Department of Biological Sciences
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Item Regulation of Oxidative Stress by Nitric Oxide Defines Lung Development and Diseases(Springer, 2019-11-24) Majumder, SyamantakDevelopment and maturation of the lung airways primarily take place in two different phases: first during embryonic days and second during postnatal days. During postnatal development, rapid angiogenesis and alveolarization are necessary to attain the capacity of the lung to support the need of the baby. During lung development, alteration in ROS level may significantly compromise maturation of the alveolar structure. We have employed a unique approach to achieve alteration in ROS level in the chick embryos to ascertain ROS function in early lung development. We have used a known ROS quenching nitric oxide (NO) donor and a ROS inducer called thalidomide, a known teratogen. Using next-generation high-throughput sequencing (NGS) analysis, we have performed the transcriptomic analysis of the NO- and thalidomide-treated chick embryos. Using STRING database, we have identified a set of lung-associated developmental genes that were significantly altered upon NO and/or thalidomide treatment and thus providing evidence that interplaying with cellular ROS level could possible alter the set of genes involved in early lung development. In conclusion, the current study shed light that alteration of ROS level could modulate the expression of early genes which are required for normal lung development and maturation.Item Evaluation of the role of nitric oxide in acid sensing ion channel mediated cell death(Elsiever, 2010-04-01) Majumder, SyamantakAcid sensing ion channels (ASICs) are widely expressed in central and peripheral nervous system. They are involved in a variety of physiological and pathophysiological processes: synaptic transmission, learning and memory, pain perception, ischemia, etc. During ischemia, metabolic acidosis causes the drop of extracellular pH (pHe) which in turn activates ASICs. Activation of calcium permeable ASIC1a has been implicated in neuronal death. ASICs are modulated by several redox reagents, divalent cations and nitric oxide (NO). Although NO potentiates ASIC mediated currents, the physiological significance of such modulation has not been studied in detail. We have evaluated the role of endogenous NO in cell death at different pH, mediated by the activation of ASICs. At pH 6.1, death rates of ASIC1 expressing Neuro2A (N2A) cells are significantly higher in comparison to the cells that do not express ASICs. Amiloride, a blocker of ASICs protects the cell from acid-injury. Sodium nitroprusside, a potent NO donor not only increases the ASIC mediated currents but also increases cell death at low pH. l-Arg, the precursor of NO also potentiates ASICs in a pH dependent manner. l-Arg-induced NO production and potentiation of ASICs were observed at pHs 7.4, 7.2, 7.0 and 6.8. Lowering the pH below 6.8 did not result in significant production of NO or potentiation of ASICs upon l-Arg stimulation. Our results suggest that potentiation of ASICs by NO and subsequent cell death in vivo depends on the severity of acidosis. During mild and moderate acidosis, NO promotes cell death by potentiating ASICs, whereas this potentiation subsides in severe acidosis due to inhibition of NO synthase.Item Shear stress promotes nitric oxide production in endothelial cells by sub-cellular delocalization of eNOS: A basis for shear stress mediated angiogenesis(Elsiever, 2010-05) Majumder, SyamantakThis study aims to investigate the role of shear stress in cellular remodeling and angiogenesis with relation to nitric oxide (NO). We observed a 2-fold increase in endothelial cell (EC) migration in relation to actin re-arrangements under 15 dyne/cm2 shear stress. Blocking NO production inhibited the migration and ring formation of ECs by 6-fold and 5-fold, respectively under shear stress. eNOS-siRNA knockdown technique also ascertained a 3-fold reduction in shear stress mediated ring formation. In ovo artery ligation model with a half and complete flow block for 30 min showed a reduction of angiogenesis by 50% and 70%, respectively. External stimulation with NO donor showed a 2-fold recovery in angiogenesis under both half and complete flow block conditions. NO intensity clustering studies by using Diaminofluorescein diacetate (DAF-2DA) probed endothelial monolayer depicted pattern-changes in NO distribution and cluster formation of ECs under shear stress. Immunofluorescence and live cell studies revealed an altered sub-cellular localization pattern