Live Imaging and Analysis of Vasoactive Properties of Drugs Using an in-ovo Chicken Embryo Model: Replacing and Reducing Animal Testing.

Vasodilation occurs as a result of the relaxation of the smooth muscle cells present in the walls of blood vessels. Various suitable models are available for the analysis of the vasoactive properties of drugs with therapeutic applications. But all these models have limitations, such as ethical issues and high cost. The purpose of this study is to develop an alternative model for studying the vasoactive properties of drugs using an in-ovo chicken embryo model. In the preliminary experiment, we used a well-known vasoconstrictor (adrenaline) and a vasodilator (spermine NoNoate) in the chick embryo area vasculosa and evaluated their concentration-response curve. Adrenaline (10 µM) and spermine NoNoate (10 µM) were administered in different arteries and veins and different positions of the right vitelline artery of the chick embryo. Results showed the middle of the vessel bed of the right vitelline artery having the best vasoactive effect compared to others. Finally, anti-hypertensive drugs, calcium channel blockers, and NOS agonists were administered in the chick embryo area vasculosa to validate the model. Results demonstrate that the chick embryo area vasculosa can be an alternative, robust, and unique in-ovo model for screening of anti-hypertensive drugs in real time.

Thalidomide and Its Analogs Differentially Target Fibroblast Growth Factor Receptors: Thalidomide Suppresses FGFR Gene Expression while Pomalidomide Dampens FGFR2 Activity.

Thalidomide is an infamous teratogen and it is continuously being explored for its anticancer properties. Fibroblast growth factor receptors (FGFRs) are implicated in embryo development and cancer pathophysiology. With striking similarities observed between FGFR implicated conditions and thalidomide embryopathy, we hypothesized thalidomide targets FGFRs. We utilized three different cell lines and chicken embryo model to investigate the effects of thalidomide and analogs on FGFR expression. We performed molecular docking, KINOMEscan analysis, and kinase activity assays to study the drug-protein interactions. The expression of FGFR1 and FGFR2 was differentially regulated by all the three drugs in cells as well as in developing organs. Transcriptome analysis of thalidomide-treated chick embryo strongly suggests the modulation of FGFR signaling and key transcription factors. Corroboration with previous studies suggests that thalidomide might affect FGFR expression through the transcription factor, E2F1. At the protein level, molecular docking predicted all three analogs to interact with lysine residue at 517th and 508th positions of FGFR2 and FGFR3, respectively. This lysine coordinates the ATP binding site of FGFR, thus hinting at the possible perturbation of FGFR activity by thalidomide. Kinome analysis revealed that kinase activities of FGFR2 and FGFR3 (G697C) reduced by 31% and 65%, respectively, in the presence of 10 µM thalidomide. Further, we checked and confirmed that the analogs inhibited the FGFR2 kinase activity in a dose-dependent manner. This study suggests that FGFRs could be potential targets of thalidomide and the two analogs, and also endorses the link between the teratogenicity and antitumor activities of the drugs.

Hypoxia perturbs endothelium by re-organizing cellular actin architecture: Nitric oxide offers limited protection.

Exposure to hypoxia causes structural changes in the endothelial cell (EC) monolayer that alter its permeability. There was a report earlier of impairment of nitric oxide (NO) production in endothelium. The intervention of NO in the altered cellular arrangements of actin cytoskeleton in endothelium for rectification of paracellular gaps in endothelium under hypoxia was observed. The present study demonstrates hypoxia inducing paracellular gaps in hypoxia-exposed blood capillaries in chick embryo extravascular model. Phalloidin staining confirmed significant polymerization of actin and unique cellular localization of the F-actin bands under hypoxia treatments. Addition of spermine NONOate (SPNO), a NO donor, or reoxygenation to endothelial monolayer attenuated hypoxia-mediated effects on endothelial permeability with partial recovery of endothelial integrity through actin remodeling. The present study indicates link of hypoxia-induced actin-associated cytoskeletal rearrangements and paracellular gaps in the endothelium with a low NO availability in the hypoxia milieu. The author concludes that NO confers protection against hypoxia-mediated cytoskeletal remodeling and endothelial leakiness.

sFRP4 signalling of apoptosis and angiostasis uses nitric oxide-cGMP-permeability axis of endothelium.

Nitric oxide (NO) plays a critical role in endothelial functions such as cellular migration, vascular permeability and angiogenesis. Angiogenesis, the formation of new blood vessels from "pre-existing" ones is a carefully regulated process and essential during reproduction, development and wound healing. Previously our lab group reported that Secreted Frizzled-Related Protein 4 (sFRP4) could inhibit angiogenesis in both in vitro and in vivo conditions. sFRP4 belongs to a family of secreted glycoproteins that function as antagonists of the canonical Wnt signalling pathway. Although the pro-apoptotic role of sFRP4 is well discussed in literature, little is known in regards to its anti-angiogenic property. The objective of this study was to elucidate sFRP4 implications in NO biology of the endothelium. Results demonstrate that sFRP4 causes endothelial dysfunction by suppressing NO-cGMP signaling and elevating corresponding ROS levels. The imbalance between NO and ROS levels results in apoptosis and subsequent leakiness of endothelium as confirmed in vivo (Texas red/Annxin - CAM assay) and in vitro (Monolayer permeability assay) conditions. Furthermore utilizing peptides synthesized from the CRD domain of sFRP4, our results showed that while these peptides were able to cause endothelial dysfunctions, they did not cause apoptosis of the endothelial cells. Thereby confirming that sFRP4 can mediate its anti-angiogenic effect independent of its pro-apoptotic property. In conclusion, the current study reports that sFRP4-mediated anti-angiogenesis occurs as a result of impaired NO-cGMP signaling which in turn allow for elevation of redox levels and promotion of apoptosis of endothelial cells.

