Department of Biological Sciences
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Item Developing an Ex Vivo Model of Ischemia Using Early Chick-Embryo: A Model to Study Ischemia Related Angiogenesis(Springer, 2012-06-19) Majumder, SyamantakIschemia created in the animals were long been used as a model to study ischemia mediated effects in vivo. Pathological angiogenesis is the key hallmark of various ischemic diseases where blood vessel formation was compromised due to low blood flow. New blood vessels form in order to compensate the low blood perfusion in the ischemic area. This neovascularization and remodeling of the existent vessels protect from the consequences of ischemia associated diseases like myocardial infarction and stroke. A better understanding of the mechanisms of functional vessel formation is a pre-requisite to improve the treatment of ischemic pathologies. Therefore, the research area warrants an easily accessible model in which vessel formation can be both manipulated and studied. However, a limited number of efforts have been put forward yet to develop an ischemia models where ischemia mediated remodeling of vessels can be studied in real time. In present study, we used 4 day grown chick embryo to ligate right vitelline artery and create partial ischemia in the vascular bed of the embryo. The model has been developed based on the principle that blocking blood flow in the vascular bed will stop the nutrient and oxygen supply to the adjacent vessels and thus creating an ischemia like condition. Additionally, ischemia related changes in angiogenesis can be followed and tracked in real time in the vascular bed of the chick embryo. The present ex vivo model can be utilized in studying ischemia related angiogenesis in specific and hypoxia and/or low oxygen mediated angiogenesis in general.Item Use of Stem Cells to Block the Activation of Hepatic Stellate Cells in Diseased Liver(Springer, 2014-11-12) Majumder, SyamantakLiver is an important organ in vertebrates and performs major functions such as digestion, drug detoxification, and protein synthesis. Chronic liver fibrosis is a major threat to human life. The etiology of liver fibrosis includes chronic hepatitis infection, alcohol abuse, and nonalcoholic steatohepatitis. The pathophysiology of liver fibrosis shows that there is accumulation of extracellular matrix (ECM) proteins including collagen, proteoglycan, and adhesive glycoproteins. Activated hepatic stellate cells (HSCs) are the major collagen-producing cells in the liver. The present in vitro study demonstrates that bipotential murine oval liver (BMOL) stem cells secrete soluble factors, which are capable of inducing apoptosis in activated HSCs and inhibit the formation of collagen. Further, the study can be extended to identify the soluble factors capable of attenuating activated HSCs and opens a new research direction to control liver fibrosis.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 Activated pericyte attenuates endothelial functions: nitric oxide – cGMP rescues activated pericyte-associated endothelial dysfunctions(CSP, 2007-11-23) Majumder, SyamantakHepatic stellate cells are liver-specific pericytes and exist in close proximity with endothelial cells. The activation of liver pericytes is intrinsic to liver pathogenesis, and leads to endothelial dysfunction, including the low bioavailability of nitric oxide (NO). However, the role of nitric oxide in pericyte–endothelium cross-talk has not yet been elucidated. This work examines the cellular mechanism of action of NO in pericyte-mediated endothelial dysfunction. We used in vitro coculture and conditioned medium systems to study the effects of activated liver pericytes on endothelial function, and an egg yolk vascular bed model was used to study the effects of activated pericytes on angiogenesis. This study also demonstrates that activated pericytes attenuate the migration, proliferation, permeability, and NO production of endothelial cells. Our results demonstrate that activated pericytes restrict angiogenesis in egg yolk vascular bed models, and NO supplementation recovers 70% of the inhibition. Our results also demonstrate that supplementation with NO, sildenafil citrate (phosphodiesterase inhibitor), and 8-bromo-cGMP (cGMP analog) partially recovers activated-pericyte-mediated endothelium dysfunction. We conclude that NO–cGMP alleviates activated-pericyte-associated endothelial dysfunction, including angiogenesis, in a cGMP-dependentItem Dose dependent effects of cadmium on tumor angiogenesis(Oncotargot, 2017) Majumder, SyamantakAngiogenesis is crucial for tumor growth and metastasis. Cadmium (Cd) exposure is associated with elevated cancer risk and mortality. Such association is, at least in part, attributable to Cd-induced tumor angiogenesis. Nevertheless, the reported effects of Cd on tumor angiogenesis appear to be either stimulatory or inhibitory, depending on the concentrations. Ultra-low concentrations of Cd (<0.5 μM) inhibit endothelial nitric oxide synthase activation, leading to reduced endothelial nitric oxide production and attenuated tumor angiogenesis. In contrast, low-lose Cd (1-10 μM) up-regulates vascular endothelial growth factor (VEGF)-mediated tumor angiogenesis by exerting sub-apoptotic levels of oxidative stress on both tumor cells and endothelial cells (ECs). The consequent activation of protein kinase B/Akt, nuclear factor-κB, and mitogen-activated protein kinase signaling cascades mediate the increased secretion of VEGF by tumor cells and the up-regulated VEGF receptor-2 expression in ECs. Furthermore, Cd in high concentrations (>10 μM) induces EC apoptosis via the activation of caspase-3, resulting in destruction of tumor vasculature. In this review, we summarize the current knowledge concerning the roles of Cd in tumor angiogenesis, with a focus on molecular mechanisms underlying the dose dependent effects of Cd on