of eNOS and phospho-eNOS under shear stress. In conclusion, shear-induced angiogenesis is mediated by nitric oxide dependent EC migration.Item Simulated microgravity promotes nitric oxide-supported angiogenesis via the iNOS-cGMP-PKG pathway in macrovascular endothelial cells(Wiley, 2020-07-01) Majumder, SyamantakAngiogenesis is a physiological process involving the growth of blood vessel in response to specific stimuli. The present study shows that limited microgravity treatments induce angiogenesis by activating macrovascular endothelial cells. Inhibition of nitric oxide production using pharmacological inhibitors and inducible nitric oxide synthase (iNOS) small interfering ribo nucleic acid (siRNA) abrogated microgravity induced nitric oxide production in macrovascular cells. The study further delineates that iNOS acts as a molecular switch for the heterogeneous effects of microgravity on macrovascular, endocardial and microvascular endothelial cells. Further dissection of nitric oxide downstream signaling confirms that simulated microgravity induces angiogenesis via the cyclic guanosine monophosphate (cGMP)–PKG dependent pathway.Item NO (nitric oxide): The ring master(Elsiever, 2011-01) Majumder, SyamantakThe migration and proliferation of endothelial cells affect the process of angiogenesis or the formation of blood vessels. Endothelial cells interact with each other to form ring-like structures in monolayers and tubular structures in matrigels. However, the transit phase between the individual endothelial cells and fully formed tubular structures is yet to be established. Guided by imaging, Western blot analysis, drug perturbation studies and siRNA studies we validate that endothelial ring structures are the fundamental and monomeric units of capillary tubes and nitric oxide is implicated in their fabrication. Giving input from experimental data, we used bagging classifier and information-gain to determine some of the physical and chemical parameters that define these biological structures. Further, we elucidated the implications of endothelial nitric oxide synthase and the NO/sGC/cGMP pathway in the formation of endothelial rings. We conclude that, formation of endothelial ring structure is important for angiogenesis and is mediated by the NO/sGC/cGMP pathway; and further endothelial rings can be used as in vitro models to study angiogenesis.Item l-Theanine promotes nitric oxide production in endothelial cells through eNOS phosphorylation(Elsiever, 2013) Majumder, SyamantakConsumption of tea (Camellia sinensis) improves vascular function and is linked to lowering the risk of cardiovascular disease. Endothelial nitric oxide is the key regulator of vascular functions in endothelium. In this study, we establish that l-theanine, a non-protein amino-acid found in tea, promotes nitric oxide (NO) production in endothelial cells. l-theanine potentiated NO production in endothelial cells was evaluated using Griess reaction, NO sensitive electrode and a NO specific fluorescent probe (4-amino-5-methylamino-2',7'-difluororescein diacetate). l-Theanine induced NO production was partially attenuated in presence of l-NAME or l-NIO and completely abolished using eNOS siRNA. eNOS activation was Ca2 + and Akt independent, as assessed by fluo-4AM and immunoblotting experiments, respectively and was associated with phosphorylation of eNOS Ser 1177. eNOS phosphorylation was inhibited in the presence of ERK1/2 inhibitor, PD-98059 and partially inhibited by PI3K inhibitor, LY-294002 and Wortmanin suggesting PI3K-ERK1/2 dependent pathway. Increased NO production was associated with vasodilation in ex ovo (chorioallantoic membrane) model. These results demonstrated that l-theanine administration in vitro activated ERK/eNOS resulting in enhanced NO production and thereby vasodilation in the artery. The results of our experiments are suggestive of l-theanine mediated vascular health benefits of tea.Item Rho-kinase as a therapeutic target in vascular diseases: Striking nitric oxide signaling(Elsiever, 2014-12) Majumder, SyamantakRho GTPases are a globular, monomeric group of small signaling G-protein molecules. Rho-associated protein kinase/Rho-kinase (ROCK) is a downstream effector protein of the Rho GTPase. Rho-kinases are the potential therapeutic targets in the treatment of cardiovascular diseases. Here, we have primarily discussed the intriguing roles of ROCK in cardiovascular health in relation to nitric oxide signaling. Further, we highlighted the biphasic effects of Y-27632, a ROCK inhibitor under shear stress, which acts as an agonist of nitric oxide production