Pharmacology and Toxicology
Permanent URI for this collectionhttp://hdl.handle.net/10342/99
Browse
Recent Submissions
Item Embargo Kininase I Mediated Signaling Evidenced in Hypertension and Target Organ Damage(East Carolina University, May 2025) White, Acacia Mahogany NecoleCarboxypeptidase N (CPN) and Carboxypeptidase M (CPM) are isoforms of kininase I, with CPN restricted to the plasma and CPM located on the plasma membrane. Kininase I is a critical pleiotropic regulator of inflammation, as it cleaves several inflammatory mediators such as anaphylatoxins, creatine kinase MM, stromal cell-derived factor-1alpha and kinins. Kinins are important vasoactive peptides which activate the kinin B2 receptor (B2R), and cause vasodilation. However, kininase I converts these kinins into agonists for the kinin B1 receptor (B1R), a receptor associated with vasoconstriction and hypertension. However, the role of kininase I in hypertension has not been identified. Male and female carboxypeptidase N1 (Cpn1) whole-body gene knockout (CPNKO) mice were utilized to investigate the role of CPN in the development of angiotensin II (Ang II)-induced hypertension and target organ damage. CPNKO and wild-type (WT) mice were implanted with radiotelemetry probes to measure blood pressure, and later with mini osmotic pumps to infuse mice with either saline or angiotensin II (Ang II; 600/ng/kg/min -1) for 4 weeks. In a separate experiment, we used tail cuff plethysmography to measure blood pressure in WT male and female mice infused with either saline or a combined dosage of the non-selective kininase I pharmacological inhibitor (Mergetpa; 10 mg/day) and Ang II (600/ng/kg/min -1) over 4 weeks. Aortic segments isolated from these mice were subjected to wire myography, to assess the effect of CPN gene deletion on vascular function. The activity and expression of kininase I and B1R were analyzed using various protein detection techniques in the brain, heart, and aorta. Proximity ligation assays were utilized to identify alterations in protein-protein interactions between B1R and other protein partners, such as CPM and aminopeptidase N (CD13), in the hypothalamic paraventricular nucleus (PVN) of WT and CPNKO mice. CPN activity assays revealed that overstimulation with Ang II in WT mice promoted CPN activity in plasma. Interestingly, Ang II infusions did not cause a significant modulation in CPM expression specifically in the PVN, heart, and aorta of CPNKO mice, compared to saline treated CPNKO and WT mice. Alternatively, CPM expression was robustly increased following Ang II infusions in WT mouse PVN, heart and aortic tissues. Mean arterial pressure (MAP), measured using radiotelemetry, was not significantly different in saline treated CPNKO and WT mice in both sexes. However, MAP was significantly elevated in WT mice infused with Ang II, but this effect was attenuated in Ang II infused CPNKO mice. Furthermore, the expression of B1R in the brain, heart and aorta was significantly enhanced by Ang II in WT mice compared to CPNKO counterparts. Based on the extent of hypertrophy and picrosirius red staining observed in Ang II infused WT and CPNKO mice, the genetic deletion of CPN protects against cardiac hypertrophy and fibrosis caused by Ang II in both sexes. Interestingly, markers associated with inflammation (IL-1β, IL-6, MCP-1/CCL2, and TNF) and oxidative stress (superoxide, 3-nitrotyrosine) were also found elevated in the heart, aorta, and PVN of Ang II infused WT mice, and these outcomes were mitigated in Ang II infused CPNKO counterparts. Furthermore, the expression CD13 in the PVN was enhanced following Ang II infusion in WT mice, while CPNKO exhibited significantly lower levels of CD13 expression in the PVN. Astonishingly, proximity ligation assays revealed that protein-protein interactions between both CPM and B1R, as well as CD13 and B1R were heightened in WT mice infused with Ang II but diminished in CPN deficient mice. Mergetpa, the non-selective kininase I inhibitor, was associated with a reduction in MAP and the expression of CPM and B1R in the PVN of WT mice, compared to WT mice infused with Ang II alone. Lastly, vascular function was assessed using wire myography revealed that male Ang II infused CPNKO and Mergetpa treated mice have preserved endothelial dependent relaxation mechanisms, compared to WT mice overstimulated with Ang II. Based on our results, kininase I blockade prevented Ang II induced hypertension and targeted end-organ damage by mitigating B1R activation. B1R mediates inflammation and oxidation and CPM/B1R or CD13/B1R protein-protein interactions may promote Ang II-induced hypertension. Specifically, the