Browsing by Author "Chappell, John C."

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    Aberrant hippocampal neurogenesis contributes to learning and memory deficits in a mouse model of repetitive mild traumatic brain injury
    Greer, Kisha (Virginia Tech, 2019-10-02)
    Adult hippocampal neurogenesis, or the process of creating new neurons in the dentate gyrus (DG) of the hippocampus, underlies learning and memory capacity. This cognitive ability is essential for humans to operate in their everyday lives, but cognitive disruption can occur in response to traumatic insult such as brain injury. Previous findings in rodent models have characterized the effect of moderate traumatic brain injury (TBI) on neurogenesis and found learning and memory shortfalls correlated with limited neurogenic capacity. While there are no substantial changes after one mild TBI, research has yet to determine if neurogenesis contributes to the worsened cognitive outcomes of repetitive mild TBI. Here, we examined the effect of neurogenesis on cognitive decline following repetitive mild TBI by utilizing AraC to limit the neurogenic capacity of the DG. Utilizing a BrdU fate-labeling strategy, we found a significant increase in the number of immature neurons that correlate learning and memory impairment. These changes were attenuated in AraC-treated animals. We further identified endothelial cell (EC)-specific EphA4 receptor as a key mediator of aberrant neurogenesis. Taken together, we conclude that increased aberrant neurogenesis contributes to learning and memory deficits after repetitive mild TBI.
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    Applications of Ultrasound to Stimulate Therapeutic Revascularization
    Gorick, Catherine M.; Chappell, John C.; Price, Richard J. (MDPI, 2019-06-24)
    Many pathological conditions are characterized or caused by the presence of an insufficient or aberrant local vasculature. Thus, therapeutic approaches aimed at modulating the caliber and/or density of the vasculature by controlling angiogenesis and arteriogenesis have been under development for many years. As our understanding of the underlying cellular and molecular mechanisms of these vascular growth processes continues to grow, so too do the available targets for therapeutic intervention. Nonetheless, the tools needed to implement such therapies have often had inherent weaknesses (i.e., invasiveness, expense, poor targeting, and control) that preclude successful outcomes. Approximately 20 years ago, the potential for using ultrasound as a new tool for therapeutically manipulating angiogenesis and arteriogenesis began to emerge. Indeed, the ability of ultrasound, especially when used in combination with contrast agent microbubbles, to mechanically manipulate the microvasculature has opened several doors for exploration. In turn, multiple studies on the influence of ultrasound-mediated bioeffects on vascular growth and the use of ultrasound for the targeted stimulation of blood vessel growth via drug and gene delivery have been performed and published over the years. In this review article, we first discuss the basic principles of therapeutic ultrasound for stimulating angiogenesis and arteriogenesis. We then follow this with a comprehensive cataloging of studies that have used ultrasound for stimulating revascularization to date. Finally, we offer a brief perspective on the future of such approaches, in the context of both further research development and possible clinical translation.
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    Blood Vessel Patterning on Retinal Astrocytes Requires Endothelial Flt-1 (VEGFR-1)
    Chappell, John C.; Darden, Jordan; Payne, Laura Beth; Fink, Kathryn; Bautch, Victoria L. (MDPI, 2019-09-07)
    Feedback mechanisms are critical components of many pro-angiogenic signaling pathways that keep vessel growth within a functional range. The Vascular Endothelial Growth Factor-A (VEGF-A) pathway utilizes the decoy VEGF-A receptor Flt-1 to provide negative feedback regulation of VEGF-A signaling. In this study, we investigated how the genetic loss of flt-1 differentially affects the branching complexity of vascular networks in tissues despite similar effects on endothelial sprouting. We selectively ablated flt-1 in the post-natal retina and found that maximum induction of flt-1 loss resulted in alterations in endothelial sprouting and filopodial extension, ultimately yielding hyper-branched networks in the absence of changes in retinal astrocyte architecture. The mosaic deletion of flt-1 revealed that sprouting endothelial cells flanked by flt-1−/− regions of vasculature more extensively associated with underlying astrocytes and exhibited aberrant sprouting, independent of the tip cell genotype. Overall, our data support a model in which tissue patterning features, such as retinal astrocytes, integrate with flt-1-regulated angiogenic molecular and cellular mechanisms to yield optimal vessel patterning for a given tissue.