various EC phenotypesItem Nitric oxide/cGMP protects endothelial cells from hypoxia-mediated leakiness(Elsiever, 2008-03-17) Majumder, SyamantakLeakiness of the endothelial bed is attributed to the over-perfusion of the pulmonary bed, which leads to high altitude pulmonary edema (HAPE). Inhalation of nitric oxide has been successfully employed to treat HAPE patients. We hypothesize that nitric oxide intervenes in the permeability of the pulmonary macrovascular endothelial bed to rectify the leaky bed under hypoxia. Our present work explores the underlying mechanism of ‘hypoxia-mediated’ endothelial malfunction by using human umbilical cord-derived immortalized endothelial cells, ECV-304, and bovine pulmonary artery primary endothelial cells. The leakiness of the endothelial monolayer was increased by two-fold under hypoxia in comparison to cells under normoxia, while optical tweezers-based tethering assays reported a higher membrane tension of endothelial cells under hypoxia. Phalloidin staining demonstrated depolymerization of F-actin stress fibers and highly polarized F-actin patterns in endothelial cells under hypoxia. Nitric oxide, 8-Br-cGMP and sildenafil citrate (phosphodiesterase type 5 inhibitor) led to recovery from hypoxia-induced leakiness of the endothelial monolayers. Results of the present study also suggest that ‘hypoxia-induced’ cytoskeletal rearrangements and membrane leakiness are associated with the low nitric oxide availability under hypoxia. We conclude that nitric oxide-based recovery of hypoxia-induced leakiness of endothelial cells is a cyclic guanosine monophosphate (cGMP)-dependent phenomenon.Item The Role of Calreticulin Transacetylase in the Activation of Human Platelet Nitrite Reductase by Polyphenolic Acetates(J-Stage, 2009) Majumder, SyamantakOur earlier investigations demonstrated the remarkable activation of cytochrome P-450 reductase and nitric oxide synthase by 7,8-diacetoxy-4-methylcoumarin, a model polyphenolic acetate by way of acetylation, catalyzed by the Calreticulin. Protein acetyltransferase action of Calreticulin was hence termed Calreticulin transacetylase (CRTAase). Nitric oxide synthase and nitrite reductase are now considered as parts of nitric oxide cycle. The activation of platelets nitric oxide synthase by 7,8-diacetoxy-4-methylcoumarin has already been demonstrated by us. Also, there are reports that certain proteins such as cytochrome P-450 reductase and cytochrome P-450 are endowed with the nitrite reductase activity in mammalian cells. Keeping these facts in view, we turned our attention to probe whether 7,8-diacetoxy-4-methylcoumarin could alter the levels of nitric oxide independent of the action of nitric oxide synthase in the human platelets model. The incubation of 7,8-diacetoxy-4-methylcoumarin and nitrite with platelets caused significant elevation of nitric oxide and cyclic guanosine monophosphate levels possibly due to the activation of nitrite reductase. Several polyphenolic acetates were similarly found to activate the nitrite reductase in tune with their affinities as substrate to CRTAase. N-ω-Nitro-L-arginine methyl ester, the inhibitor of nitric oxide synthase, failed to reverse such an effect of 7,8-diacetoxy-4-methylcoumarin. Clotrimazole which is known to be an inhibitor of nitrite reductase, effectively abolished the 7,8-diacetoxy-4-methylcoumarin mediated enhancement of nitric oxide levels in platelets as well as the nitric oxide mediated effects; such as cyclic guanosine monophosphate levels as well as adenosine diphospate induced platelets aggregation due to nitrite.Item Cadmium attenuates bradykinin-driven nitric oxide production by interplaying with the localization pattern of endothelial nitric oxide synthase(CSP, 2009-06-17) Majumder, SyamantakCadmium, a ubiquitous heavy metal, interferes with endothelial functions and angiogenesis. Bradykinin is a Ca-mobilizing soluble peptide that acts via nitric oxide to promote vasodilation and capillary permeability. The objective of the present study was to explore the Cd implications in bradykinin-dependent endothelial functions. An egg yolk angiogenesis model was employed to evaluate the effect of Cd on bradykinin-induced angiogenesis. The results demonstrate that 100 nmol/L Cd attenuated bradykinin-dependent angiogenesis. The results of the in vitro wound healing and tube formation assays by using EAhy 926, a transformed endothelial cell line, suggest that Cd blocked bradykinin-mediated endothelial migration and tube formation by 38% and 67%, respectively, while nitric oxide supplementation could reverse the effect of Cd on bradykinin-induced endothelial migration by 94%. The detection of nitric oxide by using a DAF-2DA fluorescent probe, Griess assay, and ultrasensitive electrode suggests that Cd blocked bradykinin-induced nitric oxide production. Fluorescence imaging of eNOS-GFP transfected endothelial cells, immunofluroscence, and Western blot studies of Cd and bradykinin-treated cells show that Cd interfered with the localization pattern of eNOS, which possibly attenuates nitric oxide production in part. Additionally, Ca imaging of Cd- and bradykinin-treated cells suggests that Cd blocked bradykinin-dependent Ca influx into the cells, thus partially blocking Ca-dependent nitric oxide production in endothelial cells. The results of this study conclude that Cd blunted the effect of bradykinin by interfering with the Ca-associated NOS activity specifically by impeding subcellular trafficking of eNOS.Item Thalidomide attenuates nitric oxide-driven angiogenesis by interacting with soluble guanylyl cyclase(Wiley, 2009-11-30) Majumder, SyamantakNitric oxide (NO) promotes angiogenesis by activating endothelial cells. Thalidomide arrests angiogenesis by interacting with the NO pathway, but its putative targets are not known. Here, we have attempted to identify these targetsItem 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.