Browsing by Author "Hochstetler, Alexandra"

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    Activation of TRPV4 stimulates transepithelial ion flux in a porcine choroid plexus cell line
    (American Journal of Physiology, 2018-09-01) Preston, Daniel; Simpson, Stefanie; Halm, Dan; Hochstetler, Alexandra; Schwerk, Christian; Schroten, Horst; Blazer-Yost, Bonnie L.; Biology, School of Science
    The choroid plexus (CP) epithelium plays a major role in the production of cerebrospinal fluid (CSF). A polarized cell line, the porcine CP-Riems (PCP-R) line, which exhibits many of the characteristics of the native epithelium, was used to study the effect of activation of the transient receptor potential vanilloid 4 (TRPV4) cation channel found in the PCP-R cells as well as in the native epithelium. Ussing-style electrophysiological experiments showed that activation of TRPV4 with a specific agonist, GSK1016790A, resulted in an immediate increase in both transepithelial ion flux and conductance. These changes were inhibited by either of two distinct antagonists, HC067047 or RN1734. The change in conductance was reversible and did not involve disruption of epithelial junctional complexes. Activation of TRPV4 results in Ca2+ influx, therefore, we examined whether the electrophysiological changes were the result of secondary activation of Ca2+-sensitive channels. PCP-R cells contain two Ca2+-activated K+ channels, the small conductance 2 (SK2) and the intermediate conductance (IK) channels. Based on inhibitor studies, the former is not involved in the TRPV4-mediated electrophysiological changes whereas one of the three isoforms of the IK channel (KCNN4c) may play a role in the apical secretion of K+. Blocking the activity of this IK isoform with TRAM34 inhibited the TRPV4-mediated change in net transepithelial ion flux and the increased conductance. These studies implicate TRPV4 as a hub protein in the control of CSF production through stimulation by multiple effectors resulting in transepithelial ion and subsequent water movement.
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    Characterization of TRPV4-mediated signaling pathways in an optimized human choroid plexus epithelial cell line
    (APS, 2022-12) Hulme, Louise; Hochstetler, Alexandra; Schwerk, Christian; Schroten, Horst; Ishikawa, Hiroshi; Tung, Chun-Yu; Perrin, Benjamin; Blazer-Yost, Bonnie; Biology, School of Science
    The objectives of these studies were twofold: 1) to characterize the human choroid plexus papilloma (HIBCPP) cell line as a model of the blood-cerebrospinal fluid barrier (BCSFB) via morphology, tightness, and polarization of transporters in choroid plexus epithelia (CPe), and 2) to utilize Ussing-style electrophysiology to elucidate signaling pathways associated with the activation of the transient receptor potential vanilloid 4 (TRPV4) channel involved in cerebrospinal fluid (CSF) secretion. RT-PCR was implemented to determine gene expression of cell fate markers, junctional complex proteins, and transporters of interest. Scanning electron microscopy and confocal three-dimensional renderings of cultures grown on permeable supports were utilized to delineate the morphology of the brush border, junctional complexes, and polarization of key transporters. Electrophysiology was used to understand and explore TRPV4-mediated signaling in the HIBCPP cell line, considering both short-circuit current (Isc) and conductance responses. HIBCPP cells grown under optimized culture conditions exhibited minimal multilayering, developed an intermediate resistance monolayer, retained differentiation properties, and expressed, and correctly localized, junctional proteins and native transporters. We found that activation of TRPV4 resulted in a robust, multiphasic change in electrogenic ion flux and increase in conductance accompanied by substantial fluid secretion. This response appears to be modulated by a number of different effectors, implicating phospholipase C (PLC), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K) in TRPV4-mediated ion flux. The HIBCPP cell line is a representative model of the human BCSFB, which can be utilized for studies of transporter function, intracellular signaling, and regulation of CSF production.