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    Building a Better Scar: Re-engineering Extracellular Matrix Structure in Dermal Scars
    Montgomery, Jade (Virginia Tech, 2020-01-27)
    Introduction Cutaneous scars represent a common surgical complication, yet no effective drug therapy for scar treatment currently exists despite huge patient and physician demand. A connexin 43 (Cx43) carboxyl terminus (CT) mimetic peptide, alpha Connexin Carboxy-Terminus 1 (αCT1), has demonstrated efficacy in improving long-term scar appearance in pre-clinical and clinical trials. However, current understanding of the mechanism-of-action by which αCT1 improves long-term scar appearance with early intervention treatment is not well understood. Methods In vivo: Scar biopsies from 1) human, 2) Sprague-Dawley rat, and 3) IAF Hairless guinea pig trials of αCT1 were examined for collagen matrix structure at 4 weeks (all models), and 2 and 6 weeks (rat and guinea pig models only). Collagen matrix variables examined included local disorganization of the fibers, a variable that is higher in unwounded skin compared to scar tissue, and density of the fibers, which is higher in scar tissue but can also be used as an early temporal marker of the rate of healing. In vitro: Primary murine dermal fibroblasts were isolated from the whole dermis of 3-4 week old transgenic mice expressing collagen 1(α2) GFP-tpz. Cells were sorted for expression via FACS and plated on prealigned collagen substrate for 7 days under conditions favorable to generating extracellular matrix. Results: All in vivo scar biopsies demonstrated some level of altered collagen matrix structure with αCT1 treatment. Treated scars had higher local disorganization of the collagen fibers within the wound, and an increase in collagen matrix density compared to control at certain earlier timepoints that tended to decrease or disappear at later timepoints. The IAF Hairless guinea pig, a novel splinted wound healing model presented herein, was found to closely replicate the human dermal collagen profile and changes in collagen profile spurred by αCT1, significantly outperforming the traditional rat model. Primary dermal murine fibroblasts treated in vitro with αCT1 significantly increased synthesis of procollagen 1, the precursor of collagen 1 necessary for constructing the extracellular matrix, suggesting that at least part of the reason for higher collagen density at early in vivo timepoints is due to increased collagen synthesis by fibroblasts. Conclusion: αCT1 treatment in the early stages of wound healing prompts individual fibroblasts to increase their output of collagen and create a more disorganized early collagen matrix. These early changes potentially spur the long-term scar appearance improvements seen in clinical trials, and provide a basis for future work to discover the cellular pathways to alter in order to improve wound healing and cutaneous scarring outcomes.
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    Cell-specific roles for CASK in the pathology of Optic Nerve Hypoplasia
    Kerr, Alicia Marie (Virginia Tech, 2019-06-25)
    Optic Nerve Hypoplasia (ONH) is the leading cause of childhood blindness in developed nations and its prevalence has been rising. Yet, we know little about the genetic, molecular, or cellular mechanisms underlying ONH. A previous study described ONH in a cohort of patients with mutations in CASK, an X-linked gene with established roles in neural development and synaptic function. I have demonstrated that heterozygous deletion of CASK in mice (Cask+/-) recapitulates many of the phenotypes observed in patients with CASK mutations, including ONH. This includes reduced optic nerve size, reduced numbers of retinal ganglion cells (RGCs), reduced RGC axonal diameter, and deficits in vision-related tasks. Further analysis on a homozygous partial loss of function variant (Caskfl/fl) also displayed ONH with reduced numbers of RGCs. In order to understand the mechanisms underlying CASK-associated ONH, I explored whether RGCs, the projection neurons of the retina and the cells whose axons comprise the optic nerve, generate CASK. Indeed, mRNA analysis revealed expression of CASK by a large cohort of RGCs. In order to assess whether loss of CASK from a majority of RGCs leads to ONH, I crossed a conditional allele of CASK (CASKfl/fl) with transgenic mice that express Cre Recombinase (Cre) in RGCs. Deletion of CASK from RGCs did not further alter ONH size nor RGC survival. These results demonstrate that loss of CASK signaling in this discrete neuronal populations is not sufficient to lead to further disruption in the assembly of the subcortical visual circuit, suggesting a non-cell autonomous mechanism for loss of CASK in ONH.
