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No description available.

Pulmonary arterial hypertension (PAH) is a devastating lung disease characterized by arterial pruning, occlusive vascular remodeling, and inflammation contributing to increased pulmonary vascular resistance with resultant right heart failure. Endothelial cell (EC) injury and apoptosis are commonly considered triggers for PAH, the mechanisms leading from injury to complex arterial remodeling are incompletely understood. While current therapies can improving symptoms, with the exception of parenteral prostacyclin, they do not significantly prolong transplant free survival. As well, there are no therapies that can regenerate the damaged lung short of transplantation. In this project, I sought to both advance the understanding of disease pathogenesis and explore regenerative therapeutic options for PAH. To this end, I first employed single cell RNA sequencing (scRNA-seq) at multiple time points during the Sugen 5416 (SU) – chronic hypoxia (CH) model of PAH, to provide new insights into PAH pathogenesis both during onset and progression of disease. We also employed microCT analysis to visualize and quantify the arterial pruning associated with PH and found significant loss up to 65% of the healthy arteriolar volume in this model. Through scRNA-seq analysis performed at four timepoints spanning the onset and progression of disease, two disease-specific EC cell types emerged as key drivers of PAH pathogenesis. The first was the emergence of capillary ECs with a de-differentiated gene expression profile, which we termed dedifferentiated capillary (dCap) ECs, with enrichment for the Cd74 gene. Interestingly, RNA velocity analysis suggested that these cells may be undergoing endothelial to mesenchymal transition during PAH development. At later times, a second arterial EC population became apparent, which we termed activated arterial ECs (aAECs), since it uniquely exhibited persistently elevated levels of differential gene expression consistent with a migratory, invasive and proliferative state. Interestingly, the aAECs together with the smooth muscle (SM)-like pericytes, a population which was also greatly expanded in PAH, expressed Tm4sf1, a gene previously associated with a number of cancers and abnormal cell growth. Furthermore, by immunostaining, TM4SF1 was found to be spatially localized to sites of complex and occlusive arterial remodeling, associated with both endothelial cells and pericytes in these lesions, suggesting an important role for the aAECs and SM-like pericytes in arterial remodeling and PH progression. Together, these findings suggest that aAECs, dCap ECs, and SM-like pericytes are emerging cell populations responsible for lung arterial remodeling in PAH, which drives disease progression, and that TM4SF1 may be a novel therapeutic target for this disease. As a first step in trying to develop approaches to regenerate lung arterial bed that is lost in PAH, we investigated the potential role of endothelial colony forming cells (ECFCs) and mesenchymal stromal cell (MSC) derived extracellular vesicles (EVs) as novel therapeutics, on the premise that these stem/progenitor cells would stimulate lung regeneration by mainly paracrine mechanisms. Additionally, we used biomaterials to microencapsulate cells and EVs to improve their local delivery and retention. While ECFCs were found to be ineffective in treating the monocrotaline model on their own, they were poorly retained in the lung and microencapsulation of ECFCs led to enhanced lung delivery within the first 72 hours, with resultant hemodynamic improvements in this model of PAH. MSCs are well known to be immunomodulatory and proangiogenic, largely acting through paracrine mechanisms, including by the release of EVs. Yet, following intravenous administration, nano sized EVs are rapidly cleared from circulation, potentially limiting their therapeutic potential. I adapted our microencapsulation strategy for EVs, and demonstrated significantly greater retention of microgel-loaded EVs were within the lung, resulting in enhanced local cell uptake. Interestingly, the hydrogel used for microencapsulation induced a local immune response which made it unsuitable for testing any potential therapeutic benefits of MSC-EVs in this study. Nonetheless, this work demonstrated proof-of-principle for the utility of microencapsulation as a strategy to enhance EV lung delivery. Overall, this work has identified novel lung cell populations (aAECs, dCap ECs, SM-like pericytes) driving arterial remodeling associated with PH progression, demonstrated the potential of ECFCs as a regenerative cell for the treatment of PAH, and illustrated the utility of microencapsulation as a tool to enhance lung targeting of both cells and EVs.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第24549号 / 薬科博第166号 / 新制||薬科||18(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23837号 / 薬科博第152号 / 新制||薬科||17(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