pro-inflammatory effects of kininase I initiate B1R activation, leading to the generation of reactive oxygen species (ROS) and subsequent immune cell infiltration/activation and endothelial dysfunction. Alternatively, we suggest that B1R activation mediated by CD13 signaling may contribute to Ang II-induced inflammation and fibrosis, to a lesser extent. Within the context of this study, we did not identify any clear potential sex differences associated with kininase I blockade, in WT and CPNKO mice. We have evidenced for the first time that kininase I mediated B1R activation is correlated with pathogenesis of Ang II and may serve as a potential therapeutic target for the treatment of an overactive RAAS in hypertension.Item Embargo DEVELOPMENTAL NEUROTOXIC EFFECTS OF SELECT PER- AND POLYFLUOROALKYL SUBSTANCES (PFAS) IN A MOUSE MODEL(East Carolina University, July 2024) Trowse, Kellyn AmberConnections between the nervous and immune systems, particularly those formed during development, can be perturbed by exogenous agents such as per- and polyfluoroalkyl substances (PFAS). PFAS have been used extensively in industrial and consumer products to resist heat, oil, and water. Their production and use have led to widespread environmental contamination, with a notable occurrence in the Cape Fear River in North Carolina of the United States (U.S.). Many PFAS studied to date have been implicated in adverse health effects in humans, including increased risks of some cancers, weakened immune response, liver damage, and developmental effects. Despite recent regulatory efforts for some PFAS, gaps remain in knowledge about less well studied PFAS, especially those found in the Cape Fear River and with respect to neuroimmune effects. In this study, a series of experiments to investigate developmental neuroimmune effects of PFO5-DoA (0, 0.005, 0.05, or 0.5 mg/kg) or GenX (0, 1, 5, 10 mg/kg) in B6C3F1 mice exposed from gestational day one through 17 were conducted. A homing behavior test was conducted on postnatal day (PND) 10 in GenX pups and tissue samples were collected at PND 21 and PND 56. Brain sections collected from the prefrontal/frontal cortex were stained with ionized calcium binding adaptor molecule 1 (IBA1) to visualize microglial numbers and percent area covered brain and interleukin 1 beta (IL-1) to estimate pro-inflammatory cytokine staining intensity. Average brain weights and relative brain and body weights were not statistically different for either PFO5-DoA or GenX groups. In the homing behavior test, pups exposed to 5 or 10 mg/kg had less success in finding home bedding squares than counterparts exposed to 0 or 1 mg/kg. In PND21 brains, the average number of microglia and percent area covered by microglia in mice exposed to 0.05 mg/kg of PFO5DoA were significantly decreased compared to control mice. Additionally, the average percent area covered by IL-1 staining for mice exposed to 0.05 mg/kg of PFO5DoA were significantly decreased in both PND21 and PND56 brains. Overall, while this study did not find statistically significant developmental neuroimmune effects of GenX or PFO5DoA, the potential for these PFAS to affect neuroimmune development cannot be ruled out as effects may be long-term, affected by sex hormones, and/or occur outside of the 56-day experimental lifespan of this study. Further research is needed to analyze differences between microglial abundance and phenotype early in life (from PND0-30) compared to late in life (PND60-112).Item Open Access Molecular Mechanisms Underlying Cannabidiol-Improved Vocal Recovery Following Damage to a Songbird Vocal Pre-Motor Cortical-Like Region(East Carolina University, 2023-04-27) Tripson, Mark AndrewThe non-euphorigenic phytocannabinoid CBD has been found to be effective in treating childhood-onset epilepsies, conditions that are often associated with developmental delays, including vocal communication. Zebra finch song is a complex behavior that is learned during a sensitive period of vocal development, making it a promising model for understanding the mechanisms responsible for potential CBD-related improvements in vocal learning. Like language, the quality of adult zebra finch song is maintained through continuous sensorimotor maintenance and refinement, involving brain regions that control vocal learning and production. One of these brain regions, HVC, is a pre-vocal motor cortical-like region that when partially lesioned temporarily disrupts vocal behavior. Recovery from HVC microlesions typically takes about seven days without treatment. However, treatment with CBD has been shown to both speed recovery and reduce the acute magnitude