The disruption of the blood-brain barrier (BBB) occurs after ischemic and hemorrhagic stroke and contributes to secondary brain damage. Matrix metalloproteinase-9 (MMP9) has been identified to be the main mediator of post-stroke BBB disruption. It is unknown whether deposition of heme/iron in the brain following stroke would affect MMP9 expression. In this study, I have demonstrated that heme/iron up-regulated MMP9 expression in rat astrocytes and that this upregulation was most likely due to reactive oxygen species (ROS) generated by heme/iron deposition on cells. ROS can activate AP-1 and NFκB signaling pathways which were responsible for increased MMP9 expression. Inhibiting AP-1 and NFκB decreased MMP9 expression. Heme/iron deposition also activated Nrf-2 and increased the expression of neuroprotective heme oxygenase-1. My study suggests that heme and iron deposition generates ROS and increases MMP9 expression through AP-1 and NFκB signaling pathways and that targeting these pathways or clearance of heme and iron may modulate MMP9 expression for reduced damage.

Disruption of the blood-brain barrier (BBB) is hypothesized to play an important role in the disease biology of schizophrenia (SZ). Brain microvascular endothelial cells (BMECs) have paracellular and transcellular proteins, transporters, as well as important extracellular matrix proteins, which collectively contribute to maintaining proper BBB function. While previous studies have provided some insights into the role of the BBB in SZ pathophysiology, there is a significant gap in our understanding of the cellular-molecular underpinnings of its major component, BMECs. Human induced pluripotent stem cells (hiPSCs) provide an exciting new avenue for exploring the role of BMECs in SZ. We hypothesize that BMECs have intrinsic deficits that lead to BBB dysfunction in SZ. In this study, we first aimed to test whether the existing hiPSC-derived BMEC protocols work with our patient-specific hiPSC samples. Secondly, we sought to investigate any potential deficits between BMECs derived from healthy control (HC) and SZ subjects. We successfully adapted the established protocol and confirmed the identity of these hiPSC-derived BMECs with relevant cell markers such as CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. We also evaluate barrier function by measuring trans-endothelial electrical resistance (TEER) and efflux transporters activity of ABCB1 and ABCC1. We observed evidence of poor cellular adhesion and disrupted tight junctions in a subset of SZ hiPSC-derived BMECs, where approximately 70% of them demonstrated extensive BBB disruption (reduced TEER). These findings suggest that there may be cell-autonomous disease-specific deficits in BMECs in SZ that result in BBB dysfunction. / 2022-06-07T00:00:00Z

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lucia Tomášková Supervisor: prof. PharmDr. Tomáš Šimůnek, Ph.D. Title of diploma thesis: Study of exosomes as a drug delivery system in the treatment of glioblastoma Central nervous system disorders are among the most serious diseases affecting humans. They affect not only the patient's life, but also his/her surroundings. Therefore, their therapy, whether at the level of complete cure or alleviation of accompanying symptoms, is a challenge for scientific research. In our research, we focused on glioblastoma multiforme, a brain cancer not yet treatable. The main drawback in therapy is overcoming the blood-brain barrier. Exosomes, such as the body's natural nano-vesicles, have been shown to be a suitable system for delivering drugs to brain tissue. Our research has shown that by a suitable method we are able to obtain sufficient quality exosomes from macrophage and fill them very efficiently with antitumor agents paclitaxel, doxorubicin and temozolomide, while the delivered substances show higher efficacy and fewer side effects than the free form.