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    Computational modeling of interacting VEGF and soluble VEGF receptor concentration gradients
    Hashambhoy, Yasmin L.; Chappell, John C.; Peirce, Shayn M.; Bautch, Victoria L.; Gabhann, Feilim Mac (Frontiers, 2011-10-04)
    Experimental data indicates that soluble vascular endothelial growth factor (VEGF) receptor 1 (sFlt-1) modulates the guidance cues provided to sprouting blood vessels by VEGF-A. To better delineate the role of sFlt-1 in VEGF signaling, we have developed an experimentally based computational model. This model describes dynamic spatial transport of VEGF, and its binding to receptors Flt-1 and Flk-1, in a mouse embryonic stem cell model of vessel morphogenesis. The model represents the local environment of a single blood vessel. Our simulations predict that blood vessel secretion of sFlt-1 and increased local sFlt-1 sequestration of VEGF results in decreased VEGF–Flk-1 levels on the sprout surface. In addition, the model predicts that sFlt-1 secretion increases the relative gradient of VEGF–Flk-1 along the sprout surface, which could alter endothelial cell perception of directionality cues. We also show that the proximity of neighboring sprouts may alter VEGF gradients, VEGF receptor binding, and the directionality of sprout growth. As sprout distances decrease, the probability that the sprouts will move in divergent directions increases. This model is a useful tool for determining how local sFlt-1 and VEGF gradients contribute to the spatial distribution of VEGF receptor binding, and can be used in conjunction with experimental data to explore how multi-cellular interactions and relationships between local growth factor gradients drive angiogenesis.
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    Connexin 43 Across the Vasculature: Gap Junctions and Beyond
    Sedovy, Meghan W.; Leng, Xinyan; Leaf, Melissa R.; Iqbal, Farwah; Payne, Laura Beth; Chappell, John C.; Johnstone, Scott R. (Karger Publishers, 2022-10-03)
    Connexin 43 (Cx43) is essential to the function of the vasculature. Cx43 proteins form gap junctions that allow for the exchange of ions and molecules between vascular cells to facilitate cell-to-cell signaling and coordinate vasomotor activity. Cx43 also has intracellular signaling functions that influence vascular cell proliferation and migration. Cx43 is expressed in all vascular cell types, although its expression and function vary by vessel size and location. This includes expression in vascular smooth muscle cells (vSMC), endothelial cells (EC), and pericytes. Cx43 is thought to coordinate homocellular signaling within EC and vSMC. Cx43 gap junctions also function as conduits between different cell types (heterocellular signaling), between EC and vSMC at the myoendothelial junction, and between pericyte and EC in capillaries. Alterations in Cx43 expression, localization, and post-translational modification have been identified in vascular disease states, including atherosclerosis, hypertension, and diabetes. In this review, we discuss the current understanding of Cx43 localization and function in healthy and diseased blood vessels across all vascular beds.
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    The Effects of Aging on EGFR/pSTAT3-Dependent Gliovascular Structural Plasticity
    Mills, William A. III (Virginia Tech, 2021-05-28)
    Astrocytes comprise the most abundant cell population in human brain (1). First described by Virchow as being 'glue' of the brain (2), modern research has truly extended our knowledge and understanding regarding the vast array of roles these cells execute under normal physiological conditions. Examples include neurotransmitter reuptake at the synapse (3), the regulation of blood flow at capillaries to meet neuronal energy demand (4), and maintenance/repair of the blood-brain barrier (BBB) (5), which is comprised, in part, of tight junction proteins such zonula-occludens-1 (ZO1) (6) and Claudin-5 (7). Underlying the execution of these processes is the morphological and spatial arrangement of astrocytes between neurons and endothelial cells comprising blood vessels, where comprehensively speaking, these cells form what is known as the gliovascular unit (8). Astrocytes extend large processes called endfeet that intimately associate with and enwrap up to 99% of the cerebrovascular surface (9). Disruptions to this association can occur in the form of retracted endfeet, and this has been characterized in several disease states such as major depressive disorder (10-12), ischemia (13-15), and normal biological aging (16-18). Disruption can also take the form of cellular/protein aggregate intercalation, which our lab previously characterized in a human-derived glioma model (19) and vascular amyloidosis human Amyloid Precursor Protein J20 (hAPPJ20) animal model (20). In both models, focal astrocyte-vascular disruptions coincided with perturbations to astrocyte control of blood flow, with deficits in BBB integrity present in the glioma model as well. These findings lead to the preliminary work in this dissertation where we aimed to extend BBB findings in the glioma model to the hAPPJ20 vascular amyloidosis model. Immunohistochemical analysis in two-year old hAPPJ20 animal arterioles revealed that indeed in locations of vascular amyloid buildup and endfoot separation, there was a significant reduction in a tight junction protein critical for BBB maintenance, ZO1. This reduction in ZO1 expression was accompanied by extravasation of 70kDa FITC and the ~1kDa Cadaverine, suggesting that BBB integrity was compromised. These findings led to the objective of this dissertation, which was to determine if focal ablation of an astrocyte is sufficient to disrupt BBB integrity. By utilizing the in vivo 2Phatal single-cell apoptosis induction method (21), we found that 1) focal loss of astrocyte-vascular coverage does not result in barrier deficits, but rather induces a plasticity response whereby surrounding astrocytes extend processes to reinnervate vascular vacancies no longer occupied by previously ablated astrocytes. 