Disturbed flow mediated modulation of shear forces on endothelial plane: A proposed model for studying endothelium around atherosclerotic plaques.

Disturbed fluid flow or modulated shear stress is associated with vascular conditions such as atherosclerosis, thrombosis, and aneurysm. In vitro simulation of the fluid flow around the plaque micro-environment remains a challenging approach. Currently available models have limitations such as complications in protocols, high cost, incompetence of co-culture and not being suitable for massive expression studies. Hence, the present study aimed to develop a simple, versatile model based on Computational Fluid Dynamics (CFD) simulation. Current observations of CFD have shown the regions of modulated shear stress by the disturbed fluid flow. To execute and validate the model in real sense, cell morphology, cytoskeletal arrangement, cell death, reactive oxygen species (ROS) profile, nitric oxide production and disturbed flow markers under the above condition were assessed. Endothelium at disturbed flow region which had been exposed to low shear stress and swirling flow pattern showed morphological and expression similarities with the pathological disturbed flow environment reported previously. Altogether, the proposed model can serve as a platform to simulate the real time micro-environment of disturbed flow associated with eccentric plaque shapes and the possibilities of studying its downstream events.

Mechanical perturbations trigger endothelial nitric oxide synthase activity in human red blood cells.

Nitric oxide (NO), a vascular signaling molecule, is primarily produced by endothelial NO synthase. Recently, a functional endothelial NO synthase (eNOS) was described in red blood cells (RBC). The RBC-eNOS contributes to the intravascular NO pool and regulates physiological functions. However the regulatory mechanisms and clinical implications of RBC-eNOS are unknown. The present study investigated regulation and functions of RBC-eNOS under mechanical stimulation. This study shows that mechanical stimuli perturb RBC membrane, which triggers a signaling cascade to activate the eNOS. Extracellular NO level, estimated by the 4-Amino-5-Methylamino-2', 7'-Difluorofluorescein Diacetate probe, was significantly increased under mechanical stimuli. Immunostaining and western blot studies confirmed that the mechanical stimuli phosphorylate the serine 1177 moiety of RBC-eNOS, and activates the enzyme. The NO produced by activation of RBC-eNOS in vortexed RBCs promoted important endothelial functions such as migration and vascular sprouting. We also show that mechanical perturbation facilitates nitrosylation of RBC proteins via eNOS activation. The results of the study confirm that mechanical perturbations sensitize RBC-eNOS to produce NO, which ultimately defines physiological boundaries of RBC structure and functions. Therefore, we propose that mild physical perturbations before, after, or during storage can improve viability of RBCs in blood banks.

Cadmium-induced embryopathy: nitric oxide rescues teratogenic effects of cadmium.

Although Cadmium (Cd) is a well-known heavy metal pollutant and teratogen, the mechanism behind Cd-mediated teratogenicity remains unknown. Previously, we have reported of the protective role of Nitric oxide (NO), a key signaling molecule in the embryonic developmental process, against Thalidomide-induced teratogenicity. The objective of this study was to obtain a mechanistic in-sight of the antiteratogenic potential of NO against Cd-mediated teratogenicity. To achieve this goal, we first studied the effect of Cd on the vasculature of developing embryos and then we investigated whether Cd mediated its effects by interfering with the redox regulation of NO signaling in the early development milieu. We used a chick embryonic model to determine the time and dose-dependent effects of Cd and NO recovery against Cd assault. The effects of Cd and NO recovery were assessed using various angiogenic assays. Redox and NO levels were also measured. Results demonstrated that exposure to Cd at early stage of development caused multiple birth defects in the chick embryos. Exposure to Cd suppressed endogenous NO levels and cGMP signaling, inhibiting angioblast activation and subsequently impairing yolk sac vascular development. Furthermore, Cd-induced superoxide and lipid peroxidation mediated activation of proapoptotic markers p21 and p53 in the developing embryo. Cd also caused the down-regulation of FOXO1, and up-regulation of FOXO3a and Caspase 3-mediated apoptosis. Addition of exogenous NO through a NO donor was able to blunt Cd-mediated effects and restore normal vascular and embryonic development. In conclusion, Cd-mediated teratogenicity occurs as a result of impaired NO-cGMP signaling, increased oxidative stress, and the activation of apoptotic pathways. Subsequent addition of exogenous NO through NO donor negated Cd-mediated effects and protected the developing embryo.

Nitrites derived from Foneiculum vulgare (fennel) seeds promotes vascular functions.

Recent evidence has demonstrated that nitrites play an important role in the cardiovascular system. Fennel (Foneiculum vulgare) seeds are often used as mouth fresheners after a meal in both the Indian sub-continent and around the world. The present study aims to quantify the nitrite and nitrates in fennel seeds as well as elucidating the effect of fennel derived-nitrites on vascular functions. Results from our study show that fennel seeds contain significantly higher amount of nitrites when compared to other commonly used post-meal seeds. Furthermore our study confirmed the functional effects of fennel derived-nitrites using in vitro and ex vivo models that describe the promotion of angiogenesis, cell migration, and vasorelaxation. We also showed that chewing fennel seeds enhanced nitrite content of saliva. Thus our study indicates the potential role of fennel derived-nitrites on the vascular system.