<div>Neurotherapeutics are an essential drug class that is often forgotten or neglected due to the difficulties associated with pharmaceutical development and approval. These compounds face high rates of attrition in clinical trials and late stage development predominantly due to the restrictiveness of the blood brain barrier (BBB). The inherent role of the BBB is to protect and maintain the homeostatic environment around the neuronal cells in the brain parenchyma. This is accomplished by the BBB posing not only as a physical barrier through its restrictive tight junctions that prevent paracellular permeation, but also through the high expression levels of efflux transporters and drug metabolizing enzymes that prevent transcellular permeation of potential drug compounds. In attempting to deliver compounds to the brain the intended outcome is often over-shot to the point of causing neurotoxic implications. One way to mitigate the difficulties associated with drug delivery to the brain and early evaluation of potential toxic compounds is to develop in vitro cell-based models that mimic the in vivo BBB and neurovascular unit (NVU). The mainstays of the BBB phenotype are presented in the brain microvessel endothelial cells (BMECs) and are regulated and influenced by the close contacts of supporting cells of the NVU such as astrocytes, pericytes, and neurons. An in vitro model that can mimic the close contacts between these four cell types and is capable of being implemented in pharmaceutical development for BBB permeability and neuroactivity screening could lead to better selection of hit and lead candidates, and ultimately reduce the attrition rates of neurotherapeutics.</div><div>Direct contact coculture and triculture models have been developed in our laboratory that mimic the in vivo cell-cell contacts between the different cell types of the NVU and provide increased barrier properties in comparison to other models utilizing indirect contact between cell types. Early development and optimization of these models was accomplished using the human cerebral endothelial cell line hCMEC/D3. Although this cell line proved useful in early validation stages, it was decided that a different endothelial cell source would be sought out. Work was done using iPSC derived endothelial cells (iCell® endothelial cells) and an alternative immortalized human brain endothelial cell line (HBEC-5i). Both cell lines proved to be amenable to the direct contact coculture and triculture models, with the iCell® models showing greater barrier properties in comparison to those using the HBEC-5i cell line. However, drawbacks of the iCell® model were observed in extending culturing of the cells causing the cells to “roll” to the middle of the filter and proving to be cost prohibitive for extensive optimization. Ultimately, the HBEC-5i cell line was chosen for continued development and optimization due to its immortalized origin and potential for replacing the hCMEC/D3 cell line in the direct contact models.</div><div>Optimization of the direct contact triculture using the HBEC-5i cell line was required as all of the previous development was performed using the hCMEC/D3 cells. Typically, optimization of in vitro systems is performed in a one factor at a time manner or not at all. Given the large number of factors that can influence the outcome of this model, a design of experiments (DOE) based optimization approach was taken. DOEs are traditionally used in process optimization of non-biologically based systems; however, the production of the direct contact triculture is a process that could greatly benefit from extensive optimization. The seeding densities of all three cell types used in the triculture (astrocytes, pericytes, and HBEC-5i), the extracellular matrix used, and the length of culture time post endothelial cell plating were the factors chosen for the optimization process given the observations made during early development of the model. The conditions were optimized for barrier tightness by measuring the permeability of a 4 kD dextran as a paracellular marker because the model would have limited utility without adequate tight junction formation. Based on the results of this work, optimized conditions were determined in a significantly reduced amount of time as compared to traditionally used cell model optimization methods and an in vitro BBB screening tool that mimics the physiology of the NVU was developed. Given the outcomes of these studies it can be seen that a DOE optimization approach should be considered for development of biologically based systems to understand interactions between key system factors and to reduce the time to develop these necessary systems.</div><div>BBB permeability is not the only factor that slows development of neurotherapeutics. The intent of many of these compounds is to elicit an effect on the neuronal environment; however, permeability and neuroactivity are often evaluated separately even though they are inherently linked in vivo. Further enhancement of the optimized direct contact triculture was done to develop a screening tool that could assess neuroactivity of a compound as it is related to its brain permeability. The in vitro NVU model was developed by adding human neurons to the basolateral chamber of the direct contact triculture so permeating compounds would accumulate in the receiver chamber and their neuronal effects could be measured. During development of this model it was seen that the addition of neurons both increases tightness of the apical BBB model, but also increases viability of the neurons themselves. This is likely due to the facilitation of cross-talk between the four cell types of the NVU due to the proximity of the cells in the model system. The BBB permeability linked neuroactivity of marker compounds was measured by neuronal viability and neurite outgrowth in response to compound accumulation over the neurons during the course of BBB permeation. The results of this assay showed that the model is capable of being used to assess both BBB permeability and the subsequent neuroactivity of a given compound, and that the inclusion of additional cell types from the NVU further increases the physiological relevancy of the model. This work shows that the NVU model is an enhancement of the direct contact triculture model and can be easily implemented in the early development stages of neurotherapeutic compounds. Ultimately, this model has the potential to increase the number of brain targeting compounds by facilitating early, predictive assessment and rank ordering of large compound libraries for continued development. </div><div><br></div>

Philosophiae Doctor - PhD / The blood-brain barrier (BBB) is a restrictive interface located between the blood circulation and the central nervous system (CNS), regulating the homeostatic environment of the neuronal milieu, by controlling the permeability of the cerebrovasculature. Currently, we cannot fully comprehend the regulatory features and the complexity of BBB morphology to allow for intervention clinically. The thesis consists of four publications. The methodology paper proposes a novel experimental design to visualize the morphological architecture of immortalized mouse brain endothelial cell lines (bEnd3/bEnd5). The brain endothelial cells (BECs) were grown on cellulose matrices and fixed in 2.5 % glutaraldehyde in preparation for visualization of the paracellular (PC) spaces between adjacent BECs, employing high-resolution electron microscopy (HREM), with vested interest in the morphological profile of the developing BEC.

No description available.

