Proinflammatory signaling mechanism of endocan in macrophages: Involvement of TLR2 mediated MAPK-NFkB pathways.

Endocan is an endothelial cell-specific proteoglycan that contributes to vascular dysfunction by impairing endothelial function and inducing vascular smooth muscle cell migration. However, its role in regulating macrophage inflammation, a key pathological feature of vascular dysfunction, is not well understood. In this study, we investigated the effect of endocan on macrophage inflammation to better understand its contribution to vascular dysfunction. We found that endocan upregulated pro-inflammatory cytokines including IL-1ß, IL-6 and TNF-α in RAW 264.7 cells and activated MAPK/NFkB signaling pathways. Inhibiting these pathways reduced endocan-induced cytokine levels, while inhibiting TLR2 compromised the MAPK/NFkB regulation. Additionally, LPS-induced HUVEC conditioned medium stimulated cytokine levels in RAW 264.7 cells, which were reduced by endocan siRNA treatment in HUVEC. These results suggest that endocan positively regulates pro-inflammation in macrophages through the TLR2-MAPK-NFkB axis, highlighting the potential of targeting endocan to reduce inflammation in vascular dysfunction.

Exposure to zinc oxide nanoparticles (ZnO-NPs) induces cardiovascular toxicity and exacerbates pathogenesis - Role of oxidative stress and MAPK signaling.

The precise toxico-pathogenic effects of zinc oxide nanoparticles (ZnO-NPs) on the cardiovascular system under normal and cardiovascular disease (CVD) risk factor milieu are unclear. In this study, we have investigated the dose-dependent effects of ZnO-NPs on developing chicken embryo and cell culture (H9c2 cardiomyoblast, HUVEC and aortic VSMC) models. In addition, the potentiation effect of ZnO-NPs on simulated risk factor conditions was evaluated using; 1. Reactive oxygen species (ROS) induced cardiac remodeling, 2. Angiotensin-II induced cardiac hypertrophy, 3. TNF-α induced HUVEC cell death and 4. Inorganic phosphate (Pi) induced aortic VSMC calcification models. The observed results illustrates that ZnO-NPs exposure down regulates vascular development and elevates oxidative stress in heart tissue. At the cellular level, ZnO-NPs exposure reduced the cell viability and increased the intracellular ROS generation, lipid peroxidation and caspase-3 activity in a dose-dependent manner in all three cell types. In addition, ZnO-NPs exposure significantly suppressed the endothelial nitric oxide (NO) generation, cardiac Ca2+ - ATPase activity and enhanced the cardiac mitochondrial swelling. Moreover, inhibition of p38 MAPK and JNK signaling pathways influence the cytotoxicity. Overall, ZnO-NPs exposure affects the cardiovascular system under normal conditions and it exacerbates the cardiovascular pathogenesis under selected risk factor milieu.

Endocan alters nitric oxide production in endothelial cells by targeting AKT/eNOS and NFkB/iNOS signaling.

Endocan, a secretary proteoglycan, known to induce vascular inflammation. Nitric oxide (NO) produced by endothelial cells is an important signaling molecule in maintaining the vascular homeostasis. However, the precise effect of endocan in regulating NO pathway is not known. The present study explores the effect of endocan on eNOS-iNOS-NO and ROS production in cultured endothelial cells. Results showed that recombinant endocan treatment in HUVEC could increase NO and nitrite levels. However, pharmacological inhibition of iNOS using 1400W significantly decreased these effects. Furthermore, protein expression analysis showed that endocan could inhibit AKT/eNOS pathway and activate NF-κB/iNOS pathway. The production of superoxide, hydrogen peroxide, peroxynitrite and total ROS were also significantly increased with endocan treatment supported by decreased activity of superoxide dismutase and catalase. Moreover, selective inhibition of NOX reduced the ROS formation. In addition, mRNA expression analysis demonstrated that endocan can upregulate the expression of NOX1, NOX2 and NOX4. These findings suggest that endocan alters the NO production and their by enhances oxidative stress in endothelial cells. Thus, inhibition of endocan-NO signaling could be a one of the strategy to reduce oxidative stress in vascular disease.

Molecular mechanism involved in epithelial to mesenchymal transition.

Epithelial to mesenchymal transition (EMT) is a biological process that plays an important role during embryonic development. During this process, the epithelial cells lose their polarity and acquire mesenchymal properties. In addition to embryonic development, EMT is also well-known to participate in tissue repair, inflammation, fibrosis, and tumor metastasis. In the present review, we address the basics of epithelial to mesenchymal transition during both development and disease conditions and emphasize the role of various transcription factors and miRNAs involved in the process.

Trehalose protects the endothelium from cadmium-induced dysfunction.

