Browsing by Department "Biomedical Engineering"
- Results Per Page
- Sort Options
Item Open Access 3D dynamic in vivo imaging of joint motion: application to measurement of anterior cruciate ligament function(2019) Englander, Zoë AlexandraMore than 400,000 anterior cruciate ligament (ACL) injuries occur annually in the United States, 70% of which are non-contact. A severe consequence of ACL injury is the increased risk of early-onset of osteoarthritis (OA). Importantly, the increased risk of OA persists even if the ACL is surgically reconstructed. Thus, due to the long term physical consequences and high financial burden of treatment, injury prevention and improved reconstruction techniques are critical. However, the causes of non-contact ACL injuries remain unclear, which has hindered efforts to develop effective training programs targeted at preventing these injuries. Improved understanding of the knee motions that increase the risk of ACL injury can inform more effective injury prevention strategies. Furthermore, there is presently limited in vivo data to describe the function of ACL under dynamic loading conditions. Understanding how the ACL functions to stabilize the knee joint under physiologic loading conditions can inform design criteria for grafts used in ACL reconstruction. Grafts that more accurately mimic the native function of the ACL may help prevent these severe long term degenerative changes in the knee joint after injury.
To this end, measurements of in vivo ACL function during knee motion are critical to understanding how non-contact ACL injuries occur and the function of the ACL in stabilizing the joint during activities of daily living. Specifically, identifying the knee motions that increase ACL length and strain can elucidate the mechanisms of non-contact ACL injury, as a taut ligament is more likely to fail. Furthermore, measuring ACL elongation patterns during dynamic activity can inform the design criteria for grafts used in reconstructive surgery. To obtain measurements, 3D imaging techniques that can be used to measure dynamic in vivo ACL elongation and strain at high temporal and spatial resolution are needed.
Thus, in this dissertation a method of measuring knee motion and ACL function during dynamic activity in vivo using high-speed biplanar radiography in combination with magnetic resonance (MR) imaging was developed. In this technique, 3D surface models of the knee joint are created from MR images and registered to high-speed biplanar radiographs of knee motion. The use of MR imaging to model the joint allows for visualization of bone and soft tissue anatomy, in particular the attachment site footprints of the ligaments. By registering the bone models to biplanar radiographs using software developed in this dissertation, the relative positions of the bones and associated ligament attachment site footprints at the time of radiographic imaging can be reproduced. Thus, measurements of knee kinematics and ligament function during dynamic activity can be obtained at high spatial and temporal resolution.
We have applied the techniques developed in this dissertation to obtain novel dynamic in vivo measurements of the mechanical function of the knee joint. Specifically, the physiologic elongation and strain behaviors of the ACL during gait and single-legged jumping were measured. Additionally, the dynamic function of the patellar tendon during single legged jumping was measured. The findings of this dissertation have helped to elucidate the knee kinematics that increase ACL injury vulnerability by identifying the dynamic motions that result in elongation and strain in the ACL. Furthermore, the findings of this dissertation have provided critical data to inform design criteria for grafts used in reconstructive surgery such that reconstructive techniques better mimic the physiologic function of the ACL.
The methodologies described in this dissertation can be applied to study the mechanical behavior of other joints such as the spine, and other soft tissues, such as articular cartilage, under various loading conditions. Therefore, these methods may have a significant impact on the field of biomechanics as a whole, and may have applicability to a number of musculoskeletal applications.
Item Open Access 3D Human Skeletal Muscle Model for Studying Satellite Cell Quiescence and Pompe disease(2021) Wang, JasonTissue-engineered skeletal muscle presents promising opportunities for developing high-fidelity in vitro models for investigating human muscle biology in the areas of regeneration and disease. In muscle regeneration, satellite cells (SCs) are essential for new muscle fiber formation; however, they lose their native quiescent state upon isolation, making in vitro studies of human SC function challenging. To optimally promote SC quiescence and enable exploration of SC dynamics in vitro, engineered muscle needs to recapitulate the native muscle microenvironment, which is comprised of muscle fibers, extracellular matrix, and other biochemical and mechanical cues. In disease modeling, mechanistic studies and therapeutic development are still extensively evaluated in animal models, which have limited translational relevance to patients. Specifically, Pompe disease is caused by a variety of mutations in the lysosomal enzyme acid alpha-glucosidase (GAA) that varyingly affect residual GAA activity and cannot be captured in the current GAA-/- mouse model. Therefore, human in vitro models are needed to enhance our mechanistic understanding of diseases and stimulate the development of effective therapies. To overcome these limitations, we set the dissertation goals to: 1) generate a pool of quiescent SCs and explore the mechanisms governing their formation and activation using an engineered skeletal muscle microenvironment and 2) develop a high-fidelity tissue-engineered skeletal muscle model for Pompe disease to investigate pathological mechanisms and test candidate therapies. To achieve these goals, we first compared methods for primary human myoblast expansion and found that p38 inhibition significantly increases the formation of Pax7+ cells in engineered 3D skeletal muscle tissues (“myobundles”). Gene expression analysis suggested that within the myobundle environment the Pax7+ cells adopt a quiescent phenotype (3D SCs), characterized by increased Pax7 expression, cell cycle exit, and Notch signaling activation relative to the original 2D expanded myoblasts. We then compared 3D SCs to previously described satellite-like cells that form alongside myotubes in 2D culture, termed reserve cells (RCs). Compared to RCs, 3D SCs showed an advanced quiescent phenotype characterized by a higher Pax7, Spry1, and Notch3 expression, as well as increased functional myogenesis demonstrated by formation of myobundles with higher contractile strength. To examine 3D SC activation, we tested several myobundle injury methods and identified treatment with a bee toxin, melittin, to robustly induce myofiber fragmentation, functional decline, and 3D SC proliferation. To further investigate the transcriptional processes describing how 2D myoblasts acquire 3D SC phenotype (i.e. deactivate) and how 3D SCs respond to injury (i.e. reactivate), we applied single cell RNA-sequencing (scRNA-seq) from which we discovered the existence of two subpools of 3D SCs—“quiescent” (qSC) and “activated” (aSC). The qSC subpool possessed greater expression of quiescence genes Pax7, Spry1, and Hey1, whereas the aSC subpool exhibited increased expression of inflammatory and differentiation markers. Furthermore, we performed trajectory inference along the deactivation process from 2D myoblasts to qSCs and identified deactivation-associated genes, included downregulated genes for proliferation, cytoskeletal reorganization, and myogenic differentiation. In response to tissue injury, we observed a decrease in the proportion of qSCs and an increase in the proportion of aSCs and committed myogenic progenitor cells suggestive of myogenic differentiation. In addition, we observed transcriptional changes within the aSC population reflective of SC activation in vivo, namely increased TNF- signaling, proliferation, and glycolytic and oxidative metabolism. These results strongly suggested that 3D SC heterogeneity and function recapitulate several aspects of native human SCs and could be applied to study human muscle regeneration and disease-associated SC dysfunction. To evaluate the myobundle system in the context of