2) Replacement astrocytes are capable of inducing vasocontractile responses in blood vessels, and that 3) aging significantly attenuates the kinetics of this process. We then tested the hypothesis that focal loss of astrocyte-vascular coverage leads to a gliovascular structural plasticity response, in part, through the phosphorylation of signal transducer and activator of transcription 3 (STAT3) by Janus Kinase 2 (JAK2). This dissertation found that 4), this was indeed the case, and finally, 5) we determined that gliovascular structural plasticity occurs after reperfusion post-focal photothrombotic stroke. Together, the work presented in this dissertation sheds light on a novel plasticity response whereby astrocytes maintain continual cerebrovascular coverage and therefore physiological control. Future studies should aim to determine if 1) astrocytes also replace the synaptic contacts with neighboring neurons once held by a previous astrocyte, and 2) what therapeutic opportunity gliovascular structural plasticity may present regarding BBB repair following stroke.
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    Evaluating cell viability, capillary perfusion, and collateral tortuosity in an ex vivo mouse intestine fluidics model
    Willi, Caroline E.; Abdelazim, Hanaa; Chappell, John C. (Frontiers, 2022-12-09)
    Numerous disease conditions involve the sudden or progressive loss of blood flow. Perfusion restoration is vital for returning affected organs to full health. While a range of clinical interventions can successfully restore flow to downstream tissues, the microvascular responses after a loss-of-flow event can vary over time and may involve substantial microvessel instability. Increased insight into perfusion-mediated capillary stability and access-to-flow is therefore essential for advancing therapeutic reperfusion strategies and improving patient outcomes. To that end, we developed a tissue-based microvascular fluidics model to better understand (i) microvascular stability and access-to-flow over an acute time course post-ischemia, and (ii) collateral flow in vessels neighboring an occlusion site. We utilized murine intestinal tissue regions by catheterizing a feeder artery and introducing perfusate at physiologically comparable flow-rates. The cannulated vessel as well as a portion of the downstream vessels and associated intestinal tissue were cultured while constant perfusion conditions were maintained. An occlusion was introduced in a selected arterial segment, and changes in perfusion within areas receiving varying degrees of collateral flow were observed over time. To observe the microvascular response to perfusion changes, we incorporated (i) tissues harboring cell-reporter constructs, specifically Ng2-DsRed labeling of intestinal pericytes, and (ii) different types of fluorescent perfusates to quantify capillary access-to-flow at discrete time points. In our model, we found that perfusion tracers could enter capillaries within regions downstream of an occlusion upon the initial introduction of perfusion, but at 24 h tissue perfusion was severely decreased. However, live/dead cell discrimination revealed that the tissue overall did not experience significant cell death, including that of microvascular pericytes, even after 48 h. Our findings suggest that altered flow conditions may rapidly initiate cellular responses that reduce capillary access-to-flow, even in the absence of cellular deterioration or hypoxia. Overall, this ex vivo tissue-based microfluidics model may serve as a platform upon which a variety of follow-on studies may be conducted. It will thus enhance our understanding of microvessel stability and access-to-flow during an occlusive event and the role of collateral flow during normal and disrupted perfusion.
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    Extracellular Matrix Contributions to Early Vascular Development and Pericyte Precursor Dynamics
    Hoque, Maruf M. (Virginia Tech, 2023-07-24)
    The vasculature is a highly intricate system of "highways" that shuttles blood from the heart to every tissue and organ in the human body. These vessels are responsible for carrying oxygen, trafficking hormones, delivering nutrients, and removing waste products from the body. The formation of a functioning vascular system depends on the close coordination of many cell types and, on the capillary level, specifically endothelial cells and pericytes as well as the surrounding protein microenvironment, known as the extracellular matrix (ECM). Impaired coordination amongst the cellular and protein constituents results in the improper functioning of the vascular network and can eventually contribute to the failure of organ systems. This dissertation research focuses on how improper ECM deposition affects vascular assembly. We utilized several approaches to affect ECM composition, specifically: 1) hypoxia exposure and 2) reducing ECM pharmacologically and utilizing lentiviral-mediated silencing of Type IV Collagen (Col-IV, gene Col4a1) expression. In these experimental settings, we observed downstream changes in the coordination between endothelial cells and pericytes while forming vascular networks. In short, this dissertation work suggests that excess ECM deposition, and particularly that of Col-IV, has unique deleterious effects on the developing vasculature as compared to reduced ECM deposition. The findings from this work suggest mechanisms underlying how the vasculature may be destabilized in hypoxia-associated pathologies, such as preeclampsia.