The objective of the present study is to identify the possible regulatory role of trehalose (Tre) against cadmium chloride (CdCl2 )-induced endothelial cell dysfunction. To screen the dose-dependent effect of both Tre and CdCl2 , a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was performed. Interestingly, MTT assay results have shown that co-incubation of Tre (1 mM) with CdCl2 significantly decreased the CdCl2 (5 µM) cytotoxicity. Nitric oxide (NO) measurement using Griess assay and 4-amino-5-methylamino-2',7'-difluorofluorescein fluorescence probe results have shown that CdCl2 decreases NO production in endothelial cells. Western blotting analysis results showed that CdCl2 decreases endothelial nitric oxide synthase (eNOS) and phospho endothelial nitric oxide synthase (peNOS) expression. The present study results have also observed that CdCl2 treatment increases reactive oxygen species (ROS) production. However, combination treatment (Tre + CdCl2 ) could restore the NO production in CdCl2 -treated cells. In addition, combination treatment could also restore eNOS and peNOS expression in endothelial cells. Moreover, Tre treatment was found to decrease CdCl2 -induced ROS production. Collectively, the present study results demonstrate that Tre possesses a significant protective action against CdCl2 -mediated endothelial dysfunction by increasing NO production, eNOS and peNOS expression, and by decreasing oxidative stress.

Levan production from sucrose using chicken feather peptone as a low cost supplemental nutrient source.

Chicken feather peptone (CFP) derived from poultry waste is a rich source of essential minerals and amino acids. This, along with suitable carbon source, can be used as a low cost complex supplemental nutrient source for microbial fermentation. In the present work, CFP blended with sucrose was evaluated for the production of levan using Bacillus subtilis MTCC 441. Amount of CFP added to the medium significantly influenced levan production and it was found that at a concentration 2 g/L, maximum levan yield of 0.26 ±â€¯0.04 g/g sucrose was obtained. The levan yield obtained with CFP as a low cost supplemental nutrient source was comparable with that obtained from commercial medium (0.31 ±â€¯0.02 g/g sucrose). Levan produced using CFP was tested on primary cell lines at various concentrations (100-1000 µM) and found to be non-toxic and bio-compatible in nature. This indicates that CFP could be used as low cost nutrient source for levan production.

In-silico and in-vitro analysis of endocan interaction with statins.

Endocan known as a cardiovascular inflammatory biomarker, found to be elevated in atherosclerosis. However, the 3D structure and the stimulatory effect of endocan on macrophages are unknown. Hence, we predicted the three-dimensional structure of human endocan and calculated the binding efficiency of statins towards endocan and determined their inhibition potential. Molecular docking studies of simvastatin (-9.64 kcal/mol) showed that binding is stabilized by the hydrogen bonds with Cys60, Cys54 residues, and several hydrophobic interactions. Moreover, MD simulations and pull-down assay results confirmed that simvastatin binding is stable with human endocan. In-silico results obtained in the present study were validated under in-vitro condition by analysing the effect of endocan under simvastatin treatment. Western blot results have shown that simvastatin could reduce endocan expression in LPS-treated endothelial cells. Further, endocan treatment in RAW 264.7 macrophages stimulates NO, ROS production and increases iNOS, CRP expression. However, endocan and simvastatin combination treatment could suppress NO, ROS production and iNOS, CRP activation. The present study results suggest that endocan could induce vascular inflammation in macrophages. In addition, the results showed that simvastatin could interact with endocan and thereby suppress the stimuli-induced effect. Thus, endocan may play a role in atherogenesis by activating macrophages.

Studies on solvent precipitation of levan synthesized using Bacillus subtilis MTCC 441.

Levan is a water soluble biopolymer widely used in food, pharma, personal care and aquaculture industries. In this work, levan was synthesized by Bacillus subtilis MTCC 441 using sucrose as a sole carbon source. Effects of pH, sucrose concentration, nitrogen source, nitrogen concentration, inoculum size and agitation speed on levan production were studied. Yeast extract (YE) was found to be the best nitrogen source. Sucrose concentration - 100 g/L, pH - 7, YE concentration - 2 g/L, inoculum size 10% (v/v) and RPM - 150 were found to be optimal values for levan production. Effects of precipitation pH (3-12), choice of solvent (ethanol, isopropanol, acetone, and methanol) and supernatant to solvent ratio (1:1 to 1:6) on levan yield were also studied. Isopropanol resulted in maximum levan recovery among the four solvents considered. Optimal pH and supernatant to solvent ratio for levan precipitation were found to be 11 and 1:5, respectively. Corresponding levan yield was 0.395 g/g of sucrose supplied. The product obtained was characterized using FTIR, 1H-NMR, 13C-NMR, and GPC. The cytotoxicity of the precipitated levan was studied on EA.hy926 cell line using MTT assay and the compound was proven to be non-toxic to the cells.

Corrigendum to "Studies on solvent precipitation of levan synthesized using Bacillus subtilis MTCC 441" [Heliyon 5 (9) (September 2019) e02414].