Extracellular vesicles (EVs) are small membrane-surrounded structures containing transmembrane proteins and enclosing cytosolic proteins and nucleic acids. They are released in the extracellular space by both normal and neoplastic cells and play an important role in cell-cell communication in numerous physiological processes and pathological conditions, through the transfer of their functional cargo to recipient cells. EVs are highly abundant in biological fluids, and even more represented in cancer patients’ biofluids, therefore many studies suggested that they can be instrumental in liquid biopsies as prognostic markers or for early detection of tumors. Moreover, being secreted by potentially all the cells, they can serve in oncology to represent the tumor heterogeneity, which is underestimated by the current diagnostic tools. Given their small size, EVs are difficult to isolate in a high-throughput way and, therefore, one of the main obstacles to their clinical application, is that the existing isolation methods are impractical. During these years, I worked at the development and optimization of a novel technique that allows purification of heterogeneous EVs from biological fluids in an efficient, fast and reproducible way. This technique, named Nickel-Based Isolation (NBI), is a biochemical assay that allows obtaining polydisperse EVs in a physiological pH solution, therefore, preserving their morphology, heterogeneity, and stability. We tested and optimized this assay in protein-enriched systems and comparing it to the techniques currently used to characterize and measure EVs, such as flow cytometry and Tunable Resistive Pulse Sensing. We challenged the reproducibility of this method by isolating EVs from different biological fluids. Interestingly, the EVs purified with NBI result more intact and stable compared to the ones obtained with other methods, and can be studied in a clinical setting and used as an innovative tool for detection of molecules associated with diseases. We demonstrated the specificity of the procedure by using individual isolated vesicles in biochemical and molecular assay, optimized to characterize the biological content of EVs. We were able to detect picomolar concentration of PSMA on 105 EVs isolated from plasma of prostate cancer patients and BRAF-V600E transcript in just 103 EVs from the plasma of colon cancer patients, reaching unprecedented matching with tissue biopsy results. We also investigated the transcriptome of EVs isolated from glioblastoma cancer stem cells, in order to exploit the potential of EVs as diagnostic markers.

[ES] El infarto agudo de miocardio (IAM) es una de las principales causas de morbilidad y mortalidad en los países desarrollados. A lo largo de los últimos años se ha visto que la respuesta inflamatoria que ocurre tras desencadenarse el IAM es muy importante en el desarrollo clínico de esta patología. Si se produce una respuesta inflamatoria exacerbada aumenta el riesgo de remodelado cardiaco adverso y fallo cardiaco, pero el hecho de que no se desencadene la respuesta inflamatoria también tiene consecuencias negativas. Debido a la importancia de la respuesta inflamatoria en el IAM, recientemente se han intentado desarrollar terapias dirigidas frente componentes celulares o moleculares que participan en esta respuesta. Dentro de estas terapias, la terapia celular con células mesenquimales estromales (MSC) se ha postulado como un buen candidato. Las MSC se caracterizan fundamentalmente por su capacidad inmunomoduladora, lo que ha conducido a su empleo como agentes terapéuticos en diferentes enfermedades que cursan con procesos inflamatorios. Sin embargo, a lo largo de los últimos años, numerosos estudios han mostrado que el efecto terapéutico de las MSC está mediado fundamentalmente por las vesículas extracelulares (EVs) que liberan. Estas EVs recapitulan los efectos terapéuticos de las células de origen, por lo que también presentan efectos inmunomoduladores. El empleo de las EVs de MSC como agentes terapéuticos presenta ventajas respecto al uso de las MSC como, por ejemplo, una mayor bioseguridad. No obstante, el uso clínico de las EVs todavía tiene que hacer frente a retos como la obtención de grandes cantidades de EVs que constituyan un producto clínico estable y homogéneo. En este trabajo se ha querido evaluar, por un lado, si las EVs obtenidas de diferentes biopsias de la misma fuente tisular de MSC pueden constituir un producto biológico homogéneo que presente las mismas características y funcionalidad. Por otro lado, se ha evaluado si estas EVs se pueden emplear como agente terapéutico frente a la respuesta inflamatoria que se desencadena tras el IAM. Para ello se ha estudiado el efecto inmunomodulador de las EVs sobre células del sistema inmune, fundamentalmente macrófagos, in vitro y en un modelo in vivo de IAM en ratas. Los resultados mostraron que las EVs favorecen la diferenciación de los macrófagos M1 proinflamatorios hacia un fenotipo similar al M2, aumentando la expresión de marcadores M2 y reduciendo la secreción de citocinas proinflamatorias. Además, las