays the foundation for development of a representative 3D in vitro model of the human NVU constructed from iPSCs.

  View details for DOI 10.1016/j.stemcr.2019.01.009

  View details for PubMedID 30773484

  View details for PubMedCentralID PMC6409430