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    Microvascular bioengineering: a focus on pericytes
    Zhao, Huaning; Chappell, John C. (2019-03-29)
    Capillaries within the microcirculation are essential for oxygen delivery and nutrient/waste exchange, among other critical functions. Microvascular bioengineering approaches have sought to recapitulate many key features of these capillary networks, with an increasing appreciation for the necessity of incorporating vascular pericytes. Here, we briefly review established and more recent insights into important aspects of pericyte identification and function within the microvasculature. We then consider the importance of including vascular pericytes in various bioengineered microvessel platforms including 3D culturing and microfluidic systems. We also discuss how vascular pericytes are a vital component in the construction of computational models that simulate microcirculation phenomena including angiogenesis, microvascular biomechanics, and kinetics of exchange across the vessel wall. In reviewing these topics, we highlight the notion that incorporating pericytes into microvascular bioengineering applications will increase their utility and accelerate the translation of basic discoveries to clinical solutions for vascular-related pathologies.
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    Pericyte heterogeneity identified by 3D ultrastructural analysis of the microvessel wall
    Abdelazim, Hanaa; Payne, Laura Beth; Nolan, Kyle; Paralkar, Karan; Bradley, Vanessa; Kanodia, Ronak; Gude, Rosalie; Ward, Rachael; Monavarfeshani, Aboozar; Fox, Michael A.; Chappell, John C. (Frontiers, 2022-12-16)
    Confident identification of pericytes (PCs) remains an obstacle in the field, as a single molecular marker for these unique perivascular cells remains elusive. Adding to this challenge is the recent appreciation that PC populations may be heterogeneous, displaying a range of morphologies within capillary networks. We found additional support on the ultrastructural level for the classification of these PC subtypes—“thin-strand” (TSP), mesh (MP), and ensheathing (EP)—based on distinct morphological characteristics. Interestingly, we also found several examples of another cell type, likely a vascular smooth muscle cell, in a medial layer between endothelial cells (ECs) and pericytes (PCs) harboring characteristics of the ensheathing type. A conserved feature across the different PC subtypes was the presence of extracellular matrix (ECM) surrounding the vascular unit and distributed in between neighboring cells. The thickness of this vascular basement membrane was remarkably consistent depending on its location, but never strayed beyond a range of 150–300 nm unless thinned to facilitate closer proximity of neighboring cells (suggesting direct contact). The density of PC-EC contact points (“peg-and-socket” structures) was another distinguishing feature across the different PC subtypes, as were the apparent contact locations between vascular cells and brain parenchymal cells. In addition to this thinning, the extracellular matrix (ECM) surrounding EPs displayed another unique configuration in the form of extensions that emitted out radially into the surrounding parenchyma. Knowledge of the origin and function of these structures is still emerging, but their appearance suggests the potential for being mechanical elements and/or perhaps signaling nodes via embedded molecular cues. Overall, this unique ultrastructural perspective provides new insights into PC heterogeneity and the presence of medial cells within the microvessel wall, the consideration of extracellular matrix (ECM) coverage as another PC identification criteria, and unique extracellular matrix (ECM) configurations (i.e., radial extensions) that may reveal additional aspects of PC heterogeneity.