disease modeling, we developed the first 3D tissue-engineered skeletal muscle model of infantile onset Pompe disease (IOPD), the most severe form of Pompe disease. Diseased myobundles demonstrated characteristic GAA enzyme deficiency, accumulation of the GAA target glycogen, and lysosome enlargement. Despite exhibiting these key biochemical and structural hallmarks of disease, IOPD myobundles did not show deficits in contractile force generation or autophagic buildup. We therefore identified metabolic stress conditions that acutely targeted disease-associated abnormalities in the lysosomes and glycogen metabolism, which revealed impairments in contractile function and glycogen mobilization. To further elucidate the biological mechanisms underlying the phenotype of IOPD myobundles, we applied RNA sequencing (RNA-seq) and observed enrichment for terms consistent with Pompe disease phenotype including downregulation of gene sets involved in muscle contraction, increased endoplasmic reticulum stress, and reduced utilization of specific metabolic pathways. We then compared the transcriptomic profiles of GAA-/-¬ and wild-type mice to identify a Pompe disease signature and confirmed the presence of this signature in IOPD myobundles. Finally, treating IOPD myobundles with clinically used recombinant protein (rhGAA) therapy resulted in increased GAA activity, glycogen clearance, and a partial reversal of the disease signature, further confirming the utility of the myobundle system for studies of Pompe disease and therapy. In summary, this dissertation describes novel strategies for the formation and characterization of quiescent human SCs using the myobundle system. We present first-time application of scRNA-seq to engineered skeletal muscle, and uncover transcriptional descriptors of human myoblast deactivation and SC heterogeneity and activation. When utilizing human myobundles as a novel model of Pompe disease, we identified disease hallmarks and responses to therapy consistent with observations in Pompe patients. We anticipate that the findings and methods developed in this work will serve as a useful framework for the future engineering of regenerative human muscle for therapeutic and disease modeling applications.
Item Open Access A 3-D Multiparametric Ultrasound Elasticity Imaging System for Targeted Prostate Biopsy Guidance(2023) Chan, Derek Yu XuanProstate cancer is the most common cancer and second-leading cause of cancer death among men in the United States. Early and accurate diagnosis of prostate cancer remains challenging; following an abnormal rectal exam or elevated levels of prostate-specific antigen in serum, clinical guidelines recommend transrectal ultrasound-guided biopsy. However, lesions are often indistinguishable from noncancerous prostate tissue in conventional B-mode ultrasound images, which have a diagnostic sensitivity of about 30%, so the biopsy is not typically targeted to suspicious regions. Instead, the biopsy systematically samples 12 pre-specified regions of the gland. Systematic sampling often fails to detect cancer during the first biopsy, and while multiparametric MRI (mpMRI) techniques have been developed to guide a targeted biopsy, fused with live ultrasound, this approach remains susceptible to registration errors, and is expensive and less accessible.
The goal of this work is to leverage ultrasound elasticity imaging methods, including acoustic radiation force impulse (ARFI) imaging and shear wave elasticity imaging (SWEI), to develop and optimize a robust 3-D elasticity imaging system for ultrasound-guided prostate biopsies and to quantify its performance in prostate cancer detection. Towards that goal, in this dissertation advanced techniques for generating ARFI and SWEI images are developed and evaluated, and a deep learning framework is explored for multiparametric ultrasound (mpUS) imaging, which combines data from different ultrasound-based modalities.
In Chapter 3, an algorithm is implemented that permits the simultaneous imaging of prostate cancer and zonal anatomy using both ARFI and SWEI. This combined sequence involves using closely spaced push beams across the lateral field of view, which enables the collection of higher signal-to-noise (SNR) shear wave data to reconstruct the SWEI volume than is typically acquired. Data from different push locations are combined using an estimated shear wave propagation time between push excitations to align arrival times, resulting in SWEI imaging of prostate cancer with high contrast-to-noise ratio (CNR), enhanced spatial resolution, and reduced reflection artifacts.
In Chapter 4, a fully convolutional neural network (CNN) is used for ARFI displacement estimation in the prostate. A novel method for generating ultrasound training data is described, in which synthetic 3-D displacement volumes with a combination of randomly seeded ellipsoids are used to displace scatterers, from which simulated ultrasonic imaging is performed. The trained network enables the visualization of in vivo prostate cancer and prostate anatomy, providing comparable performance with respect to both accuracy and speed compared to standard time delay estimation approaches.
Chapter 5 explores the application of deep learning for mpUS prostate cancer imaging by evaluating the use of a deep neural network (DNN) to generate an mpUS image volume from four ultrasound-based modalities for the detection of prostate cancer: ARFI, SWEI, quantitative ultrasound, and B-mode. The DNN, which was trained to maximize lesion CNR, outperforms the previous method of using a linear support vector machine to combine the input modalities, and generates mpUS image volumes that provide clear visualization of prostate cancer.
Chapter 6 presents the results of the first in vivo clinical trial that assesses the use of ARFI imaging for targeted prostate biopsy guidance in a single patient visit, comparing its performance with mpMRI-targeted biopsy and systematic sampling. The process of data acquisition, processing, and biopsy targeting is described. The study demonstrates the feasibility of using 3-D ARFI for guiding a targeted biopsy of the prostate, where it is most sensitive to higher-grade cancers. The findings also indicate the potential for using 2-D ARFI imaging to confirm target location during live B-mode imaging, which could improve existing ultrasonic fusion biopsy workflows.
Chapter 7 summarizes the research findings and considers potential directions for future research. By developing advanced ARFI and SWEI imaging techniques for imaging the prostate gland, and combining information from different ultrasound modalities, prostate cancer and zonal anatomy can be imaged with high contrast and resolution. The findings from this work suggest that ultrasound elasticity imaging holds great promise for facilitating image-guided targeted biopsies of clinically significant prostate cancer.
Item Embargo A bidirectional switch to treat colonic dysmotility(2023) Barth, Bradley BrighamSevere constipation can be life-threatening and disproportionately affects patients who may not benefit from conventional treatments. Sacral nerve stimulation (SNS) is an alternative to laxatives and pharmaceuticals, and it modulates propulsive action in the colon. Conventional SNS failed to treat slow-transit constipation. I hypothesized bursts of nerve stimulation interleaved by quiescent periods increase colonic transit more effectively than continuous nerve stimulation. I electrically stimulated the colon directly in computational models and the isolated mouse colon to characterize properties of the colonic motor complex (CMC), and I used optical and fluorescent imaging, electromyography, and manometry to compare the effect of pelvic and sacral nerve stimulation on colonic motility. I developed a computational model of colonic motility and compared the effects of burst and conventional nerve stimulation on pellet velocity and colonic emptying under normal and slow transit conditions. Burst nerve stimulation evoked more frequent calcium and pressure waves, and increased fecal pellet output than continuous nerve stimulation in the isolated mouse colon, anesthetized rat, and computational model, respectively. Burst nerve stimulation with optimized burst frequency, duration, and interval more effectively produced prokinetic motility than continuous nerve stimulation, suggesting that burst SNS may be a viable clinical treatment for severe and slow transit constipation.