The blood-brain barrier (BBB) is regulated by factors that can be secreted by multiple cell types, including astrocytes, that maintain the BBB in health and promote repair after injury. However, astrocyte contributions to the BBB are largely assumed from transplantation studies in which astrocyte progenitor grafts conferred BBB-like properties to tissues that normally lack a BBB. To determine if astrocytes contribute an essential and non-redundant function in maintaining the healthy BBB, I genetically ablated a small number of astrocytes using a conditional, tamoxifen-inducible mouse model. Within 2 hours after induction, I observed sparse astrocyte death in the cortex and leakage of the small molecule Cadaverine and large plasma protein fibrinogen, which are normally contained by a functional BBB. Vessels within regions of ablated astrocytes showed reduced expression of the tight junction protein zonula occludens-1, indicating impairment of the physical barrier formed between endothelial cells. Cadaverine leakage persisted for weeks, a feature I also found in mice after mild concussive traumatic brain injury (cTBI), thus highlighting the potential for revealing astrocyte roles in post-injury repair. Unlike the genetic ablation model, astrocytes within Cadaverine leakage areas did not undergo cell death after cTBI and instead downregulated homeostatic proteins. Our preliminary results show this atypical phenotype appearing 10 minutes after cTBI, along with severe vessel rupture, BBB leakage, and disruption of endfoot and basement membrane proteins. This damage persists for months, suggesting that the BBB fails to repair in these areas. Our results provide direct in-vivo evidence for essential astrocyte roles in the maintenance of the healthy BBB. Maintenance and/or repair fail after mild concussive cTBI, possibly contributing to irreversible progression to neurodegenerative diseases. / Doctor of Philosophy / The blood-brain barrier (BBB) is a unique property of blood vessels in the Central Nervous System (CNS) different from other vessels in the body. The physically tight barrier of the BBB is formed by tight junction proteins between endothelial cells and limits paracellular diffusion. The metabolic barrier is formed by concentrations of glucose transporters that promote transport of essential nutrients to the brain. Lastly, a transport barrier limits the passage of molecules and cells across the endothelial cell layer, preventing the entry of non-essential molecules, including pathogens and immune cells found in the blood. The BBB is thought to be induced and maintained by factors secreted by nearby cells in the brain. Among these cells are astrocytes, a type of glial cell that nearly completely cover blood vessels with their processes called endfeet. This strategic positioning led the field to assume that astrocytes are responsible for generating the unique properties of the BBB. Yet little direct evidence exists to support this conclusion, and newer evidence calls into question if astrocytes are even needed for BBB functions. To test this, I used a genetic mouse model to induce death of small numbers of astrocytes in adult mice. This caused leakage of blood contents of various sizes into the brain. In addition, the tight junction proteins responsible for forming the physical BBB were disrupted. These effects remained for weeks, a feature I also found after mild concussive traumatic brain injury (cTBI). This suggests that astrocytes may have an additional function in repairing the injured BBB. Our results demonstrate an essential role for astrocytes in the maintenance of the healthy adult BBB. Maintenance and/or its repair fail after cTBI, possibly contributing to the cascade into irreversible progression to neurodegenerative diseases.

Mild traumatic brain injury (mTBI)/ concussion accounts for 70-90% of all reported TBI cases in the United States and can cause long-term neurological outcomes that negatively impact quality of life. Previous studies revealed that increased blood-brain barrier (BBB) leakage is correlated with poor neurological outcomes after mTBI, yet the biological mechanisms linking BBB damage to the onset of neurological deficits after mTBI are not well understood. Previously, we found that astrocytes lose expression of homeostatic proteins after mTBI, characterizing the changes in astrocytic protein expression as an "atypical astrocyte response." Yet, the upstream mechanisms that induce this atypical astrocyte response after mTBI have yet to be elucidated. In models of more severe TBI, exposure to blood-borne factors triggers astrogliosis via upregulation in markers, such as glial fibrillary acidic protein (GFAP), but how exposure to blood-borne factors affects astrocyte protein expression in the context of mTBI is not well understood. Therefore, we hypothesized that mTBI-induced BBB damage causes atypical astrocytes via exposure to blood-borne factors. To test this hypothesis, we use a mTBI mouse model, two-photon microscopy, an endothelial cell-specific genetic ablation model, and serum-free primary astrocyte cultures. Here, we found that mTBI causes BBB damage through the loss of proteins involved in maintaining the BBB's physical and metabolic barriers, and BBB damage is sustained long-term after injury. Also, we demonstrated that leakage of blood-borne factors is sufficient to trigger atypical astrocytes, and plasma exposure triggers a similar response in vitro. Overall, these findings suggest that mTBI induces long-term BBB damage, and exposure to blood-borne factors triggers the loss of key homeostatic astrocytic proteins involved in maintaining healthy neuronal function. / Doctor of Philosophy / Mild traumatic brain injury (mTBI)/ concussion makes up 70-90% of all TBI cases reported in the United States and is commonly observed after car crashes, sports-related tackles, and blast exposure during military combat. People who experience mTBI develop debilitating long-term neurological consequences, such as sleep disturbances, depression, and dementia. Clinical data suggests mTBI causes damage to the barrier between the brain and blood, known as the blood-brain barrier (BBB). This damage has been correlated to the onset of poor neurological deficits, yet how damage to this barrier is causally linked to long-term neurological consequences remains to be fully understood. In our lab, we found that mTBI causes loss of proteins important for maintaining a healthy environment in the brain in specialized cells called astrocytes. However, the biological events that trigger the loss of protein expression in astrocytes after mTBI have yet to be fully investigated. Thus, we hypothesized that mTBI causes loss of these proteins via leakage of blood-borne factors. To test this hypothesis, we used a mTBI mouse model, two-photon microscopy, genetic manipulation, and cell cultures. In our studies, we found that mTBI triggers BBB damage via loss of proteins that make up its protective properties. Also, we demonstrated that leakage of blood-borne factors is sufficient to cause loss of astrocyte-specific proteins both in brain and cell cultures. Altogether, we show that a single mTBI is sufficient to cause loss of astrocyte-specific protein expression via exposure to blood-borne factors. These findings may point to targeting either the blood-borne factor(s) or their corresponding receptor pathways in astrocytes to halt the progression of long-term neurological deficits after mTBI.

