Studies of the molecular anatomy of stroma vascular cells in murine adipose and renal tissues

<p dir="ltr">Adipose tissue plays a central role in metabolism and has during the last decades gained significant attention from the scientific community due the growing health concerns caused by the increased prevalence of obesity. Obesity-related diseases, including those affecting the vascular system, cancer, and diabetes, are among the most prevalent non-communicable diseases today. However, the issue may not solely lie within obesity itself since there are claims of metabolic "healthy" individuals with obesity, although the time for which one can live with obesity and still be health is limited. Nevertheless, this implies that the fundamental contributor to these diseases may rather be a dysfunctional adipose tissue rather than obesity. The primary contributors to associated morbidities to obesity may in fact be attributed to the enlarged adipocytes that are known to cause inflammation and reduced insulin sensitivity in adipose tissue. This, in turn, may redirect fatty acids from adipose tissue to other organs, leading to dyslipidemia, unhealthy systemic insulin resistance and elevated blood glucose levels.</p><p dir="ltr">Current incretin-mimetic therapies, such as semaglutide and tirzepatide, aim to reduce food intake, thereby decreasing body weight and improving insulin sensitivity and blood glucose levels. The medicine possesses several physiological properties, and the reduction of blood glucose can be attributed to several mechanisms such as decreased production of glucose by the liver, increased secretion of insulin, and slowed down digestion of food, as well as the reduced food intake accompanied by body weight loss. However, the reduction of body weight comes with desirable reduction in fat mass and an undesired loss of lean mass, potentially increasing the risk of adverse effects. An alternative therapeutic strategy could therefore focus on enhancing the function and fat storage capacity of adipose tissue, thereby minimizing ectopic fat and improving insulin sensitivity. In this context, adipose stem cells could play a pivotal role, as they have the ability to differentiate into mature adipocytes and by so increasing the fat storage capacity of the tissue. Nonetheless, to develop medications aimed at enhancing the overall function of adipose tissue, such as by stimulating adipogenesis, a more detailed characterization of the cell types involved in adipogenic differentiation and adipose tissue homeostasis is needed. To this end, significant interest has been directed towards the single-cell RNA sequencing technology and for its potential to more accurately define cell types within adipose tissue. Previous methods, like bulk RNA sequencing of whole tissue or cells isolated by antibodies, have not fully captured the complexity and heterogeneity of tissue composition. Bulk RNA sequencing can overlook rare cell types, overshadowed by mRNA from more prevalent cell types, and the absence of specific cell-type markers suitable for antibody enrichment has made isolating certain cell populations nearly impossible.</p><p dir="ltr">In this thesis, we utilized single-cell RNA sequencing to characterize cells within the stromal vascular fraction of white adipose tissue in lean healthy wild-type mice (<b>Paper I</b>), during obesity/diabetes and treatment (<b>Paper II</b>), and to study endothelial cells in kidney nephrons during disease progression of a common complication to diabetes, namely diabetic kidney disease (<b>Paper III</b>). Our work has led to a better characterization of adipose stem cells (<b>Paper I</b>) by comparing their gene expression profile to other cell types in adipose tissue and similar cell types from other organs. Our analysis identified adipose stem cells as fibroblast- like cells with a unique gene expression profile distinct from mural and endothelial cells. We found that the gene expression of adipose stem cells in mice is strongly influenced by anatomical location and sex, with male perivascular adipose stem cells showing a higher propensity for adipogenic differentiation than their female counterparts.</p><p dir="ltr">In <b>Paper II</b>, we explored how the transcriptomic landscape of adipose tissue is affected by obesity/type-2-diabetes and treatment using a db/db mice model treated with the PPARG agonist pioglitazone. The treatment improved the metabolic profile, decreasing glucose and lipid levels in blood. Our analysis indicated that adipose tissue was more affected by the disease than skeletal muscle and heart, with a gene expression profile suggesting inflammation in adipose tissue. The transcriptomic data suggested that the treatment resolved inflammation, potentially through increased adipogenic differentiation of adipose stem cells into mature adipocytes and/or improved function of the vascular system in adipose tissue.</p><p dir="ltr">In <b>Paper III</b>, we aimed to identify endothelial cell subtypes in kidney nephrons using single-cell RNA sequencing technology and explore their transcriptomic changes during the progression of diabetic kidney disease using the BTBRob/ob mice model. We provided a single-cell RNA sequencing technology atlas covering major endothelial cell subtypes in kidney nephrons of lean mice as well as during the progression of the disease. Paper III highlights novel marker genes for endothelial cell subtypes and confirms their anatomical locations through tissue imaging. Our data suggest that glomerular endothelial cells and peritubular capillaries are most affected during diabetic kidney disease, with gene expression profile analysis indicating inactivation of oxidative phosphorylation pathways.</p><p dir="ltr">In conclusion, our work underscores the utility of single-cell RNA sequencing in bridging previous knowledge gaps regarding the stromal vascular cell types of adipose tissue and renal endothelial cells, both in healthy and diseased states.</p><h3>List of scientific papers</h3><p dir="ltr">I. <b>Uhrbom, M.</b> Muhl, L. Genové, G. Liu, J. Palmgren, H. Alexandersson, I. Karlsson, F. Zhou A-X. Lunnerdal, S. Gustafsson, S. Buyandelger, B. Petkevicius, K. Ahlstedt, I. Karlsson, D. Aasehaug, L. He, L. Jeansson, M. Betsholtz, C. & Peng, X-R. Adipose stem cells are sexually dimorphic cells with dual roles as preadipocytes and resident fibroblasts. Nat Commun. 2024, 15, 7643. <a href="https://doi.org/10.1038/s41467-024-51867-9" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41467-024-51867-9</a></p><p dir="ltr">II. <b>Uhrbom, M.</b> Leke, R. Andréasson, A-C. Alexandersson, I. Collin, J. Ahlstedt, Xiang, Z, I. Aasehaug, L. Baker, T. Zarrouki, B. Betsholtz, C. and Peng, X-R. Single cell analysis of adipose tissue response to pioglitazone in a type-2 diabetes model. [Manuscript]</p><p dir="ltr">III. Zhou, A-X. Jeansson, M. He, L. Wigge, L. Tonelius, P. Tati, R. Cederblad, L. Muhl, L. <b>Uhrbom, M.</b> Liu, J. Granqvist, AB. Lerman, L, O. Betsholtz, C. Hansen, P, B, L. Renal Endothelial Single-Cell Transcriptomics Reveals Spatiotemporal Regulation and Divergent Roles of Differential Gene Transcription and Alternative Splicing in Murine Diabetic Nephropathy. Int. J. Mol. Sci. 2024, 25, 4320. <a href="https://doi.org/10.3390/ijms25084320" rel="noreferrer" target="_blank">https://doi.org/10.3390/ijms25084320</a></p>