Item Open Access A Comprehensive Framework for Adaptive Optics Scanning Light Ophthalmoscope Image Analysis(2019) Cunefare, DavidDiagnosis, prognosis, and treatment of many ocular and neurodegenerative diseases, including achromatopsia (ACHM), require the visualization of microscopic structures in the eye. The development of adaptive optics ophthalmic imaging systems has made high resolution visualization of ocular microstructures possible. These systems include the confocal and split detector adaptive optics scanning light ophthalmoscope (AOSLO), which can visualize human cone and rod photoreceptors in vivo. However, the avalanche of data generated by such imaging systems is often too large, costly, and time consuming to be evaluated manually, making automation necessary. The few currently available automated cone photoreceptor identification methods are unable to reliably identify rods and cones in low-quality images of diseased eyes, which are common in clinical practice.
This dissertation describes the development of automated methods for the analysis of AOSLO images, specifically focusing on cone and rod photoreceptors which are the most commonly studied biomarker using these systems. A traditional image processing approach, which requires little training data and takes advantage of intuitive image features, is presented for detecting cone photoreceptors in split detector AOSLO images. The focus is then shifted to deep learning using convolutional neural networks (CNNs), which have been shown in other image processing tasks to be more adaptable and produce better results than classical image processing approaches, at the cost of requiring more training data and acting as a “black box”. A CNN based method for detecting cones is presented and validated against state-of-the-art cone detections methods for confocal and split detector images. The CNN based method is then modified to take advantage of multimodal AOSLO information in order to detect cones in images of subjects with ACHM. Finally, a significantly faster CNN based approach is developed for the classification and detection of cones and rods, and is validated on images from both healthy and pathological subjects. Additionally, several image processing and analysis works on optical coherence tomography images that were carried out during the completion of this dissertation are presented.
The completion of this dissertation led to fast and accurate image analysis tools for the quantification of biomarkers in AOSLO images pertinent to an array of retinal diseases, lessening the reliance on subjective and time-consuming manual analysis. For the first time, automatic methods have comparable accuracy to humans for quantifying photoreceptors in diseased eyes. This is an important step in the long-term goal to facilitate early diagnosis, accurate prognosis, and personalized treatment of ocular and neurodegenerative diseases through optimal visualization and quantification of microscopic structures in the eye.
Item Open Access A Discrete Monolayer Cardiac Tissue Model for Tissue Preparation Specific Modeling(2010) Kim, JongmyeongEngineered monolayers created by using microabrasion and micropatterning methods have provided a simplified in vitro system to study the effects of anisotropy and fiber direction on electrical propagation. Interpreting the behavior in these culture systems has often been performed using classical computer models with continuous properties. Such models, however, do not account for the effects of random cell shapes, cell orientations and cleft spaces inherent in these monolayers on the resulting wavefront conduction. Additionally when the continuous computer model is built to study impulse propagations, the intracellular conductivities of the model are commonly assigned to match impulse conduction velocity of the model to the experimental measurement. However this method can result in inaccurate intracellular conductivities considering the relationship among the conduction velocity, intracellular conductivities and ion channel properties. In this study, we present novel methods for modeling a monolayer cardiac tissue and for estimating intracellular conductivities from an optical mapping. First, in the proposed method for modeling a monolayer of cardiac tissue, the factors governing cell shape, cell-to-cell coupling and the degree of cleft space are not constant but rather are treated as spatially random with assigned distributions. This approach makes it possible to simulate wavefront propagation in a manner analogous to performing experiments on engineered monolayer tissues. Simulated results are compared to reported experimental data measured from monolayers used to investigate the role of cellular architecture on conduction velocities and anisotropy ratios. We also present an estimate for obtaining the electrical properties from these networks and demonstrate how variations in the discrete cellular architecture affect the macroscopic conductivities. The simulation results agree with the common assumption that under normal ranges of coupling strengths, tissues whose cell shapes and connectivity show relatively uniform distributions can be represented using continuous models with conductivities derived from random discrete cellular architecture using either estimates. The results also reveal that in the presence of abrupt changes in cell orientation, local estimates of tissue properties predict smoother changes in conductivities that may not adequately predict the discrete nature of propagation at the transition sites. Second, a novel approach is proposed to estimate intracellular conductivities from the optical mapping of the monolayer cardiac tissue under subthreshold stimulus. This method uses a simplified membrane model, which represents the membrane as a second order polynomial of the membrane potential. The simplified membrane model and the intracellular conductivities are estimated from the optical mapping of the monolayer tissue under the subthreshold stimulus. We showed that the proposed method provides more accurate intracellular conductivities compared to a method using a constant membrane resistance.
Item Open Access A Framework for Dissecting and Applying Bacterial Antibiotic Responses(2017) Meredith, Hannah Ruth BrittanyAn essential property of microbial communities is the ability to survive a disturbance. This is readily observed in bacteria, which have developed the ability to survive every antibiotic treatment at an alarming rate, considering the timescale at which new antibiotics are developed. Thus, there is a critical need to use antibiotics more effectively, extend the shelf life of existing antibiotics and minimize their side effects. This requires understanding the mechanisms underlying bacterial drug responses. Past studies have focused on survival in the presence of antibiotics by individual cells, such as genetic mutants. Also important, however, is the fact that a population of bacterial cells can collectively survive antibiotic treatments lethal to individual cells. This tolerance can arise by diverse mechanisms, including resistance-conferring enzyme production, titration-mediated bistable growth inhibition, swarming and interpopulation interactions. These strategies can enable rapid population recovery after antibiotic treatment and provide a time window during which otherwise susceptible bacteria can acquire inheritable genetic resistance.
To further explore bacterial antibiotic responses, I focused on bacteria producing β-lactamase, an enzyme that has drastically limited the use of our most commonly prescribed antibiotics: β-lactams. Through the characterization of clinical isolates and a computational model, my Ph.D. thesis has three implications:
First, survival can be achieved through resistance, the ability to absorb effects of a disturbance without a significant change, or resilience, the ability to recover after being perturbed by a disturbance. Current practices for determining the antibiotic sensitivity of bacteria do not characterize a population as resistant and/or resilient, they only report whether the bacteria can survive the antibiotic exposure. As resistance and resilience often depend on different attributes, distinguishing between these two modes of survival could inform treatment strategies. These concepts have long been applied to the analysis of ecological systems, though their interpretations are often subject to debate. This framework readily lends itself to the dissection of the bacterial response to antibiotic treatment, where both terms can be unambiguously defined.
