Pharmacy

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Research from the School of Pharmacy. The website may be found at https://www.ndsu.edu/pharmacy/

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Apelin-APJ Signaling in Hypertensive Coronary Arteries
(North Dakota State University, 2022) Anto, Santo Kalathingal
Apelin is an endogenous ligand for APJ receptors, which are highly expressed throughout the cardiovascular system, including coronary arteries. Apelin causes endothelium‐derived nitric oxide (NO) –dependent relaxation of coronary arteries under physiological conditions, but little is known about regulation of coronary vasomotor tone by apelin under pathological conditions. This research addresses the critical gap in understanding of the apelin signaling in coronary circulation under normal and pathological conditions. Evidence from this study suggests that apelin could not provide beneficial vasodilatory effects in hypertensive coronary arteries. Moreover, apelin impairs endothelium-dependent relaxations to vasodilator, acetylcholine. Impaired apelin-APJ signaling in hypertensive coronary arteries is possibly through defective production or release of NO from coronary endothelial cells rather than inhibiting effects of NO in coronary arterial smooth muscle cells. My next aim was to understand the mechanisms involved in the loss of apelin response in hypertensive arteries. The results suggested that the APJ receptor signaling via GRK2 pathway is possibly responsible for the impaired apelin response in hypertensive coronary arteries. Interestingly, APJ receptor biased agonist, CMF-019, -induced relaxation in hypertensive coronary arteries and showed no effects on vasodilatory response to acetylcholine. My results also suggest the possible impairment of PI3K/AKT/eNOS pathway mediated by GRK2 activation in hypertensive coronary arteries. My final aim was to check whether apelin signaling is impaired in coronary arteries only under hypertensive conditions or if it occurs in other disease conditions. My data suggests that apelin signaling is impaired in coronary arteries exposed to cigarette smoke extract (CSE), a model for secondhand smoke exposure. Interestingly, similar to hypertensive coronary arteries, apelin lost its beneficial vasodilatory effects possibly through the GRK2 activation in CSE treated coronary arteries. Overall, this research provides evidence that apelin behaves differently under physiological and pathological conditions. As a point of fact, apelin not only lost its beneficial effects but also might have negative effects under pathological conditions such as hypertension and secondhand smoke exposure. I anticipate that the results from this approach will be useful in improving the therapeutic strategies with apelin and other APJ receptor agonists that are aimed to alleviate different cardiovascular disorders.
BKCa-IP3R Decoupling in Hypertension
(North Dakota State University, 2022) Niloy, Sayeman Islam
Hypertension is a significant risk factor for cardiovascular diseases and a leading cause of worldwide morbidity and mortality. Dysregulation of intracellular Ca2+ in vascular smooth muscle (VSM) cells is one major contributor to the development of vascular hypercontractility and remodeling in hypertension. Plasma membrane (PM)-localized large-conductance, Ca2+-activated K+ (BKCa) channels prevent hypercontractility through membrane hyperpolarization in response to vasoconstrictor-induced activation of inositol trisphosphate receptors (IP3Rs), localized on the sarcoplasmic reticulum (SR). However, loss of close contact or coupling between BKCa and IP3R may diminish the BKCa-mediated protection against hypercontractility and hypertrophy and contribute to the development of hypertension. The overall goal of this study was to understand the role of BKCa-IP3R coupling in the development of vascular hypercontractility and remodeling. I used a hypertensive animal model, spontaneously hypertensive rat (SHR), to study the impact of the loss of this coupling. My hypothesis was that there is a loss of communication between the IP3 receptors and the BKCa channels in SHR VSM cells leading to reduced BKCa current after IP3R activation. My first objective was to determine the role of functional coupling of BKCa and IP3R in vascular hypercontractility and hypertrophy development. Based on the findings, one can conclude that in SHR mesenteric VSM cells, there is a loss of functional IP3R-BKCa coupling, and it might be involved in vascular hypercontractility and hypertrophy. My second objective was to examine and compare the molecular coupling of BKCa and IP3R between normotensive and hypertensive rats. My data suggest that the molecular connection between BKCa and IP3R is disrupted in SHR VSM cells. My results also suggest that this loss of connection is not due to downregulation of junctophilin-2 (JPH2) but may be due to defective tethering of JPH2 to the PM. Together, this research provides an improved understanding of the crucial roles played by BKCa-IP3R coupling in hypertension. An understanding of ion channel coupling under disease conditions may provide relevant caveats where BKCa channels are considered a therapeutic target. I expect that the knowledge gained from my studies will fundamentally advance the field of ion channel-based therapeutics, especially in cardiovascular disorders.
