Steady Laminar Flow Decreases Endothelial Glycolytic Flux While Enhancing Proteoglycan Synthesis and Antioxidant Pathways.

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.

A Systems Approach to Biomechanics, Mechanobiology, and Biotransport.

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.

Fast-Training Deep Learning Algorithm for Multiplex Quantification of Mammalian Bioproduction Metabolites via Contactless Short-Wave Infrared Hyperspectral Sensing.

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.

Angiotensin II Increases Oxidative Stress and Inflammation in Female, But Not Male, Endothelial Cells.

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.

Interpreting metabolic complexity via isotope-assisted metabolic flux analysis.

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.

Induced pluripotent stem cell-derived cells model brain microvascular endothelial cell glucose metabolism.

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.

Isotope-Assisted Metabolic Flux Analysis: A Powerful Technique to Gain New Insights into the Human Metabolome in Health and Disease.

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.

Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity.

[Figure: see text].

13C Metabolic Flux Analysis Indicates Endothelial Cells Attenuate Metabolic Perturbations by Modulating TCA Activity.

Disrupted 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.

Sex differences in the blood-brain barrier and neurodegenerative diseases.

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.

Endothelial response to glucose: dysfunction, metabolism, and transport.

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.

A simple method to align cells on 3D hydrogels using 3D printed molds.

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.

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks.

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

A Modified Parallel Plate Flow Chamber to Study Local Endothelial Response to Recirculating Disturbed Flow.

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.

Correction to: Translating Mechanobiology to the Clinic: A Panel Discussion from the 2018 CMBE Conference.

[This corrects the article DOI: 10.1007/s12195-018-0556-5.].

A Course-Based Undergraduate Research Experience in Biofluid Mechanics.

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.

A Three-Dimensional In Vitro Coculture Model to Quantify Breast Epithelial Cell Adhesion to Endothelial Cells.

Three-dimensional (3D) in vitro culture models better recapitulate the tissue microenvironment, and therefore may provide a better platform to evaluate therapeutic effects on adhesive cell-cell interactions. The objective of this study was to determine if AD-01, a peptide derivative of FK506-binding protein like that is reported to bind to the adhesion receptor CD44, would induce a greater reduction in breast epithelial spheroid adhesion to endothelial tube-like networks in our 3D coculture model system compared to two-dimensional (2D) culture. MCF10A, MCF10A-NeuN, MDA-MB-231, and MCF7 breast epithelial cells were pretreated with AD-01 either as single cells or as spheroids. Breast epithelial cell adhesion to 2D tissue culture substrates was first measured, followed by spheroid formation (breast cell-cell adhesion) and spheroid adhesion to Matrigel or endothelial networks. Finally, CD44 expression was quantified in breast epithelial cells in 2D and 3D culture. Our results show that AD-01 had the largest effect on spheroid formation, specifically in breast cancer cell lines. AD-01 also inhibited breast cancer spheroid adhesion to and migration along endothelial networks. The different breast epithelial cell lines expressed more CD44 when cultured as 3D spheroids, but this did not universally translate into higher protein levels. This study shows that 3D coculture models can enable unique insights into cell adhesion, migration, and cell-cell interactions, thereby enhancing understanding of basic biological mechanisms. Furthermore, such 3D coculture systems may also represent a more relevant testing platform for understanding the mechanism-of-action of new therapeutic agents. Impact Statement Cell adhesion is inherently different in two dimensional (2D) compared to three dimensional (3D) culture; yet, most adhesion assays in academia and industry are still conducted in 2D because few simple, yet effective, adhesion models exist in 3D. Recently we developed a 3D in vitro coculture model to examine breast epithelial spheroid interactions with endothelial tubes. We now show that this 3D coculture model can effectively be used to interrogate and quantify drug-induced differences in breast epithelial cell adhesion that are unique to 3D cocultures. This 3D coculture adhesion model can furthermore be modified for use with other cell types to better predict drug effects on cell-vasculature adhesion.

Stiff Substrates Enhance Endothelial Oxidative Stress in Response to Protein Kinase C Activation.

Arterial stiffness, which increases with aging and hypertension, is an independent cardiovascular risk factor. While stiffer substrates are known to affect single endothelial cell morphology and migration, the effect of substrate stiffness on endothelial monolayer function is less understood. The objective of this study was to determine if substrate stiffness increased endothelial monolayer reactive oxygen species (ROS) in response to protein kinase C (PKC) activation and if this oxidative stress then impacted adherens junction integrity. Porcine aortic endothelial cells were cultured on varied stiffness polyacrylamide gels and treated with phorbol 12-myristate 13-acetate (PMA), which stimulates PKC and ROS without increasing actinomyosin contractility. PMA-treated endothelial cells on stiffer substrates increased ROS and adherens junction loss without increased contractility. ROS scavengers abrogated PMA effects on cell-cell junctions, with a more profound effect in cells on stiffer substrates. Finally, endothelial cells in aortae from elastin haploinsufficient mice (Eln+/-), which were stiffer than aortae from wild-type mice, showed decreased VE-cadherin colocalization with peripheral actin following PMA treatment. These data suggest that oxidative stress may be enhanced in endothelial cells in stiffer vessels, which could contribute to the association between arterial stiffness and cardiovascular disease.