of disruptions. Given the anti-inflammatory properties of CBD in seizure and other models, we suspected involvement of similar mechanisms in vocal recovery. To test this, we investigated CBD modulation of post-lesion expression of inflammatory cytokines, markers of neuronal stress, microglial migration, and changes in synaptic densities within relevant song control regions. Results indicate that CBD-improved vocal recovery is associated with reduced oxidative stress and anti-inflammatory activity. This decrease in inflammation and stress marker expression was associated with reduced density of microglia staining within song regions afferent to the lesion target, HVC (including learning-essential Area X [basal ganglia] and vocal motor RA [motor cortex]). Furthermore, we measured densities of excitatory synapses within Area X and RA, finding significant lesion-related decreases that were largely reversed by CBD. This synaptic protection was associated with BDNF/Arc/MSK1 upregulation, implicating mechanisms important to homeostatic synaptic scaling. Overall, this work indicates that CBD improves post-CNS damage recovery of learned vocal behavior by promoting multiple homeostatic mechanisms. This efficacy may generalize to sensorimotor skills learned by other vertebrates and suggests potential application to TBI and related disorders.Item Open Access Grouping of PFAS for Human Health Risk Assessment: Findings from an Independent Panel of Experts(2022-07-08) DeWitt, J.; Anderson, J.K.; et alItem Restricted Examining the therapeutic potential of the investigational agent, 15dPMJ2, in mismatch repair proficient colon cancer(East Carolina University, 2022-08-15) Myers, ArielDespite the success of immune checkpoint inhibitor (ICI) therapy in many cancers, colon cancer with low microsatellite instability (MSI-L), also known as proficient mismatch repair (pMMR), does not respond to these agents. This colon cancer subtype is characterized by low neoantigen production and immune cell infiltration, which results in suboptimal reactivity to ICI therapy. This highlights the need for therapeutics that increase the immunogenicity of pMMR colon cancers. Accumulating evidence suggests that the damage associated molecular pattern (DAMP) signaling pathway is a promising target for the development of these types of immunostimulatory therapeutics. DAMP signals, including the cell surface expression of calreticulin and the extracellular release of ATP, have been shown to increase tumor immunogenicity and stimulate an antitumor immune response. It is also becoming clear that inhibitory DAMPs (iDAMPs), which activate immunosuppressive cell types, also play a role in the ultimate outcome of DAMP activation. These iDAMPs also act on tumorigenic cells to promote proliferation and tumor progression. Thus, the present study sought to investigate the role of stimulatory and inhibitory DAMPs on the anti-tumor activity of the investigational agent, 15-deoxy-Δ12,14-Prostaglandin Ethanolamide J2 (15dPMJ2), in pMMR colon cancer. The results from our in vivo and in vitro experiments show that 15dPMJ2 was cytotoxic in murine CT26 cells, which are a model of pMMR colon cancer. 15dPMJ2 also increased ER stress and apoptosis, two processes needed for stimulatory DAMP expression. In addition, 15dPMJ2 increased cell surface expression of anti-tumor DAMPs: calreticulin and extracellular release of ATP. 15dPMJ2 also increased the expression of the iDAMP and proliferative signal, prostaglandin E2 (PGE2). However, PGE2 had no effect on the cytotoxic activity of 15dPMJ2. These results indicate the cytotoxicity of 15dPMJ2 against pMMR colon cancer was independent of the pro-proliferative effects of PGE2.Item Restricted Kinin B1 Receptor Mediated Mechanisms in Hypertension(East Carolina University, 2022-08-15) Parekh, Rohan UHypertension continues to cause a burden on the global population as there are growing numbers of patients who present with treatment resistant hypertension. The etiology of hypertension is considered complex and multifactorial and is often difficult to treat even with a myriad of antihypertensive therapeutics. Thus, without novel approaches to combat treatment-resistant forms of hypertension our ability to curtail this burden on the health care system will remain somewhat limited. Evidence has suggested that increased inflammation and oxidative stress, within specific cardiovascular regulatory brain regions such as the paraventricular nucleus (PVN) of the hypothalamus, can result in sympathetic overactivation, a driving force in patient populations with neurogenic hypertension, a form of resistant hypertension. Unfortunately, if