Development of Nano-Architecture Systems for High-Efficiency Tumor-Targeted Drug Delivery
(North Dakota State University, 2021) Mamnoon, Babak
Breast cancer is the most common malignancy and the second leading cause of death among women in the United States. The commonly used breast cancer treatment strategies include surgery, chemotherapy, radiation, and hormonal therapy. Since chemotherapeutic agents do not adequately differentiate between normal and cancerous cells, systemic toxicity and adverse effects associated with these anticancer drugs limit their therapeutic efficacy. In addition, uncontrolled cell proliferation and insufficient blood supply produce low oxygen partial pressure or hypoxia in almost all solid tumors. Hypoxia increases cancer cell survival through aggressiveness, metastasis, and resistance to chemotherapy, leading to poor clinical outcomes. Targeted drug delivery nanoparticles can significantly reduce off-target toxicity of chemotherapy by selectively targeting tumor tissues. Polymersomes are self-assembled drug-encapsulated polymeric nanoparticles in which an aqueous core is enclosed by a bilayer membrane. To attain the appropriate therapeutic efficacy, polymersomes need to rapidly release their anticancer drug at the tumor sites. To fulfill this requirement, stimuli-responsive polymersomes have been developed. Since most of the tumors have hypoxic areas inside, hypoxia-responsive polymersomes are one of the most effective drug delivery vehicles for cancer treatment. To prepare targeted drug delivery systems, functionalizing polymersomes with specific ligands intended to be recognized by the receptors of the cancer cells, is the most common strategy. Herein, we designed three distinct hypoxia-responsive polymersomes for targeting breast cancer tissues. More than 80% of breast cancers express estrogen receptor (ER-positive), and about 15-25% of them do not express any receptors (triple-negative). Hence, we decorated our polymersomes with three different ligands including estradiol and endoxifen for targeting ER-positive breast microtumors, and iRGD peptide for targeting triple-negative breast tumors.
Estrogen Receptors Signaling in Airway Smooth Muscles: Role in Intracellular Calcium Regulation and Contractility
(North Dakota State University, 2021) Sangeeta, Bhallamudi
Epidemiological evidence suggests higher incidence and severity of asthma in pre-menopausal women and aging men, suggesting a role of sex steroids, especially estrogen. In this regard, it is not clear whether specific estrogen receptors (ERα and ERβ) play differential roles or whether there is any imbalance in their normal signaling during asthma. Airway smooth muscle (ASM) cell is of contractile phenotype which is involved in contractions and airway hyperresponsiveness. Therefore, this research focused to understand ER signaling in the context of airway hyperresponsiveness and bridge the gaps in knowledge about the role of ERs in human ASM cells. The first aim demonstrate the long-term differential signaling of ERs in the regulation of [Ca2+]i handling in the human ASM. It was found that ERα activation increases the [Ca2+]i response in baseline conditions, while ERβ activation has neutral effect. Moreover, the differential signaling of ERs is more evident in asthma or inflammation where ERβ activation decreases the [Ca2+]i response in the presence of inflammation while ERα increases it. Further elucidation of the mechanisms of their signaling on [Ca2+]i suggests that ERβ results in decreased [Ca2+]i response through increased SERCA2 expression and function, inhibition of pathways involved in activating the voltage gated LTCC and maintenance of the morphology of mitochondria. In the next aim, it was found that ERβ causes a potentiation of the activity of β2-AR which leads to an increase in cAMP. Also, ERβ is found to be involved in dephosphorylation of contractile apparatus ultimately leading to bronchodilation. Presenting a contrasting picture, ERα causes an increase in pro-contractile machinery such as RhoA activity and phospho-MYPT leading to increased overall contractility in ASM cells. To confirm these in vitro findings in the presence of other structural and immune cells involved in inflammation, I have further evaluated the ex vivo and in vivo roles of ER signaling in airways. Interestingly, ERβ was found to be protective for the airways while ERα further aggravated the contractility of the airways. These novel findings of ER signaling in the context of contractile mechanisms of the airways can be utilized in designing novel therapeutics for bronchodilation.
