Cell & Molecular Biology

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/30806

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    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, Bruce
    Introduction: 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.
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    Increased angiogenic differentiation of HUVECs treated with pre-conditioned media from Lipedema adipocytes in vitro
    (2022) Al-Ghadban, Sara; Walczak, Samantha; Bunnell, Bruce
    Purpose: 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.
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    Molecular characterization of human adipose-tissue derived stem cells (ASCs) from the breast in the presence and absence of exogenous estrogen
    (2022) Artiles, Maria; Al-Ghadban, Sara; Bunnell, Bruce
    Purpose: Adipose-derived stem cells (ASCs) are multipotent cells of mesodermal origin with self-renewal and differentiation capabilities, isolated from adipose tissue. ASCs are located in adipose tissue depots throughout the body and maintained for life. Our group has extensively studied abdominal ASCs (aASCs) harvested from lean and obese individuals and how their contribution to the tumor microenvironment (TME) results in amplified breast cancer proliferation, tumorigenesis, and metastasis; in a series of processes that have been linked to leptin signaling cascades and estrogen-mediated pathways. Furthermore, these properties are exacerbated in aASCs from obese donors, which express increased levels of leptin and pro-inflammatory cytokines compared to those harvested from lean individuals. Of particular interest are ASCs located in breast adipose tissue (bASCs). These bASCs have unique immunomodulatory, anti-inflammatory, and antioxidative properties. They serve essential endocrine functions in healthy breast tissue, but they also play a role in the development and pathophysiology of breast cancer, being a critical component of the breast cancer TME. While bASCs influence their microenvironment, they are also altered by physiological changes associated with obesity, the circulating levels of the sex hormones such as estrogen and progesterone, and the adipokine leptin. The fluctuations in estrogen levels before and after menopause are also accompanied by a shift in the accumulation site of subcutaneous adipose tissue. This study aims to characterize bASCs from human donors and compare their gene expression, proliferation, differentiation properties, and response to exogenous hormonal variations, to those of aASCs. Methods: bASCs were characterized and compared to aASCs after being cultured in the presence or absence of exogenous estrogen. Adipogenic and osteogenic differentiation was induced and assessed with Oil Red O and Alizarin Red S staining, respectively. Alterations to colony-forming unit capabilities were assessed with crystal violet staining. Cell surface markers were evaluated using flow cytometry. The expression of hormonal receptors, adipogenic genes, and osteogenic genes was evaluated by real-time PCR and Simple Western. Results: bASCs display similar morphological characteristics, surface markers, colony-forming capabilities, proliferation potential, and differentiation potential to that of aASCs. However, bASCs express higher levels of hormone receptors and are more susceptible to changes in exogenous estrogen than aASCs. Conclusion: Given that hormone receptor positive breast cancers are more prevalent in obese women after menopause, while triple negative breast cancers (TNBC) are more common in obese pre-menopausal women, hormonal levels may play a role in the development of both hormone receptor-positive breast tumors and TNBC tumors by affecting the surrounding bASCs
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    Inositol-requiring enzyme 1 alpha (IRE1α) at the int-ER-section of astrocyte-mediated neurotoxicity: Implications in (METH)amphetamine use and HIV-associated neurocognitive disorders
    (2022) Proulx, Jessica; Borgmann, Kathleen
    Human immunodeficiency virus 1 (HIV-1) invades the central nervous system (CNS) early during infection and can persist in the CNS for life despite effective antiretroviral treatment. Infection and activation of residential glial cells leads to low viral replication and chronic inflammation, which damage neurons contributing to a spectrum of HIV-associated neurocognitive disorders (HAND). Astrocytes are the most numerous glial cells in the CNS and provide essential support to neurons to ensure CNS health and function. During a neuropathological challenge, such as HIV-1 infection, astrocytes can shift their neuroprotective functions to become neurotoxic and even serve as reservoirs for HIV-1 infection. Indeed, astrocyte-mediated neurotoxicity (astrogliosis) is a hallmark of neurodegenerative diseases and disorders provoking consequences such as neuroinflammation, oxidative stress, and glutamate