blood pressure remains uncontrolled, organ damage can ensue. Our laboratory previously showed increased kinin activity in the PVN in hypertension, however the mechanisms are still not fully understood. Kinin B1 (B1R) receptor activation has been shown to lead to increased proinflammatory and vasoconstrictive effects. Previous studies investigating the therapeutic potential of B1 receptor blockade have provided evidence of its beneficial effects in chronic inflammatory conditions but has not been fully studied in the context of hypertension. Our hypothesis is that B1R blockade reduces blood pressure, inflammation, and end organ damage in angiotensin II-induced hypertension. To explore the role of B1R, we used an in vivo Ang II infusion model coupled with in vitro primary hypothalamic neuron cultures. In the in vitro model we measured the levels of B1R expression, markers of inflammation and oxidative stress, and components of the Renin-Angiotensin System (RAS). In the in vivo model we measured B1R expression, blood pressure, levels of endogenous B1R agonist, proinflammatory cytokines, markers of target organ damage, and interactions between B1R and Angiotensin II type I receptor. Both the in vitro and in vivo findings supported B1R blockade using either a pharmacological antagonist or using genetic deletion was about to attenuate Ang II-induced inflammation, oxidative stress, end organ damage and hypertension. These studies solidify the interaction between the RAS and Kallikrein-Kinin System, specifically through interactions of B1R and AT1R through Ang II. Overall, the findings of these studies establish a critical role for hypothalamic B1 receptor in neurogenic hypertension and its potential as a novel therapeutic target.Item Open Access Kinin B1R Activation Induces Endoplasmic Reticulum Stress in Primary Hypothalamic Neurons(2022-03-08) White, Acacia; Parekh, Rohan Umesh; Theobald, Drew; Pakala, Pranaya; Myers, Ariel Lynn; Van Dross, Rukiyah; Sriramula, SrinivasItem Open Access Kinin B1 Receptor Mediates Renal Injury and Remodeling in Hypertension(2022-01-18) Basuli, Debargha; Parekh, Rohan Umesh; White, Acacia; Sriramula, Srinivas; Thayyil, AbdullahItem Open Access Invited Perspective: Key Characteristics as a Starting Point for Improved Hazard Identification of Immunotoxic Agents(2022-10-06) DeWitt, Jamie C.; Walker, Lauren M.Item Open Access The Actin Bundling Protein Fascin‑1 as an ACE2‑Accessory Protein(2022-08-31) Sriramula, Srinivas; Ogunlade, Blessing; Guidry, Jessie J.; Mukerjee, Snigdha; Lazartigues, Eric; Filipeanu, Catalin M.Item Open Access Scavenger Receptor BI Attenuates IL-17A–Dependent Neutrophilic Inflammation in Asthma(2021-06) Reece, Sky W.; Kilburg-Basnyat, Brita; Hodge, Myles X.; Luo, Bin; Gowdy, Kymberly M.; Varikuti, Sanjay; Dunigan-Russell, Katelyn; Madenspacher, Jennifer H.; Thomas, Seddon Y.; Tokarz, Debra A.; Tighe, Robert M.; Cook, Donald N.; Fessler, Michael B.Item Open Access CB1 Antagonism Increases Excitatory Synaptogenesis in a Cortical Spheroid Model of Fetal Brain Development(2021) Papariello, Alexis; Taylor, David; Soderstrom, Ken; Litwa, KarenItem Open Access Hypothalamic Kinin B1 Receptor Mediates Orexin System Hyperactivity in Neurogenic Hypertension(2021) Parekh, Rohan Umesh; White, Acacia; Lefer, Korin E.; Abdel-Rahman, Abdel A.; Sriramula, Srinivas; Biancardi, Vinicia C.; Eells, Jeffrey B.Item Open Access Damage-Associated Molecular Pattern (DAMP) Activation in Melanoma: Investigation of the Immunogenic Activity of 15-Deoxy, Δ12,14 Prostamide J2(2021) Elhassanny, Ahmed; Escobedo, Rene; Van Dross, Rukiyah; Ladin, Daniel; Burns, Colin SandersonItem Restricted Examining the therapeutic potential of the investigational agent, 15dPMJ2, in mismatch repair proficient colon cancer(East Carolina University, 2022-08-15) Myers, ArielDespite the success of immune checkpoint inhibitor (ICI) therapy in many cancers, colon cancer with low microsatellite instability (MSI-L), also known as proficient mismatch repair (pMMR), does not respond to these agents. This colon cancer subtype is characterized by low neoantigen production and immune cell infiltration, which results in suboptimal reactivity to ICI therapy. This highlights the need for therapeutics that increase the immunogenicity of pMMR colon cancers. Accumulating evidence suggests that the damage associated molecular pattern (DAMP) signaling pathway is a promising target for the development of these types of immunostimulatory therapeutics. DAMP signals, including the cell surface expression of calreticulin and the extracellular release of ATP, have been shown to increase tumor immunogenicity and stimulate an antitumor immune response. It is also becoming clear that inhibitory DAMPs (iDAMPs), which activate immunosuppressive cell types, also play a role in the ultimate outcome of DAMP activation. These iDAMPs also act on tumorigenic cells to promote proliferation and tumor progression. Thus, the present study sought to investigate the role of stimulatory and inhibitory DAMPs on the anti-tumor activity of the investigational agent, 15-deoxy-Δ12,14-Prostaglandin Ethanolamide J2 (15dPMJ2), in pMMR colon cancer. The results from our in vivo and in vitro experiments show that 15dPMJ2 was cytotoxic in murine CT26 cells, which are a model of pMMR colon cancer. 