in endothelial cells. The biphasic effects of this inhibitor raised the question of safety of the drug usage in treating cardiovascular diseases.Item A comparative study of NONOate based NO donors: Spermine NONOate is the best suited NO donor for angiogenesis(Elsiever, 2014-01-30) Majumder, SyamantakNitric oxide (NO) is a known modulator of angiogenesis. The NONOate subfamily of NO donors has long been used in experimental and clinical studies to promote angiogenesis. However, no studies have been conducted yet to compare the angiogenesis potential of these NO donors in respect to their pattern of NO release. We hypothesize that having different pattern of NO release, each of the NO donors in NONOate subfamily can promote key stages of angiogenesis in differential manner. To verify our hypothesis, NO donors with half life ranging from seconds to several hours and having very different pattern of NO release were selected to evaluate their efficacy in modulating angiogenesis. Endothelial tube formation using EAhy926 cells was maximally increased by Spermine NONOate (SP) treatment. SP treatment maximally induced both ex vivo and in vivo angiogenesis using egg yolk and cotton plug angiogenesis models respectively. Experiment using chick embryo partial ischemia model revealed SP as the best suited NO donor to recover ischemia driven hampered angiogenesis. The present study elaborated that differential release pattern of NO by different NO donors can modulate angiogenesis differentially and also suggested that SP have a unique pattern of NO release that best fits for angiogenesis.Item Inhibition of Angiogenesis and Nitric Oxide Synthase (NOS), by Embelin & Vilangin Using in vitro, in vivo & in Silico Studies(TUOMS PRESS, 2014) Majumder, SyamantakIn recent year’s anti-angiogenesis agents have been recognized as effective drugs for the treatment of solid tumors, this prompted us to conduct the present study. Methods: The anti-angiogenic activity of dimeric form of embelin (vilangin) was evaluated using endothelial cell (in vitro) and chorioallantoic membrane (CAM) egg yolk angiogenesis model (in vivo) and in addition the docking behaviour of human nitric oxide synthases (NOS) with four different ligands was evaluated along with their putative binding sites using Discovery Studio Version 3.1 (in silico) compared with the parent compound (embelin). Results: Vilangin exhibits 50% cytotoxic at 92 ± 1 µg/ml concentration level with reference to ECV 304 endothelial cells. Both vilangin and embelin, showed inhibitory effects on wound healing, single cell migration, nitric oxide production, and endothelial ring formation at 0.1 and 1.0 μg/ml concentration level. Similarly, CAM assay also showed inhibitory effect of vilangin and embelin with respect their reduction in length, size and junctions of blood capillaries compared to untreated egg yolk. Docking studies and binding free energy calculations revealed that vilangin has maximum interaction energy (-74.6 kcal/mol) as compared to the other investigated ligands. Conclusion: The results suggest that both vilangin and embelin attenuates angiogenesis in similar manner.Item Interleukin-6 secreted by bipotential murine oval liver stem cells induces apoptosis of activated hepatic stellate cells by activating NF-κB-inducible nitric oxide synthase signaling(CSB, 2016-10-09) Majumder, SyamantakLiver fibrosis is now well recognized as the causative factor for increased mortality from complications associated with liver pathologies. Activated hepatic stellate cells (HSCs) play a critical role in the progression of liver fibrosis. Therefore, targeting these activated HSCs to prevent and (or) treat liver disease is a worthwhile approach to explore. In the present in vitro study, we investigated the use of bipotential murine oval liver cells (BMOL) in regulating the functions of activated HSCs to prevent progression of liver fibrosis. We used a conditioned medium-based approach to study the effect of BMOL cells on activated HSC survival and function. Our data showed that BMOL cells block the contraction of activated HSCs by inducing apoptosis of these cells. We demonstrated that BMOL cells secrete soluble factors, such as interleukin-6 (IL-6), which induced apoptosis of activated HSCs. Using both pharmacological and molecular inhibitor approaches, we further identified that IL-6-mediated activation of NF-κB–iNOS–NO–ROS signaling in activated HSCs plays a critical role in BMOL-cell-mediated apoptosis of activated HSCs. Thus, the present study provides an alternative cell-based therapeutic approach to treat liver fibrosis.