Second, the ability to tolerate the antibiotic treatment in the short term corresponds to resistance, which primarily depends on traits associated with individual cells. In contrast, the ability to recover after being perturbed by an antibiotic corresponds to resilience, which primarily depends on traits associated with the population.
And finally, understanding the temporal dynamics of an antibiotic response could guide the design of a dosing protocol to optimize treatment efficiency for any antibiotic-pathogen combination. Ultimately, optimized dosing protocols could allow reintroduction of a repertoire of first-line antibiotics with improved treatment outcomes and preserve last-resort antibiotics.
Item Open Access A mechanistic understanding of the postantibiotic effect and treatment strategies(2017) Srimani, JaydeepAlthough antibiotics have proven to be one of the great achievements of modern medicine, their efficacy has dramatically decreased over the past several decades. This is due, in part, to the rapid pace of natural bacterial evolution, but also to the overuse and misuse of antibiotics in general. This often selects for drug-resistant pathogens, and allows them to flourish in the face of antibiotic treatment. In addition to the emergence of genetic resistance, bacteria often utilize a number of population-level behaviors to survive antibiotic treatment. This is referred to as collective antibiotic tolerance (CAT). Taken together, antibiotic resistance and tolerance have led to the re-emergence of infectious diseases throughout the world. In general, there are two strategies to combat this risk: develop novel antibiotics, and/or use existing drugs more effectively, so as to minimize the chance of resistance emergence. Novel drug development is a time- and resource-intensive process, and pharmaceutical companies are not financially incentivized to develop these types of drugs. Therefore, it is of increasing importance to understand the population dynamics underlying various bacterial survival mechanisms, and exploit this knowledge to design better antibiotic treatment protocols.
My dissertation research focuses on a prevalent phenomenon called the postantibiotic effect (PAE), which refers to the transient suppression of bacterial growth following antibiotic treatment. Although PAE has been empirically observed in a wide variety of antibiotics and microbial species, heretofore there has not been a definitive mechanistic explanation for this pervasive observation.
In this work, I use a combination of high-throughput microfluidic experiments and computational modeling to examine the relationship between dosing parameters and the degree of bacterial inhibition, quantified by population recovery time. I found that recovery time is a function of total antibiotic, regardless of how the dose profile. Moreover, a minimal model of transport and binding kinetics was sufficient to recapture this trend, suggesting a unifying explanation for historical observations of PAE in a variety of contexts. I validated this modeling using both in silico and in vitro perturbation studies.
Moreover, I showed that efflux inhibition, a common strategy in antibiotic treatment, is effective in certain dynamic-dependent situations. This work puts forth a possible mechanism for PAE, which could serve as a clinical aid in selecting effective antibiotic/adjuvant combinations, as well as in designing periodic antibiotic treatments.
Item Open Access A Model of Lung Tumor Angiogenesis in a Biomimetic Poly(ethylene glycol)-based Hydrogel System(2016) Roudsari, Laila ChristineTumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. A major outstanding challenge associated with studying tumor angiogenesis is that existing preclinical models are limited in their recapitulation of in vivo cellular organization in 3D. This disparity highlights the need for better approaches to study the dynamic interplay of relevant cells and signaling molecules as they are organized in the tumor microenvironment. In this thesis, we combined 3D culture of lung adenocarcinoma cells with adjacent 3D microvascular cell culture in 2-layer cell-adhesive, proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogels to study tumor angiogenesis and the impacts of neovascularization on tumor cell behavior.
In initial studies, 344SQ cells, a highly metastatic, murine lung adenocarcinoma cell line, were characterized alone in 3D in PEG hydrogels. 344SQ cells formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells alone in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, the engineered 2-layer tumor angiogenesis model with 344SQ and vascular cell layers was employed. Large, invasive 344SQ clusters developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed 344SQ cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration.
Two other lung adenocarcinoma cell lines were also explored in the tumor angiogenesis model: primary tumor-derived metastasis-incompetent, murine 393P cells and primary tumor-derived metastasis-capable human A549 cells. These lung cancer cells also formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media. Epithelial morphogenesis varied for the primary tumor-derived cell lines compared to 344SQ cells, with far less epithelial organization present in A549 spheroids. Additionally, 344SQ cells secreted the highest concentration of two of the three angiogenic growth factors assessed. This finding correlated to 344SQ exhibiting the most pronounced morphological response in the tumor angiogenesis model compared to the 393P and A549 cell lines.
Overall, this dissertation demonstrates the development of a novel 3D tumor angiogenesis model that was used to study vascular cell-cancer cell interactions in lung adenocarcinoma cell lines with varying metastatic capacities. Findings in this thesis have helped to elucidate the role of vascular cells in tumor progression and have identified differences in cancer cell behavior in vitro that correlate to metastatic capacity, thus highlighting the usefulness of this model platform for future discovery of novel tumor angiogenesis and tumor progression-promoting targets.
