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Differential Expression Profile of Sphingosine-1-Phosphate Receptors in Human Brain Vascular Smooth Muscle Cells and Endothelial Cells Following Hypoxia Plus Glucose Deprivation

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Sphingosine-1-phosphate receptors (S1PRs) and their signaling pathways play an important role in mediating vascular health and function. Upon ligand mediated activation, S1PRs 1-5 couple with diverse heterotrimeric G-protein subunits (Gαi, Gαq/11, Gα12/13), initiating multimodal downstream signaling pathways which result in various physiological outcomes in the vasculature, including cell proliferation and…

Sphingosine-1-phosphate receptors (S1PRs) and their signaling pathways play an important role in mediating vascular health and function. Upon ligand mediated activation, S1PRs 1-5 couple with diverse heterotrimeric G-protein subunits (Gαi, Gαq/11, Gα12/13), initiating multimodal downstream signaling pathways which result in various physiological outcomes in the vasculature, including cell proliferation and migration, barrier integrity preservation or loss, contraction, and inflammation. Specifically, S1PR2 activation has been linked to endothelial activation, barrier integrity loss, and inflammation, whereas S1PR1 activation contributes to barrier integrity preservation, vasodilation, and anti-inflammatory properties. Although the role of S1PRs during pathophysiological conditions such as acute ischemic stroke is under current investigation, the complete S1PR expression profile in the cerebrovasculature following acute ischemic injury has not yet been investigated. Therefore, the present study was aimed to characterize the expression profiles of S1PRs 1-5 in human brain microvascular endothelial cells (HBMECs) and human brain vascular smooth muscle cells (HBVSMCs) following 3h hypoxia plus glucose deprivation (HGD; in vitro ischemic injury) exposure. At the mRNA level, we observed expression of S1PRs 1-5 in HBVSMCs and S1PRs 1-4 in HBMECs. Under basal conditions, we employed real-time RT-PCR and observed that mRNA levels of S1PR1 were highest in expression followed by S1PR3 then S1PR2 in HBMECs. On the other hand, S1PR3 mRNA was the highest followed by S1PR2 then S1PR1 in HBVSMCs. In HBMECs, HGD exposure increased S1PR1 mRNA and protein levels, but decreased S1PR1 mRNA in HBVSMCs. Similarly, HGD induced increased S1PR3 mRNA in HBMECs and decreased S1PR3 mRNA in HBVSMCs. For S1PR2, HGD did not alter mRNA or protein expression in HBMECs but increased mRNA levels in HBVSMCs. These data suggest that acute exposure to HGD appears to differentially regulate expression of S1PRs in HBMECs and HBVSMCs. The differential expression in S1PRs both basally and following HGD exposure may suggest distinct signaling mechanisms at play within the two cerebrovascular cell types, implicating these receptors as potential therapeutic targets following ischemic injury.
ContributorsEghrari, Nafis (Author) / Sweazea, Karen (Thesis director) / Gonzales, Rayna (Thesis director) / Wendt, Trevor (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of International Letters and Cultures (Contributor)
Created2022-05
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Assessing the Influence of Extracellular Mitochondria on Neuroinflammation

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A prominent aspect of Alzheimer’s disease (AD) is the presence of neuroinflammation is mediated by the activation of microglial cells, which are the immune cells in the central nervous system (CNS) that express an array of cytokines that may promote an inflammatory response. The main cytokines produced are: tumor…

A prominent aspect of Alzheimer’s disease (AD) is the presence of neuroinflammation is mediated by the activation of microglial cells, which are the immune cells in the central nervous system (CNS) that express an array of cytokines that may promote an inflammatory response. The main cytokines produced are: tumor necrosis factor-alpha (TNF-), interleukin-1β (IL-1β), and interleukin-6 (IL-6). The presence of these cytokines in the CNS may lead to neuronal death, to the production of toxic chemicals (such as nitric oxide), and to the generation of amyloid beta (a major pathological feature of AD). Previous studies have shown that modulation of the inflammatory response in the nervous system can potentially prevent and/or delay the onset of neurodegenerative diseases such as AD. Therefore, it is important to identify the process that induces CNS inflammation. For example, mitochondrial lysates have been found to produce an inflammatory response due to their ability to stimulate TNF-, Aβ, and APP mRNA [10]. Interestingly, extracellular mitochondria have been detected in the brain due to neurons degrading old mitochondria extracellularly. Therefore, we set out to study the effect of whole mitochondria isolated by differential centrifugation from human neuroblastoma cells (BE(2)-M17 cells) on the neuroinflammatory response in a human microglia model (THP-1 cells). Despite our best efforts, in the end it was unclear whether the mitochondrial fraction or other cellular components induced the inflammatory response we observed. Thus, further work with an improved mitochondrial isolation method should be carried out to address this issue.
ContributorsStokes, Laura Jean (Author) / DeCourt, Boris (Thesis director) / Sweazea, Karen (Committee member) / Gonzales, Rayna (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05