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    Pericyte Progenitor Coupling to the Emerging Endothelium during Vasculogenesis via Connexin43
    Payne, Laura Beth; Tewari, Bhanu P.; Dunkenberger, Logan; Bond, Samantha; Savelli, Alyssa; Darden, Jordan; Zhao, Huaning; Willi, Caroline; Kanodia, Ronak; Gude, Rosalie; Powell, Michael D.; Oestreich, Kenneth J.; Sontheimer, Harald; Dal-Pra, Sophie; Chappell, John C. (Lippincott Williams & Wilkins, 2022-04-01)
    Background: Vascular pericytes stabilize blood vessels and contribute to their maturation, while playing other key roles in microvascular function. Nevertheless, relatively little is known about involvement of their precursors in the earliest stages of vascular development, specifically during vasculogenesis. Methods: We combined high-power, time-lapse imaging with transcriptional profiling of emerging pericytes and endothelial cells in reporter mouse and cell lines. We also analyzed conditional transgenic animals deficient in Cx43/Gja1 (connexin 43/gap junction alpha-1) expression within Ng2+ cells. Results: A subset of Ng2-DsRed+ cells, likely pericyte/mural cell precursors, arose alongside endothelial cell differentiation and organization and physically engaged vasculogenic endothelium in vivo and in vitro. We found no overlap between this population of differentiating pericyte/mural progenitors and other lineages including hemangiogenic and neuronal/glial cell types. We also observed cell-cell coupling and identified Cx43-based gap junctions contributing to pericyte-endothelial cell precursor communication during vascular assembly. Genetic loss of Cx43/Gja1 in Ng2+ pericyte progenitors compromised embryonic blood vessel formation in a subset of animals, while surviving mutants displayed little-to-no vessel abnormalities, suggesting a resilience to Cx43/Gja1 loss in Ng2+ cells or potential compensation by additional connexin isoforms. Conclusions: Together, our data suggest that a distinct pericyte lineage emerges alongside vasculogenesis and directly communicates with the nascent endothelium via Cx43 during early vessel formation. Cx43/Gja1 loss in pericyte/mural cell progenitors can induce embryonic vessel dysmorphogenesis, but alternate connexin isoforms may be able to compensate. These data provide insight that may reshape the current framework of vascular development and may also inform tissue revascularization/vascularization strategies.
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    Pericyte-Endothelial Cell Interactions during Blood Vessel Formation and in Diabetic Scenarios
    Zhao, Huaning (Virginia Tech, 2019-04-08)
    Diabetic retinopathy (DR) is an incurable, chronic disease that is the leading cause of blindness in working-age adults. A prominent characteristic of DR is the extensive dysfunction within the retina microvasculature. Specialized vascular cells known as pericytes (PCs) are lost or become dysfunctional during disease progression; a thickening of the extracellular matrix (ECM) composing the vascular basement membrane (vBM) and endothelial cell (EC) tight junction disruption are also key features of this disease and contribute to its pathogenesis. PC loss is believed to be a central cue for disease initiation. However, studies inducing PC loss and observing acute changes in the vasculature did not report severe vessel damage or vBM thickening, suggesting that the effects of PC loss occur over a longer period of time. Because the chronic effects of PC loss are more difficult to ascertain, especially in a complex condition such as DR, the mechanisms underlying microvascular defects in DR remain poorly understood. The work presented in this dissertation focuses on pericyte-endothelial cell interactions and their interplay with the ECM/vBM during a variety of physiological and pathological conditions. First, we isolated and functionally validated a primary mouse embryonic PC cell line that we then applied to a co-culture model with ECs to better understand the dynamic interactions between these two critical components of the capillary wall. In the co-culture model, we found that primary PCs promoted EC organization into vessel-like structures and enhanced EC-EC junctions. To complement these in vitro studies, we analyzed animal models and human tissue for the PC-EC interactions and ECM/vBM remodeling under different conditions (physiological and pathological). Moreover, we analyzed microglia and astrocytes to enhance our understanding of the tissue-vessel interface, bolstering our experimental results and facilitating the generation of more hypotheses for future research. Overall, our work suggests that PC-EC interactions in diabetic scenarios play a crucial role in ECM/vBM remodeling; engagement with the ECM/vBM in turn impacted PC behaviors including migration away from the endothelium and induced EC loss of tight junctions, key changes in the onset and progression of DR.