Item Embargo A multilevel high-throughput sequencing approach for identifying microbial community interactions and informing precision microbiome engineering(2023) Rodriguez, Daniel LuisA multilevel high-throughput sequencing approach for identifying microbial community interactions and informing precision microbiome engineering
Item Embargo A Multiplexed, Multi-scale Optical Imaging Platform to Quantify Tumor Metabolic Heterogeneity(2023) Deutsch, Riley JosephThe American Cancer Society reported an estimated 300,000 new cases of breast cancer and 44,000 new breast-cancer related deaths in 2022 in the United States alone. With each new successfully treated primary tumor, there is a subsequent risk of disease recurrence. Recurrence poses a risk to 10% of patients within the first 5 years post treatment and a lifetime risk of 30% across all patients. While new tools are being developed to better understand and mitigate the risk of recurrence, triple negative breast cancers, which exhibit no targetable surface markers, offer little in the way of recurrence prediction or treatment. It is understood that tumor heterogeneity is a driving force in tumor recurrence. Temporal heterogeneity is associated with therapeutic treatment, where the administration either selects for resistance subpopulations of tumor cells that are able to recur or a de novo resistant phenotype arises that leads to recurrence. Additionally, it has been well documented that tumors vary spatially across a primary tumor. This heterogeneity takes the form of genetic, epigenetic, and phenotypic heterogeneity. One such phenotype of interest is metabolic heterogeneity. Metabolism is classified as a ‘Hallmark of Cancer’ and has been studied as a driver of tumor progression for almost a century since Otto Warburg first described the phenomenon of tumors exhibiting high rates of aerobic glycolysis. Optical imaging is well poised to study metabolic heterogeneity due to its ability to image cellular level features, to multiplex multiple endpoints, and the ability to image longitudinally. Endogenous fluorescence contrast of coenzymes NADH and FAD have been used to report on the redox state of in vivo tissue and distinguish cancerous from benign lesions. The Center for Global Women’s Health Technologies (GWHT) has employed the use of exogenous fluorescent contrast agents to provide substrate-specific metabolic information. Three fluorescent agents have been validated including: 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), a glucose derivative that is able to report on glycolysis; Tetramethylrhodamine ethyl ester (TMRE), a cation that is selectively attracted to the charge gradient generated by the mitochondria during ATP synthesis, making it a reporter of OXPHOS; and Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid (Bodipy FL C16), a long chain saturated fatty acid is taken up by the cell and undergoes beta oxidation similar to native fatty acids. More recently, GWHT has begun combing these fluorescence agents for in vivo use to provide a wholistic understanding of cancer metabolism. The work here sets out to develop a novel optical imaging platform that is capable of imaging multiplexed metabolic endpoints, for quantitative intra-image analysis of metabolic gradients. This technology is built on the use of exogenous fluorescence contrast agents to report on substrate or pathway specific axes of metabolism. By simultaneously introducing multiple contrast agents, it is possible to capture a more wholistic snapshot of tissue metabolism. To encourage the adoption of this technology, a novel low-cost instrument will also be developed. Leveraging a consumer grade CMOS camera and variable focus lens, it is possible to image over multiple length scales, capturing both bulk tumor features and also single cell features. The flexibility offered by this simple innovation will allow for metabolic imaging to be applied over a variety sample type. Three specific aims were proposed to realize this goal by developing methods of multi-parametric exogenous contrast and low-cost instrumentation for multi-scale imaging of tumor metabolic heterogeneity in preclinical models. Aim 1 validated and demonstrated a method for the simultaneous injection and measurement of Bodipy FL C16 and TMRE to report on lipid uptake and mitochondrial activity, two potentially interrelated axes of metabolism. To validate this method, three sets of experiments were performed to establish that the two probes do not exhibit chemical, optical, or biological crosstalk. Chemical compatibility was established using liquid chromatography. Briefly, high molar concentration solutions of each individual probe (Bodipy FL C16, TMRE, and 2-NBDG) were created alongside a solution of all three probes at the same concentration. Chromatograms were collected immediately upon mixing, after 1 hour and after 24 hours. The area under the curve for each probe at each time point displayed an area under the curve (AUC) within 2% of the AUC of the single probe solutions, suggesting no chemical reactions. Optical crosstalk was assessed using optical spectroscopy and tissue mimicking phantoms. Optical phantoms were created with tissue mimicking optical properties and various concentration of Bodipy FL C16, TMRE, polystyrene microspheres (tissue scattering mimic), and hemoglobin (tissue absorption mimic). Leveraging an inverse Monte Carlo algorithm, we demonstrated that accurate values for each fluorescent probe could be measured regardless of the concentration of the other optical probe or level of optical scattering or absorption, indicating optical compatibility. To address biological crosstalk, two sets of 4T1 tumor bearing mice were subject to optical spectroscopy with either 1) Bodipy FL C16 alone, 2) TMRE alone, 3) a dual injection of Bodipy FL C16 and TMRE. Fluorescence spectra were measured 2-, 4-, 6-, 8-, 10-, 20-, 30-, 40-, 50-, and 60-minutes post-injection to establish uptake kinetics. It was found that the uptake kinetics of the dual probe group were not statistically different from the single probe group, indicating biological compatibility. With no observable crosstalk between Bodipy FL C16 and TMRE, the two probes method was applied to characterize murine mammary gland and two tumor of differing metastatic potential (4T1 and 67NR). In addition, to Bodipy FL C16 and TMRE, oxygen saturation and total hemoglobin were extracted from estimates of optical absorption, and these 4 endpoints were used to attempt to cluster groups of tumor and normal tissue. Difficulty clustering tumor groups of varying metastatic potential suggest a need for imaging technology. In Aim 2 a low-cost fluorescence microscope was developed capable of performing quantitative fluorescence imaging over a variety of samples. The goal of this work was to design a system that could be adapted to image a number of different sample types include core-needle tissue biopsies, preclinical window chambers, and in vitro organoids. To accomplish this a low-cost CMOS detector was used with a variable magnification lens allowing for imaging at multiple length scales. Uniform illumination was a necessary criterion for quantitative imaging. To generate uniform illumination that could be scaled across multiple length scales, an LED coupled 1:4 fanout optical fiber was employed alongside a computational model to determine the positioning of each fiber. To automate the design of illumination, a computational model was employed where each optical fiber was modeled as a Lambertian emitter in a spherical coordinate system. To determine the ideal placement of each fiber such that the individual illumination contributions of all fibers summed to a uniform distribution, a global optimizer was employed. A genetic pattern search allowed for the selection of coordinates to produce uniform illumination that could be feasibly employed at the benchtop. This integrated system is referred to as the CapCell microscope. Using this computational approach, two uniform illumination profiles were designed, one with a high aspect ratio (length ≫ width) and one with a low aspect ratio (length = width). To demonstrate the utility of optimized illumination, core needle biopsies from 4T1 tumors were stained with a tumor-specific fluorescent contrast agent, HS-27 and imaged with either optimized or unoptimized gaussian illumination. The repeatability of intra-image features was compared for the two illumination scenarios, and it was found that uniform illumination repeatedly revealed the same fluorescent features across the sample. These features were further confirmed with standard histology. Window chamber imaging demonstrated the importance of designing application specific illumination. 4T1 mammary tumors were grown orthotopically before a window chamber was surgically implanted. Animals were injected with either Bodipy FL C16, 2-NBDG, or HS-27 and imaged with both the high AR and low AR illumination platforms. As expected, the low AR, designed for window chambers, had a higher power density at the sample site and thus increased contrast compared to the low AR images. With a method and a system in place, the goal of Aim 3 was to apply the optical imaging platform to observe spatiotemporal metabolic heterogeneity. To achieve this, the CapCell microscope was upgraded to enhance contrast and improve resolution for the visualization of capillaries and single cells. This was demonstrated using 4T1 window chamber models stained with acridine orange, a nucleus specific stain, and green light reflectance to highlight hemoglobin absorption in microvessels. Given the interplay between metabolism and vasculature it was desirable to employ a vessel segmentation approach to describe vascular features within an image. A Gabor filter and Djikstra segmentation approach was employed on metabolic images to enable metabolic and vascular comparisons across an image field of view. To test the improved CapCell system, 4T1 tumors were treated with combretastatin A-1, a vascular disrupting agent. Across the course of treatment, the CapCell was able to observe bulk changes in metabolism and vascular density. Additionally, by employing high resolution imaging, it was possible to observe relationships between each metabolic probe and vessel tortuosity. This analysis allowed for the identification of metabolically unique regions within each group of animals, demonstrating the ability of this technology to parse metabolically distinct regions of tumor. In total, the work outlined here describes the development of a novel optical imaging platform capable of quantifying intratumor metabolic heterogeneity of multiple metabolic endpoints over multiple length scales. The system expands on previous work developing methods for simultaneous measurement of exogenous fluorescent contrast agents to report on lipid uptake and mitochondrial activity. The system also introduced a novel computational approach to design uniform illumination for a low-cost microscope capable of imaging across multiple sample types. Together these technologies were used to observe metabolic heterogeneity in preclinical window chamber models following chemical perturbation. The technology introduced here, is primed for future exploration. First, it would be desirable to integrate all three exogenous contrast agents for simultaneous imaging of three axes of metabolism in vivo. Once accomplished, the sample technology could be applied to study metabolic and vascular changes associated with residual disease and tumors that are entering recurrence.
