Theses and Dissertations from UMD
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Item Formulation and Delivery of Enhanced Extracellular Vesicles for Wound Repair(2021) Born, Louis Joseph; Jay, Steven M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Despite the development of a variety of therapies, complex wounds resulting from disease, surgical intervention, or trauma remain a major source of morbidity. Extracellular vesicles (EVs) have recently emerged as an alternative approach to address this issue. In particular, EVs derived from mesenchymal stem/stromal cells (MSCs) have been shown to improve wound healing, especially via enhanced wound angiogenesis. However, despite their clearly established potential, EVs have limitations that limit clinical relevancy, including low potency and rapid clearance from the body. Additionally, the ability to sustainably deliver EVs may enhance their efficacy in wound healing. Here, we leveraged the capability of EVs to be engineered via producer cell modification to investigate the therapeutic potential of EVs from MSCs transfected to overexpress a well-established pro-angiogenic long non-coding RNA HOX transcript antisense RNA (HOTAIR). We established that HOTAIR was able to be successfully loaded into MSC EVs (HOTAIR-MSC EVs) and delivered to endothelial cells in vitro with increased functional angiogenic activity. HOTAIR-MSC EVs injected intradermally around excisional wounds also showed increased angiogenic activity in vivo in two different species of rodents and improved wound healing in diabetic mice. We further report biomaterial-enabled sustained release of EVs using injectable hydrogel nanoparticles containing a composite of thiolated hyaluronic acid, maleimide functionalized poly(ε-caprolactone), and polyethylene glycol tetraacryalte as well as 3D-printed hydrogel discs composed of gelatin methacrylate for topical application. EVs released from the formulation of both of these biomaterials retained angiogenic bioactivity. Nanoparticles containing HOTAIR-MSC EVs were injected intradermally around an excisional wound in diabetic mice and were able to increase angiogenesis and improve wound healing. EVs released from 3D-printed EV-loaded GelMa hydrogels retained bioactivity in an in vitro endothelial scratch assay. Overall, these data suggest increasing the content of lncRNA HOTAIR in MSC EVs as a promising wound healing therapeutic. Additionally, establishing a biomaterial-enabled sustained release therapeutic represents a promising translational product for clinical implementation.Item INTERROGATING PROTEIN CARGOES OF MDSC-DERIVED EXOSOMES ON THE BASIS OF POST-TRANSLATIONAL MODIFICATIONS(2017) Chauhan, Sitara; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Myeloid-derived suppressor cells (MDSC) are immature myeloid cells which accumulate in cancer patients and tumor-bearing mice. Their function in the tumor microenvironment is to inactivate the immune response to cancer by suppressing both the adaptive and innate immune system. Therefore, MDSC are a major obstacle in immunotherapeutic approaches designed to cure cancer. MDSC-derived from tumor bearing mice have been found to shed exosomes. Exosomes are nano-sized vesicles that carry biologically active molecules and play a role in intercellular communication. MDSC-derived exosomes have been reported to mediate the immunosuppressive functions of the parental cells by stimulating the accumulation of MDSC and also by converting macrophages to a tumor-promoting phenotype. Recent developments in government policy have launched a goal of curing cancer using immune-based therapies (Cancer MoonShot 2020). The understanding of the mechanisms and functions of MDSC immune suppression will be crucial in the success of these therapeutic endeavors. Our current study focuses on interrogating the protein cargo carried by MDSC-derived exosomes based on differential post-translational modifications (PTMs). Post-translational modifications have important roles in functions, signaling, location and interactions of proteins. Selecting proteins based on a specific post-translational modification can aid in the identification of low-abundance proteins which may not be identified in a shotgun proteomics approach. The first aim of this work was to successfully modify an existing surface chemistry method to use on exosomes. We then used a proteomic strategy to identify glycoproteins on the surface of MDSC-derived exosomes, and then test if selected glycoproteins contribute to exosome-mediated chemotaxis and migration of MDSCs. Furthermore, we also aimed at examining the ubiquitome of the MDSC-derived exosomes, using top-down and bottom-up proteomics. Since inflammation has been reported to enhance the tumor promoting activity of the MDSC, the bottom-up analysis focused on the effects of increased inflammation on the ubiquitination of the protein cargo of MDSC-derived exosomes. Spectral counting was used to estimate differences in abundance of proteins found with ubiquitinated proteoforms in high and basal levels of inflammation. The top-down analysis aimed at characterizing the length and topology of ubiquitin linkages on substrate proteins in MDSC-exosomes.Item QUANTITATIVE ANALYSIS OF INTACT PROTEINS AND RNAS CARRIED BY IMMUNOSUPPRESSIVE EXOSOMES(2016) Geis Asteggiante, Lucia Giorgina; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment of most cancer patients. They are a major obstacle to immunotherapy because they suppress both adaptive and innate immune responses. MDSCs collected from tumor-bearing mice release nano-sized vesicles, called exosomes, which carry biologically active molecules and participate in intercellular communication. Exosomes released by MDSC stimulate migration of other MDSC towards the tumor microenvironment and convert macrophages to a tumor-promoting phenotype. Among the proteins identified in MDSC-released exosomes, S100A8 and S100A9 are low-mass, highly abundant, pro-inflammatory mediators already known to contribute directly to the immune suppressive functions of MDSC. The aim of this work was to successfully interrogate the exosomal intact protein cargo using top-down proteomics, a strategy for protein analysis that has not previously been applied to exosomes of any kind. Several protein forms (proteoforms) were fully characterized, which is critical as post-translational modifications regulate protein functions, cellular location and protein interactions. Additionally, since the tumor promoting activity of MDSC is enhanced by inflammation, we focused on evaluating the effect of increased inflammation on the proteoforms relative abundance using current top-down label-free quantitation techniques (peak intensities and peak areas), and comparing them to our recently validated spectral counting approach. Using spectral counting we were able to estimate differences in abundances of both S100A8 and S100A9 proteoforms. Furthermore, it has been previously reported that exosomes can carry micro RNAs and messenger RNAs. In order to investigate if MDSC-derived exosomes also contain RNAs, a collaborative study was carried out entailing the qualitative and quantitative analysis of miRNAs, mRNA and proteins present in MDSC and their exosomes, and evaluate their changes due to heightened inflammation. The MDSC and exosome protein cargo was analysed by bottom-up proteomics in this case, and the RNA cargo by next generation sequencing. A large number of mRNA and miRNA species were found to be carried by MDSC-derived exosomes and, strikingly, their putative functions were associated to MDSC expansion and suppressive function, and cancer development.Item INVESTIGATION BY MASS SPECTROMETRY OF THE UBIQUITOME AND PROTEIN CARGO OF EXOSOMES DERIVED FROM MYELOID-DERIVED SUPPRESSOR CELLS(2016) Adams, Katherine R.; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Exosomes released by myeloid-derived suppressor cells (MDSC) are 30 nm in diameter extracellular vesicles that have been shown to carry biologically active proteins as well as ubiquitin molecules. Ubiquitin is known to have many functions, including involvement in the formation of exosomes, although the exact role is highly contested. In the study reported here, the proteome and ubiquitome of MDSC exosomes has been investigated by bottom-up proteomics techniques. This report identifies more than 1000 proteins contained in the MDSC exosome cargo and 489 sites of ubiquitination in more than 300 ubiquitinated proteins based on recognition of glycinylglycine tagged peptides without antibody enrichment. This has allowed extensive chemical and biological characterization of the ubiquitinated cohort compared to that of the entire protein cargo to support hypotheses on the role of ubiquitin in exosomes.