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    Pericytes in Early Vascular Development
    Darden, Jordan Alexandra (Virginia Tech, 2019-04-18)
    Blood vessels are critical for the delivery of oxygen and nutrients to all cells in the body. To properly function, blood vessels and their primary components must develop and mature into a healthy network, capable of dynamic alterations to meet new needs of the body. The early genetic and molecular programs that "push" the vasculature to develop are the same programs that reactivate when there are normal changes to the body such as injury, muscle growth or decline, or aging; and when pathologies arise like cancer, stroke, and diabetes. Therefore, it is crucial to understand how the vasculature develops into a healthy system by studying all components as they mature. Endothelial cells that comprise the vessels themselves are joined by specialized partner cells called pericytes that help guide and mature vessel growth. Pericytes lie elongated along endothelial cells and have multiple points of contact with the endothelium. In this position, pericytes assist in cell-cell communication and even blood flow regulation in the microvasculature. To study the relationship between endothelial cells and pericytes during development, we observed vascular morphology in three and four dimensions, as well as the genetic and molecular mechanisms underlying how these cells are recruited and interact in several experimental models. Thus, to thoroughly analyze the morphology of these vessels, we developed a rigorous methodology using a MATLAB program to determine the colocalization and coverage of pericytes associated with vessels in large image sets. After developing analytical methods to investigate all the components of the blood vessel wall, we expanded our investigation of how pericytes and other aspects of microvasculature develop in animal models, specifically a more commonly used murine model for vascular development and for treatment of human diseases. Our findings of vascular development in mice suggest that there are important differences in how human and mouse brain blood vessels form. Therefore, studies using mice must be carefully designed to account for these discrepancies. Additionally, research into why human and mouse neurovascular development and maturation are different can aid in the development of improved experimental models to better treat human pathologies.
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    A Physio-chemical Predictive Model of Dynamic Thrombus Formation and Growth in Stenosed Vessels
    Hosseinzadegan, Hamid (Virginia Tech, 2017-11-06)
    According to the World Health Organization (WHO), Cardiovascular Disease (CVD) is the leading cause of death in the world. Biomechanics and fluid dynamics of blood flow play an important role in CVD mediation. Shear stress plays a major role in platelet-substrate interactions and thrombus formation and growth in blood flow, where under both pathological and physiological conditions platelet adhesion and accumulation occur. In this study, a three-dimensional dynamic model of platelet-rich thrombus growth in stenosed vessels using computational fluid dynamics (CFD) methods is introduced. Platelet adhesion, aggregation and activation kinetics are modeled by solving mass transport equations for blood components involved in thrombosis. The model was first verified under three different shear conditions and at two heparin levels. Three-dimensional simulations were then carried out to evaluate the performance of the model for severely damaged (stripped) aortas with mild and severe stenosis degrees. For these cases, linear shear-dependent functions were developed for platelet-surface and platelet-platelet adhesion rates. It was confirmed that the platelet adhesion rate is not only a function of Reynolds number (or wall shear rate) but also the stenosis severity of the vessel. General correlations for adhesion rates of platelets as functions of stenosis and Reynolds number were obtained based on these cases. The model was applied to different experimental systems and shown to agree well with measured platelet deposition. Then, the Arbitrary Lagrangian Eulerian (ALE) formulation was used to model dynamic growth by including geometry change in the simulation procedure. The wall boundaries were discretely moved based on the amount of platelet deposition that occurs on the vessel wall. To emulate the dynamic behavior of platelet adhesion kinetics during thrombus growth, the validated model for platelet adhesion, which calculates platelet-surface adhesion rates as a function of stenosis severity and Reynolds number, was applied to the model. The model successfully predicts the nonlinear growth of thrombi in the stenosed area. These simulations provide a useful guide to understand the effect of growing thrombus on platelet deposition rate, platelet activation kinetics and occurrence of thromboembolism (TE) in highly stenosed arteries.
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    Silencing Endothelial EphA4 Alters Transcriptional Regulation of Angiogenic Factors to Promote Vessel Recovery Following TBI
    McGuire, David Robert (Virginia Tech, 2020-07-09)
    Traumatic brain injury (TBI) can cause a number of deleterious effects to the neurovascular system, including reduced cerebral blood flow (CBF), vascular regression, and ischemia, resulting in cognitive decline. Research into therapeutic targets to restore neurovascular function following injury has identified endothelial EphA4 receptor tyrosine kinase as a major regulator of vascular regrowth. The research outlined herein utilizes an endothelial-specific EphA4 knockout mouse model (KO-EphA4flf/Tie2-Cre) to determine the extent to which this receptor may influence vascular regrowth following TBI. Analysis of the colocalization and proximity of endothelial and mural cell markers (i.e. PECAM-1 and PDGFRβ, respectively) in immunohistochemically-stained brain sections demonstrates that EphA4 silencing does not seem to affect the physical association between, nor total amounts of, endothelial cells and pericytes, between genotypes by 4 days post-injury (dpi). Nevertheless, these measures demonstrate that these cell types may preferentially proliferate and/or expand into peri-lesion tissue in both KO-EphA4flf/Tie2-Cre) and WT-EphA4fl/fl mice. These data further suggest that both genotypes experience homogeneity of PECAM-1 and PDGFRβ expression between regions of the injury cavity. Gene expression analysis using mRNA samples from both genotypes reveals that KO-EphA4flf/Tie2-Cre CCI-injured mice experience increased expression of Vegfa, Flt1, and Fn (Fibronectin) compared to sham-injured condition knockouts. These results demonstrate changes in expression of angiogenic factors in the absence of early differences in patterns of vessel formation, which may underlie improved vascular regrowth, as well as outline a potential mechanism wherein the interplay between these factors and EphA4 silencing may lead to improved cognitive outcomes following TBI.