[This corrects the article DOI: 10.1016/j.heliyon.2019.e02414.].

Nitric Oxide Reverses the Position of the Heart during Embryonic Development.

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) plays crucial roles in cardiac homeostasis. Adult cardiomyocyte specific overexpression of eNOS confers protection against myocardial-reperfusion injury. However, the global effects of NO overexpression in developing cardiovascular system is still unclear. We hypothesized that nitric oxide overexpression affects the early migration of cardiac progenitor cells, vasculogenesis and function in a chick embryo. Vehicle or nitric oxide donor DEAN (500 mM) were loaded exogenously through a small window on the broad side of freshly laid egg and embryonic development tracked by live video-microscopy. At Hamburg Hamilton (HH) stage 8, the cardiac progenitor cells (CPC) were isolated and cell migration analysed by Boyden Chamber. The vascular bed structure and heart beats were compared between vehicle and DEAN treated embryos. Finally, expression of developmental markers such as BMP4, Shh, Pitx2, Noggin were measured using reverse transcriptase PCR and in-situ hybridization. The results unexpectedly showed that exogenous addition of pharmacological NO between HH stage 7⁻8 resulted in embryos with situs inversus in 28 out of 100 embryos tested. Embryos treated with NO inhibitor cPTIO did not have situs inversus, however 10 embryos treated with L-arginine showed a situs inversus phenotype. N-acetyl cysteine addition in the presence of NO failed to rescue situs inversus phenotype. The heart beat is normal (120 beats/min) although the vascular bed pattern is altered. Migration of CPCs in DEAN treated embryos is reduced by 60% compared to vehicle. BMP4 protein expression increases on the left side of the embryo compared to vehicle control. The data suggests that the NO levels in the yolk are important in turning of the heart during embryonic development. High levels of NO may lead to situs inversus condition in avian embryo by impairing cardiac progenitor cell migration through the NO-BMP4-cGMP axis.

Establishment of pancreatic microenvironment model of ER stress: Quercetin attenuates ß-cell apoptosis by invoking nitric oxide-cGMP signaling in endothelial cells.

The involvement of endoplasmic reticulum (ER) stress in endothelial dysfunction and diabetes-associated complications has been well documented. Inhibition of ER stress represents a promising therapeutic strategy to attenuate endothelial dysfunction in diabetes. Recent attention has focused on the development of small molecule inhibitors of ER stress to maintain endothelial homeostasis in diabetes. Here we have developed a reliable, robust co-culture system that allows a study on the endothelial cells and pancreatic ß-cells crosstalk under ER stress and validated using a known ER stress modulator, quercetin. Furthermore, sensitizing of endothelial cells by quercetin (25 µM) confers protection of pancreatic ß-cells against ER stress through nitric oxide (NO∙) signaling. In addition, increased intracellular insulin and NO∙-mediated cyclic 3',5'-guanosine monophosphate (cGMP) levels in pancreatic ß-cells further confirmed the mechanism of protection under co-culture system. In addition, the potential protein targets of quercetin against ER stress in the endothelial cells were investigated through proteomic profiling and its phosphoprotein targets through Bioplex analysis. On the whole, the developed in vitro co-culture set up can serve as a platform to study the signaling network between the endothelial and pancreatic ß-cells as well as provides a mechanistic insight for the validation of novel ER stress modulators.

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.

Downregulation of dTps1 in Drosophila melanogaster larvae confirms involvement of trehalose in redox regulation following desiccation.

As a survival strategy to environmental water deficits, desiccation-tolerant organisms are commonly known for their ability to recruit stress-protective biomolecules such as trehalose. We have previously reported the pivotal role of trehalose in larval desiccation tolerance in Drosophila melanogaster. Trehalose has emerged as a versatile molecule, serving mainly as energy source in insects and also being a stress protectant. While several recent reports have revealed the unconventional role of trehalose in scavenging reactive oxygen species in yeast and plants, this aspect has not received much attention in animals. We examined the status of desiccation-induced generation of reactive oxygen species in D. melanogaster larvae and the possible involvement of trehalose in ameliorating the harmful consequences thereof. Insect trehalose synthesis is governed by the enzyme trehalose 6-phosphate synthase 1 (TPS1). Using the ubiquitous da-GAL4-driven expression of the dTps1-RNAi transgene, we generated dTps1-downregulated Drosophila larvae possessing depleted levels of dTps1 transcripts. This resulted in the inability of the larvae for trehalose synthesis, thereby allowing us to elucidate the significance of trehalose in the regulation of desiccation-responsive redox homeostasis. Furthermore, the results from molecular genetics studies, biochemical assays, electron spin resonance analyses and a simple, non-invasive method of whole larval live imaging suggested that trehalose in collaboration with superoxide dismutase (SOD) is involved in the maintenance of redox state in D. melanogaster.