excitotoxicity. Astrocyte responses that regulate these outcomes include increased release of inflammatory mediators, decreased metabolite and antioxidant provision, and impaired uptake of glutamate from the tripartite synapsis, respectively. Notably, substance use disorders, including (METH)amphetamine are disproportionately elevated among people living with HIV-1. METH use can induce neurotoxic and neurodegenerative consequences, which can increase one's risk and severity of HAND. Identifying cellular and molecular mechanisms underlying astrocyte-mediated neurotoxicity are of heightened importance to optimize the coupling between astrocytes and neurons and ensure neuronal fitness against CNS pathology, including HAND and METH use disorder. Mitochondria are essential organelles for regulating metabolic, antioxidant, and inflammatory profiles. Moreover, endoplasmic reticulum (ER)-associated signaling pathways, such as calcium and the unfolded protein response (UPR), are important messengers for cellular fate and function, including inflammation and mitochondrial homeostasis. Increasing evidence supports that the three arms of the UPR are involved in the direct contact and communication between ER and mitochondria through mitochondria-associated ER membranes (MAMs). Our previous studies in primary human astrocytes demonstrated increased UPR/MAM mediator protein expression following HIV-1 infection or chronic METH exposure, of which, inositol-requiring enzyme 1 alpha (IRE1α) was most prominently elevated. Interestingly, pharmacological inhibition of the three UPR arms, illuminated that IRE1α is a potential regulator of astrocyte mitochondrial respiration. Here, we further delve into the functional role of IRE1α in primary human astrocytes using an IRE1α overexpression plasmid followed by stimulation with proinflammatory cytokine, interleukin 1β (IL-1β). These conditions permit interpretations for both IRE1α expression and activation as IL-1β is a potent activator of ER stress. Our findings confirm IRE1α modulates astrocyte metabolic function, morphological activation, cytokine secretion, and glutamate clearance, highlighting a novel target for regulating astrocyte metabolic and inflammatory phenotypes. Therapeutic targeting of astrocyte IRE1α could help combat astrocyte-mediated neurotoxicity and potentially promote a more neuroprotective phenotype during CNS pathology.
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    Novel Kinase Inhibitors Library Screen Differentially Impacts Adipose Stem Cells (ASCs) from Lean and Obese Donors
    (2022) Rinderle, Caroline
    Purpose: Breast cancer is the second leading cause of death among women in the United States. Obesity increases the risk of developing breast cancer and ultimately leads to poorer outcomes. Obesity is defined as the excess accumulation of adipose tissue, evidenced by a BMI of 30 kg/m² or greater. Adipose tissue consists primarily of adipocytes, but the stromal vascular fraction (SVF) consists of numerous other cell types as well, including adipose-derived stem cells (ASCs). ASCs are self-renewing, multipotent, mesenchymal stem cells that have been intensely studied for their role in regenerative medicine. Our lab has shown that ASCs extracted from obese patients are recruited to breast tumors more than ASCs extracted from lean patients. This made breast cancer outcomes worse in our xenograft models, indicating a connection between ASCs in obesity and breast cancer. These changes in cancer behavior may be due to the activity of important protein kinases. Kinases are essential to cellular function, activating necessary proteins to propagate signal cascades, without which survival would be impossible. Little is known about the 538 kinases encoded in the human genome, and therefore, they need to be researched more thoroughly. If the obese ASC-breast tumor crosstalk can be interrupted via prevention of specific kinase activity, then poor breast cancer outcomes may be prevented and novel therapeutics can be uncovered. Methods: Pools of ASCs from lean and obese donors were treated 100nM of KCGS Drug Library kinase inhibitors obtained from Dr. David Drewry at the SGC at UNC Chapel Hill. After 72 hours, cells were stained with crystal violet and imaged for cellular viability and morphologic changes. Results: Fifteen kinase inhibitor drugs affected ASCs from both lean and obese donors. Nine kinase inhibitor drugs affected ASCs from obese donors only. No kinase inhibitors affected ASCs from lean donors alone. Conclusions: Obesity changes the biology of ASCs in a way that can potentially be therapeutically targeted. In the future, testing the ability of ASCs to differentiate into mature adipocytes after treatment with kinase inhibitors will give greater insight into the role specific kinases play in the biology of an obese patient. We will also study the roles of the kinase inhibitors and kinases in the biology of ASCs more thoroughly.