Recent attempts to investigate living systems from a biophysical point of view has opened new windows for development of new diagnostic methods and therapies. Pulsed electric fields (PEFs) are a new class of therapies that take advantage of biophysical properties and have proven to be effective in drug delivery and treating several disorders including tumors. While animal models are commonly being used for development of new therapies, the high cost and complexity of these models along with the difficulties to control the electric field in the animal tissue are some of the obstacles toward the development of PEFs-based therapies. Microengineered models of organs or Organs-on-Chip have been recently introduced to overcome the hurdles of animal models and provide a flexible and cost-effective platform for early investigation of a variety of new therapies. In this study microfluidic platforms with integrated micro-sensors were designed, fabricated and employed to study the consequences of PEFs at the cellular level. These platforms were specifically used to study the effects of PEFs on the permeabilization of the blood-brain barrier for enhanced drug delivery to the brain. Different techniques such as fluorescent microscopy and electrical impedance spectroscopy were used to monitor the response of the cell monolayers under investigation. Irreversible electroporation is a new focal ablation therapy based on PEFs that has enabled ablation of tumors in a non-thermal, minimally invasive procedure. Despite promising achievements and treatment of more than 5500 human patients by this technique, real-time monitoring of the treatment progress in terms of the size of the ablated region is still needed. To address that necessity we have developed micro-sensor arrays that can be implemented on the ablation probe and give real-time feedback about the size of the ablated region by measuring the electrical impedance spectrum of the tissue. / Ph. D.

Organophosphates (OP) such as malathion are organic derivatives of phosphoric acid with broad use in everyday life throughout the world, especially as insecticides. Lead particles can accumulate in soil and from there leach into our water supplies. Interaction with the environment offers opportunities for multiple exposures to combinations of different toxicants (such as lead and malathion). Thus, it is important to assess effects that these compounds exert not only on the nervous system, but also on the blood-brain barrier (BBB). The BBB consists of specialized endothelial cells that form the vasculature of the brain; it regulates passage of nutrients, while preventing potentially damaging substances from entering the brain. The main feature of the BBB is the presence of tight junctions between cells, which provide the BBB with its low permeability. The work presented in this dissertation tests the hypothesis that lead and malathion disrupt BBB integrity by affecting tight junctions of the BBB. The hypothesis suggests that disruptions involve changes in protein levels and gene expression as well as activation of transient receptor potential canonical channels (TRPC) that in turn increase intracellular calcium levels affecting tight junction structure. The hypothesis was tested by assessing lead-malathion interactions in an in vitro BBB model. This model was constructed with rat astrocytes and rat brain endothelial cells (RBE4). Assessments of cell toxicity in response to different concentrations of the neurotoxicants tested showed that concentrations between 10-5 µM and 10-6 µM were ideal to assess combinations of neurotoxicants. In general, protein levels of occludin, claudin 5, ZO1, and ZO2 decreased at all times, however, qPCR analysis of gene expression for all the proteins did not correlate with the assessments on protein levels. TRPC channel protein levels increased in response to neurotoxicant insult, which correlated with results for gene expression. This study suggests that at least one of the mechanisms that neurotoxicants lead and malathion utilize to disrupt permeability of the BBB is by affecting tight junction structure. This effect could be regulated by increases in gene expression of TRPC1 and TRPC4 that are associated with increases in the number of TRPC channels on the membrane of endothelial cells of the cerebral microvasculature. / Ph. D.