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    A distinct transcriptional profile in response to endothelial monocyte activating polypeptide II is partially mediated by JAK-STAT3 in murine macrophages
    (American Physiological Society, 2019-09-01) Lee, Daniel D.; Hochstetler, Alexandra; Murphy, Christina; Lowe, Chinn-Woan; Schwarz, Margaret A.; Pediatrics, School of Medicine
    Macrophages are important responders to environmental changes such as secreted factors. Among the secreted factors in injured tissues, the highly conserved endothelial monocyte activating polypeptide II (EMAP II) has been characterized to limit vessel formation, to be locally expressed near sites of injury labeling it a “find-me” signal, and to recruit macrophages and neutrophils. The molecular mechanisms mediated by EMAP II within macrophages once they are recruited are unknown. In this study, using a model of partially activated, recruited thioglycollate-elicited peritoneal macrophages, a transient, transcription profile of key functional genes in macrophages exposed to EMAP II was characterized. We found that EMAP II-mediated changes were elicited mainly through signal transducer and activator of transcription 3 (STAT3) as evidenced by increased Y705 phosphorylation and changes in activity and upstream of it, Janus associated kinase (JAK)1/2 upstream. Both inhibition of JAK1/2 and knockdown of Stat3 abrogated a subset of genes that are upregulated by EMAP II. Our results identify a rapid EMAP II-mediated STAT3 activation that coincides with altered pro- and anti-inflammatory gene expression in macrophages.
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    Influence of aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 on epithelial differentiation and organization during lung development
    (American Physiological Society, 2020-08) Lee, Daniel D.; Hochstetler, Alexandra; Sah, Eric; Xu, Haiming; Lowe, Chinn-Woan; Santiaguel, Sara; Thornton, Janet Lea; Pajakowski, Adam; Schwarz, Margaret A.; Anatomy and Cell Biology, School of Medicine
    Proper development of the respiratory bronchiole and alveolar epithelium proceeds through coordinated cross talk between the interface of epithelium and neighboring mesenchyme. Signals that facilitate and coordinate the cross talk as the bronchial forming canalicular stage transitions to construction of air-exchanging capillary-alveoli niche in the alveolar stage are poorly understood. Expressed within this decisive region, levels of aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1) inversely correlate with the maturation of the lung. The present study addresses the role of AIMP1 in lung development through the generation and characterization of Aimp1−/− mutant mice. Mating of Aimp1+/− produced offspring in expected Mendelian ratios throughout embryonic development. However, newborn Aimp1−/− pups exhibited neonatal lethality with mild cyanosis. Imaging both structure and ultrastructure of Aimp1−/− lungs showed disorganized bronchial epithelium, decreased type I but not type II cell differentiation, increased distal vessels, and disruption of E-cadherin deposition in cell-cell junctions. Supporting the in vivo findings of disrupted epithelial cell-cell junctions, in vitro biochemical experiments show that a portion of AIMP1 binds to phosphoinositides, the lipid anchor of proteins that have a fundamental role in both cellular membrane and actin cytoskeleton organization; a dramatic disruption in F-actin cytoskeleton was observed in Aimp1−/− mouse embryonic fibroblasts. Such observed structural defects may lead to disrupted cell-cell boundaries. Together, these results suggest a requirement of AIMP1 in epithelial cell differentiation in proper lung development.
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    Inhibition of serum- and glucocorticoid-induced kinase 1 ameliorates hydrocephalus in preclinical models
    (BMC, 2023-08-18) Hochstetler, Alexandra; Smith, Hillary; Reed, Makenna; Hulme, Louise; Territo, Paul; Bedwell, Amanda; Persohn, Scott; Perrotti, Nicola; D’Antona, Lucia; Musumeci, Francesca; Schenone, Silvia; Blazer‑Yost, Bonnie L.; Biology, School of Science
    Background: Hydrocephalus is a pathological accumulation of cerebrospinal fluid (CSF), leading to ventriculomegaly. Hydrocephalus may be primary or secondary to traumatic brain injury, infection, or intracranial hemorrhage. Regardless of cause, current treatment involves surgery to drain the excess CSF. Importantly, there are no long-term, effective pharmaceutical treatments and this represents a clinically unmet need. Many forms of hydrocephalus involve dysregulation in water and electrolyte homeostasis, making this an attractive, druggable target. Methods: In vitro, a combination of electrophysiological and fluid flux assays was used to elucidate secretory transepithelial electrolyte and fluid flux in a human cell culture model of the choroid plexus epithelium and to determine the involvement of serum-, glucocorticoid-induced kinase 1 (SGK1). In vivo, MRI studies were performed in a genetic rat model of hydrocephalus to determine effects of inhibition of SGK1 with a novel inhibitor, SI113. Results: In the cultured cell line, SI113 reduced secretory transepithelial electrolyte and fluid flux. In vivo, SI113 blocks the development of hydrocephalus with no effect on ventricular size of wild-type animals and no overt toxic effects. Mechanistically, the development of hydrocephalus in the rat model involves an increase in activated, phosphorylated SGK1 with no change in the total amount of SGK1. SI113 inhibits phosphorylation with no changes in total SGK1 levels in the choroid plexus epithelium. Conclusion: These data provide a strong preclinical basis for the use of SGK1 inhibitors in the treatment of hydrocephalus.