Kisspeptins: Airway Remodeling in Asthma
(North Dakota State University, 2022) Borkar, Niyati
Asthma is a chronic respiratory disorder that affects people of all ages. Sex and gender disparity in asthma is recognized and suggests a modulatory role for sex steroids, particularly estrogen. However, there is a dichotomous role of estrogen in airway remodeling, making it unclear whether sex hormones are protective or detrimental in asthma and suggesting a need to explore mechanisms upstream or independent of estrogen. Kisspeptins (Kp) are a novel peptide existing upstream of sex steroids and function as a crucial regulator of puberty. Kp via KISS1R are further implicated in the sex steroid biogenesis via action on the gonadotrophins. Therefore, this research hypothesizes that kisspeptin (Kp)/KISS1R signaling serves this role, thereby regulating airway remodeling and airway hyperresponsiveness (AHR).Airway smooth muscle (ASM) is a key structural cell type that contributes to remodeling in asthma. In the first aim, I report novel data indicating that Kp and KISS1R are expressed in human airways, especially ASM, with lower expression in ASM from women compared with men and lower in patients with asthma compared with people without asthma. My second aim discusses the functional mechanisms of Kp/KISS1R signaling on majorly proliferation, and in part ECM deposition studies. Proliferation studies show that Kp-10, mitigates PDGF-induced ASM proliferation. Pharmacological inhibition and shRNA knockdown of KISS1R increased basal ASM proliferation, which was further amplified by PDGF. The anti-proliferative effect of Kp-10 in ASM was mediated by inhibition of MAPK/ERK/Akt pathways, with altered expression of PCNA, C/EBP-α, Ki-67, cyclin D1, and cyclin E leading to cell cycle arrest at G0/G1 phase. ECM studies show that administration of Kp-10 can mitigate PDGF- and TGFβ- induced increase in ECM remodeling gene and protein expression, such as collagens and fibronectins. To corroborate my in vitro findings, I have further performed in vivo studies utilizing Kp-10 (a cleaved peptide of parent kisspeptin) in mixed allergen-induced mouse models of asthma. I found that Kp-10 was able to mitigate asthma by significantly improving airway structural, morphological and lung function parameters. Overall, I demonstrate the importance of Kp/KISS1R signaling in the ASM as a potential therapeutic avenue to blunt remodeling in asthma.