Item Open Access A Novel Immunoassay Platform Enabled by Non-fouling Poly(OEGMA) Surfaces(2014) Hucknall, AngusThe primary barriers to multiplexed point of care immunoassays are: (1) cost; (2) response time; and (3) sample handling. Described here is a self-contained, multiplexed immunoassay platform for point of care detection that leverages a number of enabling technologies to address these barriers. This platform is referred to as the "D4" assay, as it is composed of the following four sequential, concerted events (Figure 1): (1) Dispense (droplet of blood); (2) Dissolve (printed reagents on chip); (3) Diffuse across surface; (4) Detect binding event.
The D4 assay process begins when a finger-stick is administered and the resulting droplet of blood is applied to the surface of a detector chip. Hydrophobic ink printed onto the surface of the chip confines the blood droplet to a non-fouling region containing soluble, labile spots of detection antibodies and insoluble, non-labile spots of capture antibodies. As the soluble detection antibodies are dissolved from their printed spots by the droplet of blood, three serial events occur to generate signal (Figure 2): (1) the first half of the detection complex is formed by the binding of analytes present in blood to the stable capture agent spots; (2) diffusion of the blood laterally through the polymer brush, resulting in the dissolution and diffusion of soluble detection antibody spots; (3) solubilized detection antibodies bind to their respective analyte-capture agent spots, completing the detection complex and resulting in signal generation at the position of the non-labile capture antibody spots.
This assay relies upon the ability of labeled detection antibodies, printed into a nonfouling brush as "labile spots", to be carried by blood flow to adjacent rows of stably immobilized capture antibodies by diffusion of the analyte solution (Figure 2). Generation of signal at a given capture spot location provides identification of individual analytes (positives). Quantification of the concentration of the different analytes is carried out identically to a conventional fluorescence immunoassay by pre-calibration of the system using a dilution series of the analyte spiked into whole blood.
The D4 assay addresses several critical needs in point of care testing as follows: First, the cost of testing is reduced through miniaturization, multiplexing and one-step, on-site processing of undiluted whole blood obtained from a finger stick. Second, in order to simplify the immunoassay process, the D4 relies on diffusion to bring spatially localized reagents together to create a functional assay and thereby eliminate the need for liquid transfer steps, microfluidic manipulation of sample or reagents, and wash steps. Third, this multiplexed platform is capable of screening for a panel of markers in a single drop of blood with no sample preprocessing. Fourth, the assay is fast, which alleviates the difficulties often associated with communicating the outcome of diagnostic tests. A prototype of the D4 assay is shown in Figure 3 below.
Item Open Access A Novel Vascular Graft Diagnostic and Reversible Aptamers for the Purification of Therapeutic Cells(2017) Nichols, Michael DouglasCreation of novel tools for biomedical applications is critical for the improvement of patient diagnostics and therapeutics. Two particularly important needs lie in (1) improved in vitro testing and increased performance of prosthetic vascular grafts and (2) purification methods for cells that do not compromise their utility. Progress in these areas is urgently needed and would facilitate the availability of higher quality devices and treatments that raise the quality of patient care. This work focused on developing new approaches toward that goal.
A tremendous and immediate need exists for high-performance small-diameter synthetic vascular grafts, as a fifth of the 500,000 annual coronary artery bypass grafting (CABG) patients lack suitable autologous vessels for revascularization. This problem has driven intense research and development of increasingly diverse prosthetics that could be viable alternatives in the years to come. Evaluating these designs in vitro offers high-throughput, low-cost screening for promising graft technologies ahead of more stringent vetting in vivo.
Offering a fresh take on assessing vascular graft thrombogenicity in vitro, the buildup of pressure upstream to a clot was used as a metric to quantify the physical interaction between the graft lumen and a maturing thrombus. A closed tubing system was devised and continuously monitored as clotting solutions of fibrin glue, platelet-rich plasma or whole blood were cured to varying maturities and then purged from small-diameter ePTFE grafts or Tygon graft mimics. This approach provided insight into how blood flow resistance is influenced by a number of clinically relevant factors, such as the level of vessel occlusion and the physical nature of the resident coagulum.
Endothelialization of synthetic vascular grafts yields viable alternatives to native vessels and can be accomplished non-invasively using late-outgrowth endothelial progenitor cells (LO-EPCs) isolated from peripheral blood. However, the time required to amass sufficient cells to prepare a graft with current methods is risky for waiting CABG patients. An ambitious approach conceived to significantly decrease this wait period involved developing affinity ligands selective for LO-EPCs that would enable their capture directly from the circulation to facilitate rapid amassment. An in vitro directed evolution strategy to generate aptamers, the nucleic acid analogs of antibodies, that specifically bind these cells was carried out with initially promising but ultimately unsuccessful results. While the particular strategy executed here did not prevail, the high value and impact LO-EPC aptamers would deliver merit revisiting this work with a revised strategy such as the one proposed in this document.
Purification of autologous and allogenic cells is essential for their use in a variety of therapeutic and basic research applications in addition to augmenting graft performance. However, the antibody stains conventionally used to selectively purify cells are permanent and their continued presence can elicit an immune response in vivo and compromise native cell behavior. To avoid these issues, a cell purification strategy was crafted utilizing aptamers and matched oligonucleotide antidotes that enabled reversible cell staining. The reversible stains were robust enough for cell purification via fluorescence-activated cell sorting (FACS) yet subsequently able to be removed with gentle heat treatment and antidote. Importantly, cell function that was compromised without antidote was rescued to match the native behavior of non-stained cells following purification and antidote treatment.
Item Open Access A Point-of-Care Immunoassay for Ultra-Sensitive Detection of Ebolavirus(2020) Fontes, Cassio MendesLaboratory enabled disease diagnosis is one of the cornerstones in patient care and greatly relies in immunological techniques to detect pathogens and quantify biomarkers related to a myriad of clinical conditions. The performance of immunoassays is directly dependent on the binding affinity of the molecules that enable capture and detection of analytical targets of interest and especially on the surfaces that interact with these sensing molecules and the complex elements present in biological samples. The lack of high-quality antibodies and nonspecific protein absorption (NSPA) on test substrates have historically hindered assay sensitivity and overall performance. While antibody development has witnessed a significant evolution over time, mostly driven by the interest in antibody-based drugs, major challenges still exist in the IVD reagent generation process. Regarding NSPA, only recently protein resistant surfaces found their way into immunoassay development applications with extremely promising results.