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    Porcine choroid plexus-Riems cell line demonstrates altered polarization of transport proteins compared with the native epithelium
    (American Physiological Society, 2022) Hochstetler, Alexandra; Hulme, Louise; Delpire, Eric; Schwerk, Christian; Schroten, Horst; Preston, Daniel; Simpson, Stefanie; Blazer-Yost, Bonnie L.; Biology, School of Science
    The choroid plexus epithelium (CPe) forms a barrier between the cerebral blood supply and the cerebrospinal fluid (CSF), establishing the blood-CSF barrier (BCSFB). CSF is actively secreted by the CPe via tightly controlled processes involving multiple channels, transporters, and pumps. The importance of controlling CSF production and composition has been accentuated recently with an appreciation of CSF dysfunction in many pathologies. For mechanistic studies of CSF production, isolated CPe cell lines are valuable for the testing of hypotheses and potential drug targets. Although several continuous CPe cell lines have been described, none appear to have all the characteristics of the native epithelium and each must be used judiciously. The porcine choroid plexus-Riems (PCP-R) cell line forms a high-resistance monolayer characteristic of a barrier epithelium. Conservation of this phenotype is unusual among CPe cell lines, making this model useful for studies of the effects of infection, injury, and drugs on permeability. We have recently discovered that, although this line expresses many of the transporters expressed in the native tissue, some are mispolarized. As a result, inferences regarding fluid/electrolyte flux and the resultant CSF production should be pursued with caution. Furthermore, extended culture periods and changes in media composition result in significant morphological and functional variability. These studies provide a more detailed characterization of the PCP-R cell line concerning transporter expression, polarization, and functionality, as well as plasticity in culture, with the goal to provide the scientific community with information necessary to optimize future experiments with this model.
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    Role of Choroid Plexus TRPV4 Channel in Health and Disease
    (2022-08) Hochstetler, Alexandra; Blazer-Yost, Bonnie L; Berbari, Nicolas; Baucum II, AJ; Roper, Randall; Raskin, Jeffrey
    Pediatric hydrocephalus is a complex neurological condition associated with a pathological accumulation of cerebrospinal fluid (CSF), typically within the brain ventricular system. Pediatric hydrocephalus can be primary (due to genetic abnormalities or idiopathic causes), or secondary to injuries such as hemorrhage, trauma, or infection. The current permanent treatment paradigms for pediatric hydrocephalus are exclusively surgical and include the diversion of CSF via shunt or ventriculostomy. These surgical interventions are wrought with failures, burdening both the United States healthcare system and patients with repeat neurosurgical procedures. Thus, the development of nonsurgical interventions to treat hydrocephalus represents a clinically unmet need. To study hydrocephalus, we use a genetic rat model of primary neonatal hydrocephalus, the Tmem67P394L mutant. In several proof-of-concept studies, we identify antagonism of the transient receptor potential vanilloid 4 (TRPV4) channel and associated upstream regulatory kinase, serum-andglucocorticoid-induced kinase 1 (SGK1) as therapeutics for the treatment of hydrocephalus. Using in vitro models of the choroid plexus epithelium, the tissue which produces CSF, we show compelling proof-of-mechanism for TRPV4 antagonism and SGK1 inhibition at preventing CSF production. Therefore, the studies in this dissertation provide substantive evidence on the role of TRPV4 in the choroid plexus in health and disease.