Browsing by Subject "endothelial cell"
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(2022)Diabetes mellitus is an incurable disease caused by dysfunctional insulin signaling. The brown adipose tissue (BAT) serves as a hotspot for both lipid and glucose consumption and is thus an attractive target for treating metabolic diseases. Newly surfacing evidence suggest that the endothelial cells (ECs) lining the inner layer of vessels might regulate the morphology and function of adipose tissues. Several studies, including our own, suggest that the vessel density is negatively affected by metabolic diseases. As the BAT is an important organ for systemic lipid and glucose metabolism, and as the effects of metabolic diseases on BAT vessels are not adequately explored, I wanted to investigate how the BAT vasculature changes upon early time points of type 1 (T1D) and 2 (T2D) diabetes in this thesis work. To this end, I used mouse models with chemically induced T1D and genetic T2D and characterized these models with immunohistochemical analyses and immunoassays. To explore the transcriptomic landscapes of ECs and adipose stem cells (ASCs), I analyzed scRNAseq data of BAT stromal vascular fractions (SVF), focusing on changes in gene expression and EC-ASC interactions at a transcriptomic level. Also, by using a publicly available single-cell RNA sequencing (scRNAseq) dataset, I compared BAT SVF gene expression to complement the data resulting from our experiments. The results from this work reveal differential angiogenic responses in the T1D and T2D mouse models and open new avenues of research into how these different pathways are activated and how we can take advantage of these differences to treat diseases. All in all, this work will support the efforts in developing better options for future diabetes prevention, diagnosis, and care.
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(2019)Human umbilical vein endothelial cells are responsible for maintaining and forming new vessels from existing ones, in a biological process called sprouting angiogenesis. Sprouting angiogenesis is a crucial mechanism for the resolution of hypoxia and normal development of tissues. It also plays a key role in internal plague hemorrhages, which can lead to embolisms and other cardiovascular complications. Angiogenesis is also crucial for cancer development. Sprouting angiogenesis is initiated by hypoxic tissue excreted vascular endothelial growth factor gradient, which induces normal endothelial cells into either a proliferative stalk cell or a signal sensing tip cell phenotype. Both of these cell types depend on the rapid flow of lipids to their plasma membrane, either to form plasma membrane protrusions in tip cells or as new plasma membrane material in dividing stalk cells. This flow is envisioned to involve both vesicle-mediated and non-vesicular mechanisms. A major non-vesicular route of lipid transfer occurs at membrane contact sites via lipid transport proteins. Furthermore, lipids can be transported to the plasma membrane by the direct fusion of vesicles or endosomes with the plasma membrane This thesis set out to explore the role of two membrane contact site proteins, oxysterol-binding protein- related protein 2 and protrudin, in angiogenesis and lipid transfer. Their role was examined by RNA-sequencing transient knock-down samples of these proteins in HUVECs. The RNA-sequencing data was examined by differential expression, gene ontology overrepresentation and gene set enrichment analyses. Gene expression analysis provided almost 10 000 significantly changed transcripts (adjusted p-values < 0.05), in each silenced cell type. The distribution of differentially expressed genes in oxysterol-binding protein- related protein 2 silenced cells, is skewed toward negative fold changes, whereas the distribution of differentially expressed genes in protrudin silenced samples is normally distributed. The results also show significant changes in gene ontologies related to proliferation, cell cycle, angiogenesis as well as hypoxia in both sample types. Gene set enrichment analysis showed upregulation in angiogenesis related pathways, such as the PI3K-Akt and MAPK pathways, in both samples. Significant downregulation was present in cell cycle related pathways and cholesterol biosynthesis pathway in both ORP2 and protrudin silenced samples.
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(2020)Human induced pluripotent stem cells (hiPSC) can be propagated in a long-term culture and further differentiated into many cell types, including cardiomyocytes (CM) and endothelial cells (EC). Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) are promising tools in cardiac research, since they retain the original genotype of the individual donor and thus enable the use of patient- and disease specific cells. Crucial for the optimal use of hiPSC-CMs in experiments are methods for assessing cardiomyocyte phenotype. Contraction is a prominent feature for CMs, and it is essential that contraction can be quantified accurately. Reliable quantification is relevant when hiPSC-CMs are used for studying disease phenotypes, cardiac safety pharmacology, genotype-phenotype correlations, cardiac disease mechanisms and cardiac function over time. In this thesis project, contractile behavior of hiPSC-CMs was analyzed using video microscopy and online tool MUSCLEMOTION. Contraction parameters were obtained from hiPSC-CMs derived from patients with hypoplastic left heart syndrome (HLHS) and healthy controls on multiple timepoints during differentiation. In addition, contraction was analyzed in iPSC-CMs cocultured with induced pluripotent stem cell derived endothelial cells (iPSC-ECs), since it has been suggested that ECs can promote morphological and functional maturation of CMs in culture. Contraction duration (CD), time to peak (TTP), relaxation time (RT) and contraction amplitude (CA) was compared between different timepoints as well as between CMs cocultured with ECs and CMs cultured alone. Compared to control cell lines, HLHS patient hiPSC-CMs exhibited longer CD, TTP and RT as well as higher CA values. This difference was present in most of the timepoints, suggesting slower contractile kinetics in HLHS patient iPSC-CMs compared to control iPSC-CMs. Significant changes were also observed in contraction parameters when comparing hiPSC-CMs in coculture and monoculture. Contraction parameters of coculture iPSC-CMs changed in a relatively consistent manner over time, increasing or decreasing throughout the monitoring period whereas in hiPSC-CM monoculture there was more variation between timepoints. This project and results support the use of modern methods in detailed functional characterization of hiPSC-derived cells. In addition, it highlights the potential of coculture in disease modeling and the fact that hiPSC-CMs express variation in phenotypes. However, experiments should be repeated, and additional methods should be used in order to further validate the results and conclusions.
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