In this dissertation, we aimed to better understand the surface properties required to deliver a new generation of immunodiagnostics with high sensitivity and broad dynamic range. Our studies demonstrated that there are very specific physicochemical requirements a surface must present to enable inkjet based, simple fabrication of antigen detection tests. Interestingly, we determined that POEGMA based brushes, our prototypical surface, naturally presents a fine balance between protein resistance and hydrophilicity that enables their non-covalent biofunctionalization and use for IVD applications. Once we confirmed POEGMA as the ideal coating for the fabrication of antigen detection tests, our work evolved to address several of the challenges related to antibody generation for diagnostic test development. Towards this end, our work entailed the development of a new antibody pair that targets non-overlapping epitopes of ebolavirus secreted glycoprotein, a truncated version of the structural glycoprotein that is actively secreted from infected cells in early stages of the infection. The generation of these antibodies was achieved by associating scFv phage-display technology with the transient expression of promising scFv candidates as Fc fusions in mammalian cells followed by their seamless purification with an IsoTag based chromatography-free system. These elements when combined with a novel antibody pairing strategy, that leverages the D4 Assay’s multiplexing capabilities and ease of fabrication, warranted the rapid identification of optimal capture and detection reagents. Finally, employing these reagents, we developed and validated an ultra-sensitive ebolavirus detection test based on the D4 Assay test format, which was able to not only match but outperform the sensitivity of qRT-PCR. This exceptional sensitivity which can enable the deployment of life-saving treatment and containment efforts was demonstrated in two independent nonhuman primate models of the disease and attests to the success of this new IVD test development work-flow.
Item Open Access A Point-Of-Care Immunoassay Platform for Measuring Antibody Avidity(2021) Oshabaheebwa, SolomonSerological testing—detection of antibodies—plays a key role in the diagnosis, management, and surveillance of infectious diseases. Serological assays can detect both active and past infections which is essential in understanding epidemiological variables such as incidence and fatality rates. Another important metric, antibody avidity, provides additional insight into recency of infection, can be used to discriminate between closely related infectious species, assess vaccine efficacy and provides estimates of who is and who is not immune to certain infections. However, conventional methods of measuring antibody avidity are costly, time consuming, and utilize harsh denaturing reagents that negatively impact automated immuno-ELISA equipment. These challenges have deterred the development of point of care tests for antibody avidity. In this thesis, we investigated the performance of four assay formats for antibody detection developed by inkjet-printing assay reagents on glass surfaces coated with a non-fouling polymer brush. We then adopted the antibody detection formats to determine antibody avidity by measuring resistance of the antibody-antigen bonds to chaotropic agents. We further developed a new technique of measuring antibody avidity by reducing the concentration of capture antigen (cAg) on the immunoassay platform. In this new technique, avidity index was determined as the ratio of fluorescence intensity measured at a lower cAg concentration to intensity measured at a higher cAg concentration. This technique showed strong correlation (R > 0.8) with the conventional method of antibody avidity measurement (resistance to chaotropic agent) in three antibody-antigen systems. Additionally, we showed that the proposed platform can detect key biomarkers for identifying recent HIV1 infections. The targeted biomarkers were based on measuring titers and avidity of antibodies secreted against specific clades of HIV envelope proteins. They included clade C GP140 IgG3, transmitted/founder clade C GP140 IgG4 avidity, clade B GP140 IgG4 avidity, and GP41 immunodominant region (GP41-ID) IgG avidity. The proposed assay detected all four biomarkers with wide dynamic ranges (>103.6) and high sensitivity in diluted pooled human serum. The proposed platform for antibody avidity testing is rapid, easy to use and has high correlation with chaotropic resistance. It therefore has potential to enable measurement of antibody avidity at the point of care for clinical applications.
Item Open Access A Stem Cell-Based Strategy for Modeling Human Kidney Disease and Discovering Novel Therapeutics(2022) Burt, Morgan AlexandraChronic kidney disease (CKD) is a degenerative disorder that affects millions of people worldwide and there are no targeted therapeutics. Given the global burden and increasing prevalence of CKD, the kidneys represent an attractive target for regenerative medicine. The most severe forms of CKD involve irreversible damage to kidney glomerular podocytes - the specialized epithelial cells that encase glomerular capillaries and regulate the removal of toxins and waste from blood. Therefore, the goal of this research proposal was to develop a novel strategy to protect or promote repair of injured human kidney tissues with an initial focus on glomerular podocytes. To achieve this goal, we leveraged advances in the directed differentiation of stem cells and in vitro disease modeling techniques to develop translationally relevant human models of podocyte injury. We used these models to identify potential biomarkers of early onset podocyte dysfunction, endogenous therapeutic targets, and reno-protective drug candidates, with a particular emphasis on studying pathways implicated in biomechanical signaling. Our studies revealed that the mechanosensitive proteins YAP, CTGF, and Cyr61 may be viable endogenous therapeutic targets, while CTGF and Cyr61 expression could serve as biomarkers of podocyte mechanical integrity and cell health. Additionally, our preliminary high-throughput drug screens have identified promising podocyte-protective drug candidates, which will be the subject of future studies.
Item Open Access A sublingual vaccine strategy based on tablet delivery of molecular assemblies(2019) Opolot, Emmanuel EinyatVaccines have been a revolutionary intervention for infectious diseases over the past many decades. However, over 2 million people continue to die every year of vaccine-preventable causes, especially in low-resource areas. This is mainly due to inefficiencies in the vaccine supply chain which in entirety lead to loss of potency of vaccines worth over $250 million every year due to temperature fluctuations. Additionally, vaccines that actually reach users may lose effectiveness because of more challenges related to their direct delivery to the recipients; for example, contamination and injuries from misuse of needles. We sought to take a step in addressing these challenges by developing thermally stable vaccine tablets for the sublingual delivery of self-assembled peptide nanofibers. Tablets were engineered from a combination of supramolecular peptide nanofibers plus the excipients, dextran and mannitol. The tablet structure was characterized to assess the impact of tablet formation on the nanofiber structure, as well as for suitability of sublingual delivery. In vivo studies were then carried out in a mouse model to determine the capacity to raise antigen-specific immunogenic responses. Sublingual delivery of the tablet in the mouse model was achieved and an immunogenic response was raised in mice. This proof-of-concept study indicates a step towards improving vaccines with regard to addressing challenges of the vaccine supply chain and vaccine delivery, especially in low resource centers.