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    A Soluble Platelet-Derived Growth Factor Receptor-β Originates via Pre-mRNA Splicing in the Healthy Brain and Is Upregulated during Hypoxia and Aging
    Payne, Laura Beth; Abdelazim, Hanaa; Hoque, Maruf; Barnes, Audra; Mironovova, Zuzana; Willi, Caroline E.; Darden, Jordan; Houk, Clifton; Sedovy, Meghan W.; Johnstone, Scott R.; Chappell, John C. (MDPI, 2023-04-21)
    The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRβ) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases such as PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFRβ (sPDGFRβ) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFRβ variants, and specifically during tissue homeostasis. Here, we found sPDGFRβ protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFRβ isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFRβ by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRβ transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRβ protein was detected throughout the brain parenchyma in distinct regions, such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFRβ variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRβ variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRβ likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFRβ in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion—critical processes underlying neuronal health and function, and in turn, memory and cognition.
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    Specific labeling of synaptic schwann cells reveals unique cellular and molecular features
    Castro, Ryan W.; Taetzsch, Thomas; Vaughan, Sydney K.; Godbe, Kerilyn; Chappell, John C.; Settlage, Robert E.; Valdez, Gregorio (2020-06-25)
    Perisynaptic Schwann cells (PSCs) are specialized, non-myelinating, synaptic glia of the neuromuscular junction (NMJ), that participate in synapse development, function, maintenance, and repair. The study of PSCs has relied on an anatomy-based approach, as the identities of cell-specific PSC molecular markers have remained elusive. This limited approach has precluded our ability to isolate and genetically manipulate PSCs in a cell specific manner. We have identified neuron-glia antigen 2 (NG2) as a unique molecular marker of S100 beta+ PSCs in skeletal muscle. NG2 is expressed in Schwann cells already associated with the NMJ, indicating that it is a marker of differentiated PSCs. Using a newly generated transgenic mouse in which PSCs are specifically labeled, we show that PSCs have a unique molecular signature that includes genes known to play critical roles in PSCs and synapses. These findings will serve as a springboard for revealing drivers of PSC differentiation and function.
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    Von Hippel-Lindau Syndrome: Characterization of a Potentially Novel VEGF-A Isoform and Elucidation of Molecular and Vascular Mechanisms of Observed Phenotypic Changes
    North, Morgan Hunter (Virginia Tech, 2020-06-17)
    Von Hippel-Lindau (VHL) syndrome is an autosomal dominant predisposition to cancer in neurological tissues, the kidneys, adrenal glands, pancreas, and liver, including neurological hemangioblastoma (HB), pheochromocytoma (PCC), pancreatic neuroendocrine tumors (PNET), pancreatic and renal cysts, and clear cell renal cell carcinoma (ccRCC). The disease process follows Knudson's two-hit model, requiring spontaneous loss or mutation of a normal VHL tumor suppressor allele to induce expression of the disease. VHL syndrome principally involves dysregulation of oxygen sensing pathways including the Hypoxia Inducible Factor (HIF)-Vascular Endothelial Growth Factor-A (VEGF-A) and HIF-Erythropoietin (EPO) pathways. RNA sequencing (RNA-Seq) data from our previously published experiments revealed a potentially novel VEGF-A splice variant with excision of the VEGF Receptor-1 (VEGFR-1)/Flt-1 binding domain, rendering this isoform resistant to native down-regulation. Additionally, phenotypic changes were observed in adult VHL mutant mice, specifically very red appearing extremities with prominently visible vasculature. In order to determine the etiology of this phenotype, we observed red blood cell count, Epo gene expression levels, and arterialization of the blood vessels in these experimental mice as compared to littermate controls. Current research into the VEGF-A isoform is ongoing in the lab, and preliminary evidence for the etiology of the apparent chronic erythema phenotype is inconclusive due to lack of experimental replicates due to COVID-19 quarantine orders.