Cell & Molecular Biology
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/30806
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Browsing Cell & Molecular Biology by Author "Walczak, Samantha"
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Item Characterization of Estrogen Receptors (ERs) and ER-metabolizing enzymes in Lipedema and Non-Lipedema Adipose Stem Cells (ASCs) and differentiated adipocytes(2022) Walczak, Samantha; Al-Ghadban, Sara; Rinderle, Caroline; Bunnell, BruceIntroduction: Lipedema is a chronic, idiopathic painful disease characterized by an excess of adipose tissue in the lower extremities, commonly misdiagnosed as obesity, lymphedema, or chronic venous insufficiency. As the severity of lipedema worsens patients have reduced mobility, easy bruising, and fatigue and it is thought to resist lifestyle modifications. While treatments such as liposuction can help ease these symptoms, it is not curative, and the underlying etiology is unknown. Hypothesis: As the development of lipedema often begins or worsens during periods of hormonal change such as puberty, pregnancy, or menopause, we hypothesize that alterations in estrogen drive lipedema pathogenesis. Aim: The aim of this study is to characterize the gene expression of estrogen receptors (ER-α and ER-β), G-protein coupled estrogen receptor (GPER), and ER metabolizing enzymes: Hydroxysteroid 17-beta dehydrogenase (HSD17B1, B7, B12), Hormone-sensitive Lipase (LIPE) and Steroid Sulfatase (STS) in ASCs and differentiated adipocytes in BMI and age-matched non-lipedema and lipedema patients. Methods: Cell culture and Oil Red O stain, RNA extraction and RT-PCR assays were used to assess the expression of ERs and the estrogen metabolizing enzymes in ASCs and differentiated adipocytes. Results: ER-α, ER-β, and GPER gene expression were increased in Lipedema ASCs cultured in hormone-depleted media, as well as in differentiated adipocytes compared to non-lipedema corresponding cells. LIPE, STS, HSD17B17, and HSD17B12 gene expression were also increased in Lipedema differentiated adipocytes compared to non-lipedema differentiated adipocytes. In addition, the gene expression of HSD17B1 was increased in Lipedema ASCs cultured in hormone-depleted media compared to non-lipedema ASCs. Conclusion: These results indicate that expression of ERs and estrogen metabolizing enzymes are altered by Lipedema and suggest that estrogen may play a role in adipose tissue dysregulation in lipedema. Exploring this possible etiology further could contribute to the expansion of treatment options and management available to lipedema patients.Item Increased angiogenic differentiation of HUVECs treated with pre-conditioned media from Lipedema adipocytes in vitro(2022) Al-Ghadban, Sara; Walczak, Samantha; Bunnell, BrucePurpose: Lipedema is a connective tissue disorder characterized by an increased number of dilated blood vessels (angiogenesis), fibrosis, inflammation and accumulation of interstitial fluid in the subcutaneous adipose tissue. This project aims to gain insights into the lipedema vasculature using human umbilical vein endothelial cells (HUVECs) as an in vitro model of angiogenesis. Methods: HUVECs were cultured in conditioned media (CM) collected from lipedema adipocytes differentiated in vitro. The effects on the expression of the endothelial and angiogenic markers [CD31, von Willebrand Factor (vWF), Angiopoietin 2 (Ang2), Hepatocyte Growth Factor (HGF), Vascular endothelial growth factor (VEGF), Matrix Metalloproteinase (MMPs)] in HUVECs, as well as tube formation, were investigated. Real-time polymerase chain reaction (RT-PCR), tube formation and Western blot assays were used to assess the expression of endothelial and angiogenic markers in HUVECs. In addition, the ability of HUVECs to form capillary-like tubular structures (tubes) when seeded on reconstituted basement membrane (Matrigel) was determined by phase-contrast microscopy and the number of tubes formed (number of nodes and branches) was quantified using NIH ImageJ software. Results: The expression of CD31 and Ang2 were increased at both the gene and protein levels, while vWF expression was significantly decreased in HUVECs treated with CM from lipedema adipocytes. In addition, the gene expression of HGF and MMP9 and the number of nodes and branches were increased in treated HUVECs. No changes in VEGF gene expression were detected. Conclusion: These results indicate that lipedema adipocyte-CM promotes the vascular tube formation of endothelial cells through paracrine mechanisms. The expression of multiple factors that stimulate the process of angiogenesis was upregulated after exposure to conditioned media from lipedema adipocytes. Definition of the pathways that enhance angiogenesis in lipedema tissues will help researchers develop new therapeutic approaches to treat this disease.