Item Open Access A Synthetic-biology Approach to Understanding Bacterial Programmed Death and Implications for Antibiotic Treatment(2013) Tanouchi, YuProgrammed death is often associated with a bacterial stress response. This behavior appears paradoxical, as it offers no benefit to the individual. This paradox can be explained if the death is `altruistic': the sacrifice of some cells can benefit the survivors through release of `public goods'. However, the conditions where bacterial programmed death becomes advantageous have not been unambiguously demonstrated experimentally. Here, I determined such conditions by engineering tunable, stress-induced altruistic death in the bacterium Escherichia coli. Using a mathematical model, we predicted the existence of an optimal programmed death rate that maximizes population growth under stress. I further predicted that altruistic death could generate the `Eagle effect', a counter-intuitive phenomenon where bacteria appear to grow better when treated with higher antibiotic concentrations. In support of these modeling insights, I experimentally demonstrated both the optimality in programmed death rate and the Eagle effect using our engineered system. These findings fill a critical conceptual gap in the analysis of the evolution of bacterial programmed death, and have implications for a design of antibiotic treatment.
Item Open Access A Tissue-Engineered Blood Vessel Model for Vascular Aging(2021) Salmon, Ellen ElizabethClinical studies have identified strong correlations between aging and the development of atherosclerosis. In particular, endothelial cell senescence is implicated in age-related changes in vasoreactivity. Oxidative stress is considered the primary source of endothelial cell (EC) senescence in vivo. EC senescence leads to abnormal proliferation of vascular smooth muscle cells, reduced vasoreactivity, enhanced vascular permeability, and greater adhesion of circulating monocytes and lipids. Endothelial senescence often occurs coincident with an inflammatory response within the endothelium. Recapitulating this mechanism of inducing EC senescence in vitro will facilitate a more precise understanding of how aging contributes to endothelial dysfunction and development of vascular diseases, particularly atherosclerosis. Additionally, evidence of vascular remodeling, particularly deposition of fibronectin and stiffening of the vessel wall matrix, is found in both older patients and atheroprone regions. The independent effects of these factors on the function of endothelial cells is poorly understood due to the inability to study them in isolation in vivo. The Truskey lab developed tissue-engineered blood vessels (TEBVs) which recapitulate the structure and function of an arteriole in vitro. These vessels can be fabricated rapidly, perfused immediately after fabrication, and reach functional maturity after a week. Measurements of endothelium-mediated vascular function confirm the presence of a healthy endothelium in the vessels for several weeks after initial fabrication. This in vitro system allows more precise control over the cellular and structural components of blood vessels than is possible with in vivo experiments. Ultimately, the development of a more robust in vitro model for atherosclerosis will contribute to an increased understanding of vascular disease progression and provide a platform for the evaluation of new drugs during preclinical trials. Specific Aim 1: Evaluate the functional effects of stress-induced senescence on TEBVs. Stress-induced senescence reduced endothelium-dependent vessel function and resulted in endothelial cell inflammation with minimal effects on the surrounding hNDFs. Stress-induced senescence was induced in vitro by treatment with hydrogen peroxide. 2-D cells and TEBVs were treated for 5 or 7 days with hydrogen peroxide. Cells in 2-D were stained for p21 to evaluate senescence, as well as key immune cell adhesion markers VCAM-1, ICAM-1, and E-Selectin. To characterize the effects on TEBVs, vasoreactivity in response to an endothelium-independent vasodilator (sodium nitroprusside) and vasoconstrictor (phenylephrine) were quantified, as well as endothelium-dependent vasoreactivity (acetylcholine). Immunostaining of p21 and VCAM-1 expression was also used to confirm that senescence and inflammation were induced in the TEBVs alongside the reduction in endothelium-dependent vasodilation. Specific Aim 2: Evaluate the capacity of stress-induced senescence to increase the monocyte adhesion and foam cell formation in the TEBVs Stress-induced senescence in TEBVs increased adhesion of circulating monocytes and foam cell formation in accumulated monocytes and medial hNDFs. Senescence was induced as in Aim 1, by treating vessels with hydrogen peroxide. The resulting increase in senescence, VCAM-1, and E-selectin increased adhesion of circulating monocytes to the vessel wall. To develop an atherogenesis model, low density lipoprotein was enzymatically modified into a more inflammatory state which is often identified within atherosclerotic lesions. Introducing enzyme-modified low-density lipoprotein (eLDL) alongside hydrogen peroxide treatment further increased endothelial cell activation. There was a significant increase in the percentage of ICAM-1 positive cells when eLDL was applied to endothelial cells alongside H2O2. hNDFs absorbed and retained eLDL, even without H2O2 in the growth media. When TEBVs were exposed to a combination of eLDL, H2O2, and cell-tracker red monocytes, endothelium-dependent vasoreactivity was significantly compromised. Lipid retention within the vessel wall was significant, as was adhesion of monocytes. Specific Aim 3: Evaluate the drug-responsiveness of the TEBV senescence model and the ability of geroprotective agents to reduce senescence-induced vascular dysfunction, monocyte adhesion, and foam cell formation. Development of a physiologically relevant model for vascular senescence can provide a valuable tool for evaluating the efficacy of drugs targeting atherosclerosis, particularly a new class of drugs in development called senolytics. Senolytics, and their sister drugs senomorphics, specifically target senescent cells and transiently disable the anti-apoptotic pathways that prolong their lives, reducing the burden of senescent cells within the tissue. Senomorphics target factors within the senescence-associated secretory pathway (SASP) to reduce cytokine production and inflammation. Dasatinib and quercetin, two senolytics, and tacrolimus, a senomorphic, were tested on CBECFCs growing in 2-D to see if they were effective at reducing the percentage of p21 positive (senescent) cells. Tacrolimus was found to be the best candidate and used in TEBV trials. TEBVs treated with tacrolimus for 48 hours after induction of senescence recovered significantly more endothelium-dependent vasoreactivity compared to vessels left to recover from H2O2 in normal growth media. Additionally, addition of tacrolimus for the duration of hydrogen peroxide treatment had an atheroprotective effect. Adhesion of monocytes and foam cell formation were significantly reduced compared to vessels without tacrolimus. In summary, the work presented here demonstrates that a TEBV model of vascular senescence can be generated in under two weeks using near-physiological levels of hydrogen peroxide. This model can be capitalized upon to model atherogenesis by adding only eLDL and monocytes. We were also able to effectively use the senomorphic tacrolimus to mitigate the effects of senescence on monocyte adhesion and lipid uptake. This system could be used to investigate other senolytics or test the efficacy and toxicity of novel drugs still in development.
Item Open Access A Tissue-Engineered Microvascular System to Evaluate Vascular Progenitor Cells for Angiogenic Therapies(2015) Brown Peters, Erica ChoThe ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.