15dPMJ2 also increased ER stress and apoptosis, two processes needed for stimulatory DAMP expression. In addition, 15dPMJ2 increased cell surface expression of anti-tumor DAMPs: calreticulin and extracellular release of ATP. 15dPMJ2 also increased the expression of the iDAMP and proliferative signal, prostaglandin E2 (PGE2). However, PGE2 had no effect on the cytotoxic activity of 15dPMJ2. These results indicate the cytotoxicity of 15dPMJ2 against pMMR colon cancer was independent of the pro-proliferative effects of PGE2.Item Restricted Kinin B1 Receptor Mediated Mechanisms in Hypertension(East Carolina University, 2022-08-15) Parekh, Rohan UmeshHypertension continues to cause a burden on the global population as there are growing numbers of patients who present with treatment resistant hypertension. The etiology of hypertension is considered complex and multifactorial and is often difficult to treat even with a myriad of antihypertensive therapeutics. Thus, without novel approaches to combat treatment-resistant forms of hypertension our ability to curtail this burden on the health care system will remain somewhat limited. Evidence has suggested that increased inflammation and oxidative stress, within specific cardiovascular regulatory brain regions such as the paraventricular nucleus (PVN) of the hypothalamus, can result in sympathetic overactivation, a driving force in patient populations with neurogenic hypertension, a form of resistant hypertension. Unfortunately, if blood pressure remains uncontrolled, organ damage can ensue. Our laboratory previously showed increased kinin activity in the PVN in hypertension, however the mechanisms are still not fully understood. Kinin B1 (B1R) receptor activation has been shown to lead to increased proinflammatory and vasoconstrictive effects. Previous studies investigating the therapeutic potential of B1 receptor blockade have provided evidence of its beneficial effects in chronic inflammatory conditions but has not been fully studied in the context of hypertension. Our hypothesis is that B1R blockade reduces blood pressure, inflammation, and end organ damage in angiotensin II-induced hypertension. To explore the role of B1R, we used an in vivo Ang II infusion model coupled with in vitro primary hypothalamic neuron cultures. In the in vitro model we measured the levels of B1R expression, markers of inflammation and oxidative stress, and components of the Renin-Angiotensin System (RAS). In the in vivo model we measured B1R expression, blood pressure, levels of endogenous B1R agonist, proinflammatory cytokines, markers of target organ damage, and interactions between B1R and Angiotensin II type I receptor. Both the in vitro and in vivo findings supported B1R blockade using either a pharmacological antagonist or using genetic deletion was about to attenuate Ang II-induced inflammation, oxidative stress, end organ damage and hypertension. These studies solidify the interaction between the RAS and Kallikrein-Kinin System, specifically through interactions of B1R and AT1R through Ang II. Overall, the findings of these studies establish a critical role for hypothalamic B1 receptor in neurogenic hypertension and its potential as a novel therapeutic target.Item Open Access Activation of Kinin B1R Upregulates ADAM17 and Results in ACE2 Shedding in Neurons(2021) Parekh, Rohan Umesh; Sriramula, SrinivasItem Open Access Prohibitin-1 Is a Dynamically Regulated Blood Protein with Cardioprotective Effects in Sepsis(2021) Mattox, Taylor A.; Psaltis, Christine; Robidoux, Jacques; Kilburg-Basnyat, Brita; Gowdy, Kymberly M.; Weihbrecht, Katie; Murphy, Michael P.; Anderson, EthanItem Open Access Aldehyde Dehydrogenase Inhibition Ameliorates Cardiac Dysfunction and Exacerbates Hypotension Caused by Alcohol in Female Rats(2020-11-24) Abdel-Rahman, Abdel A.Item Open Access Estrogen Receptor ERα Plays a Major Role in Ethanol-Evoked Myocardial Oxidative Stress and Dysfunction in Conscious Female Rats(2016-02-01) Abdel-Rahman, Abdel A.