Targeted Three-Way-Junction RNA Nanoparticle Reprogrammed Cyclooxygenase-2 Catalyzed Dihomo-ϒ-Linolenic Acid Peroxidation Pattern in Lung Cancer
(North Dakota State University, 2021) Pang, Lizhi
As the second most common cancer and the first leading cause of cancer deaths, lung cancer attracts much research attention. Since cyclooxygenase-2 (COX-2) is overexpressing in nearly 80% of lung cancer patients, COX-2 inhibitors may benefit cancer patients via breaking the COX-2/arachidonic acid (AA)/ prostaglandin E2 (PGE2) axis, which is highly relevant to cancer progression. However, the COX-2 inhibitors fail to improve survival of cancer patients in clinical studies. To effectively take advantage of COX-2 overexpression in lung cancer, in this study, we advanced a novel strategy, instead of direct COX-2 inhibition, redirecting COX-2 catalyzed dihomo-γ-linolenic acid peroxidation by knocking down delta-5-desaturase (D5D) in lung cancer cells. We found that the D5D siRNA knockdown could repress the AA and PGE2 formation in lung cancer cells. While a distant free radical byproduct, 8-hydroxyoctanoic acid (8-HOA) was derived from DGLA by COX-2. Both exogenous 8-HOA and DGLA-derived endogenous 8-HOA resulted in inhibition of proliferation, survival, migration but activation of apoptosis in lung cancer cells. We believe that the inhibitory effect of 8-HOA lung cancer is possibly due to histone deacetylase (HDAC) and YAP1/TAZ inhibition. Additionally, we demonstrated the synergistic effect between 8-HOA and first-line chemo cisplatin on lung cancer. To improve the efficiency of D5D inhibition, we used innovative RNA nanotechnology to specifically deliver D5D siRNA to lung cancer in vitro and in vivo. The D5D siRNA was harbored by 3 way-junction (3WJ) RNA nanoparticle along with epithelial cell adhesion molecule (EpCAM) aptamer as targeting module, and Alexa 647 as imaging module. By using lung cancer and normal lung epithelial cell models, we demonstrated that 3WJ-EpCAM-D5D siRNA nanoparticles could suppress lung cancer cell growth by promoting 8-HOA formation in a COX-2 dependent manner. The EpCAM aptamer ensured the specific in vivo delivery of RNA nanoparticles to lung tumors, avoiding damage to other tissues and toxic/off-target effect. 3WJ-EpCAM-D5D siRNA nanoparticle significantly inhibited xenograft tumor growth in nude mice by regulating apoptosis, metastasis, and proliferation. Overall, our strategy generated an effective and safer outcome and paved the road to COX-2-based precise medicine for lung cancer therapy.
Understanding the Role of Rage in Cell Adhesion
(North Dakota State University, 2022) Thiyagarajan, Swetha
The Receptor for Advanced Glycation Endproducts (RAGE) is a mammalian specific cell surface receptor. RAGE consists of three extracellular domains (V, C1, and C2), a transmembrane domain, and an intracellular cytoplasmic tail. RAGE has a significant role in human pathogenesis, including neurodegenerative diseases, diabetic complications, and certain cancers. Deregulation of cell adhesion is one of the contributing cellular events common in many of the above listed human pathologies and might be mediated via RAGE signaling. In our study, we aimed to understand the role of RAGE in cell adhesion and to define the importance of the different domains of RAGE in mediating this phenomenon. For this study, a protein engineering approach was used to express full-length RAGE (FL-RAGE) and a panel of domain deletion constructs ((ΔV-, ΔC1-, ΔC2-, DN-, TmCyto-) RAGE) of the receptor. The necessary expression constructs were assembled in the pcDNA3 vector, and the RAGE variants were expressed in HEK293 cells. The expression and cellular localization of RAGE in HEK 293 cells were analyzed using Western blot, immunofluorescence microscopy, and flow cytometry techniques. Our results show that the cytoplasmic domain of RAGE was sufficient to contribute to cell adhesion to the extracellular matrix to a level comparable to that of the FL-RAGE expressing cells. The current mechanistic model suggests that RAGE signaling is initiated by ligand binding to the extracellular region, followed by conformational changes in the intracellular domain. Subsequently, this conformation change leads to the recruitment of RAGE-interacting proteins on the intracellular side of the plasma membrane. However, in this thesis, we present evidence of an alternative mechanism of RAGE signaling possibly involving the translocation of RAGE into the nucleus. The results from our study suggest an alternative model for RAGE signaling and will help to better understand RAGE signaling in pathophysiological conditions. Our results could contribute to the development of new small molecule drugs targeting intracellular RAGE or the intracellular RAGE domain as a novel approach for inhibiting RAGE signaling.

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Browsing Pharmacy by browse.metadata.department "Pharmaceutical Sciences"