EVs promovieron la activación de neutrófilos in vitro y la reducción de su estrés oxidativo. La administración de EVs en ratas sometidas a IAM amortiguó la caída de la función cardiaca y limitó la extensión de la zona infartada a los 7 y 21 días postinfarto. Las EVs también redujeron el número de macrófagos y neutrófilos proinflamatorios dentro de la zona infartada, favoreciendo la resolución de la inflamación. En conclusión, las EVs empleadas en este trabajo han demostrado ser un producto biológico estable con independencia de la biopsia de la que proceden, y han demostrado ser capaces de ejercer respuestas pro-resolutivas eficaces en un modelo de isquemia miocárdica, lo que las convierte en potenciales agentes terapéuticos para tratar la inflamación en el IAM. / [CA] L'infart agut de miocardi (IAM) és una de les principals causes de morbiditat i mortalitat als països desenvolupats. Al llarg dels darrers anys s'ha vist que la resposta inflamatòria que passa després de desencadenar-se l'IAM és molt important en el desenvolupament clínic d'aquesta patologia. Si es produeix una resposta inflamatòria exacerbada augmenta el risc de remodelat cardíac advers i fallada cardíaca, però el fet que no es desencadeni la resposta inflamatòria també té conseqüències negatives. A causa de la importància de la resposta inflamatòria a l'IAM, recentment s'han intentat desenvolupar teràpies dirigides davant de components cel·lulars o moleculars que participen en aquesta resposta. Dins aquestes teràpies, la teràpia cel·lular amb cèl·lules mesenquimals estromals (MSC) s'ha postulat com un bon candidat. Les MSC es caracteritzen fonamentalment per la seva capacitat immunomoduladora, cosa que ha conduït a la seva ocupació com a agents terapèutics en diferents malalties que cursen amb processos inflamatoris. Tot i això, al llarg dels últims anys, nombrosos estudis han mostrat que l'efecte terapèutic de les MSC està intervingut fonamentalment per les vesícules extracel·lulars (EVs) que alliberen. Aquestes EVs recapitulen els efectes terapèutics de les cèl·lules dorigen, per la qual cosa també presenten efectes immunomoduladors. L'ús de les EVs de MSC com a agents terapèutics presenta avantatges respecte a l'ús de les MSC com, per exemple, una bioseguretat més gran. Tot i això, l'ús clínic de les EVs encara ha de fer front a reptes com l'obtenció de grans quantitats d'EVs que constitueixin un producte clínic estable i homogeni. En aquest treball s'ha volgut avaluar, d'una banda, si les EV obtingudes de diferents biòpsies de la mateixa font tissular de MSC poden constituir un producte biològic homogeni que presenti les mateixes característiques i funcionalitat. D'altra banda, s'ha avaluat si aquestes EVs es poden fer servir com a agent terapèutic davant de la resposta inflamatòria que es desencadena després de l'IAM. Per això s'ha estudiat l'efecte immunomodulador de les EV sobre cèl·lules del sistema immune, fonamentalment macròfags, in vitro i en un model in vivo d'IAM en rates. Els resultats van mostrar que les EVs afavoreixen la diferenciació dels macròfags M1 proinflamatoris cap a un fenotip similar al M2, augmentant l'expressió de marcadors M2 i reduint la secreció de citocines proinflamatòries. A més, les VE van promoure l'activació de neutròfils in vitro i la reducció del seu estrès oxidatiu. L'administració d'EVs en rates sotmeses a IAM va esmorteir la caiguda de la funció cardíaca i va limitar l'extensió de la zona infartada als 7 i 21 dies postinfart. Les EVs també van reduir el nombre de macròfags i neutròfils proinflamatoris dins de la zona infartada, afavorint la resolució de la inflamació. En conclusió, les EVs emprades en aquest treball han demostrat ser un producte biològic estable amb independència de la biòpsia de què procedeixen, i han demostrat ser capaços d'exercir respostes pro-resolutives eficaces en un model d'isquèmia miocàrdica, cosa que les converteix en agents terapèutics potencials per tractar la inflamació a l'IAM. / [EN] Acute myocardial infarction (AMI) is one of the main causes of morbidity and mortality in developed countries. Over the last few years, it has been shown that the inflammatory response that occurs after AMI is triggered is very important in the clinical development of this pathology. If an exacerbated inflammatory response occurs, the risk of adverse cardiac remodeling and heart failure increases, but failure to trigger the inflammatory response also has negative consequences. Because of the importance of the inflammatory response in AMI, recent attempts have been made to develop therapies that target cellular or molecular components involved in this response. Within these therapies, cell therapy with mesenchymal stromal cells (MSC) has been postulated as a good candidate. MSC are mainly characterized by their immunomodulatory capacity, which has led to their use as therapeutic agents in different diseases involving inflammatory processes. However, in recent years, numerous studies have shown that the therapeutic effect of MSCs is mainly mediated by the extracellular vesicles (EVs) they release. These EVs recapitulate the therapeutic effects of the cells of origin and therefore also have immunomodulatory effects. The use of MSC-EVs as therapeutic agents has advantages over the use of MSC, such as increased biosafety. However, the clinical use of EVs still faces challenges such as obtaining large quantities of EVs that constitute a stable and homogeneous clinical product. The aim of this study was to evaluate, on the one hand, whether EVs obtained from different biopsies of the same MSC tissue source can constitute a homogeneous biological product with the same characteristics and functionality. On the other hand, we have evaluated whether these EVs can be used as a therapeutic agent against the inflammatory response triggered after AMI. To this end, the immunomodulatory effect of EVs on immune system cells, mainly macrophages, was studied in vitro and in an in vivo model of AMI in rats. The results showed that EVs favored the differentiation of proinflammatory M1 macrophages towards an M2-like phenotype, increasing the expression of M2 markers and reducing the secretion of proinflammatory cytokines. In addition, EVs promoted the activation of neutrophils in vitro and the reduction of their oxidative stress. The administration of EVs in rats subjected to AMI blunted the decline in cardiac function and limited the extent of the infarct zone at 7- and 21-days post-infarction. EVs also reduced the number of proinflammatory macrophages and neutrophils within the infarct zone, favoring the resolution of inflammation. In conclusion, the EVs used in this work have been shown to be a stable biological product regardless of the biopsy from which they are derived and have been shown to be able to exert effective pro-resolving responses in a model of myocardial ischemia, making them potential therapeutic agents to treat inflammation in AMI. / Amaro Prellezo, E. (2024). Las vesículas extracelulares derivadas de células mesenquimales estromales de pulpa dental como producto terapéutico frente a la respuesta inmune que se desencadena tras el infarto agudo de miocardio [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/202973

Brucella abortus is a zoonotic agent that primarily infects cattle and causes brucellosis. B. abortus strain RB51 is a live, attenuated vaccine licensed for cattle. However, there is no available vaccine to prevent human brucellosis. Outer membrane vesicles have been tested as potential vaccines to prevent diseases caused by bacterial species. OMV are constantly released from Gram-negative bacteria. They are comprised principally of the outer membrane components and periplasmic proteins from the bacterial cell envelope. The research in this thesis examined the adjuvant property of non-replicative, metabolically active irradiated strain RB51 and the protective ability of OMV derived from strain RB51. Irradiated B. abortus strain RB51 was assessed for its ability to act as an adjuvant to induce protection against malaria. It was found that irradiated B. abortus strain RB51 administered along with fasciclin related adhesive protein (FRAP) to mice induced a protective immune response and a significant decrease in parasitemia after challenge with Plasmodium berghei. Strain RB51 and strain RB51 over-producing Cu/Zn superoxide dismutase (Cu/Zn SOD) were used to produce OMV. Western blotting and SDS-PAGE gel staining confirmed the presence of OMV and the over-production of Cu/Zn SOD. OMV were delivered to mice using an intraperitoneal route and, in some cases, with aluminum hydroxide adjuvant. The immune response was assessed by antibody isotyping with respect to OMV and measuring splenic clearance (i.e. protection) from a B. abortus strain 2308 challenge. The results demonstrate that OMV from B. abortus strain RB51 or strain RB51 over producing Cu/Zn SOD produced a Th1 polarized immune response as measured by specific OMV antibodies and cytokines but no statistically significant protection was observed. / Master of Science

In this paper we study a methodology for the numerical simulation of stable structures of fluid membranes and vesicles in biological organisms. In particular, we discuss the effects of spontaneous curvature on vesicle cell membranes under the bending energy for given volume and surface area. The geometric modeling of the vesicle shapes are undertaken by means of surfaces generated as Partial Differential Equations (PDEs). We combine PDE based geometric modeling with numerical optimization in order to study the stable shapes adopted by the vesicle membranes. Thus, through the PDE method we generate a generic template of a vesicle membrane which is then efficiently parameterized. The parameterization is taken as a basis to set up a numerical optimization procedure which enables us to predict a series of vesicle shapes subject to given surface area and volume.

京都大学 / 新制・課程博士 / 博士(薬学) / 甲第25233号 / 薬博第868号 / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 樋口 ゆり子, 教授 寺田 智祐, 教授 山下 富義 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DGAM