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.

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.

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.

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.

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.

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.

Tipping off endothelial tubes: nitric oxide drives tip cells.

Angiogenesis, the formation of new blood vessels from pre-existing vessels, is a complex process that warrants cell migration, proliferation, tip cell formation, ring formation, and finally tube formation. Angiogenesis is initiated by a single leader endothelial cell called "tip cell," followed by vessel elongation by "stalk cells." Tip cells are characterized by their long filopodial extensions and expression of vascular endothelial growth factor receptor-2 and endocan. Although nitric oxide (NO) is an important modulator of angiogenesis, its role in angiogenic sprouting and specifically in tip cell formation is poorly understood. The present study tested the role of endothelial nitric oxide synthase (eNOS)/NO/cyclic GMP (cGMP) signaling in tip cell formation. In primary endothelial cell culture, about 40% of the tip cells showed characteristic sub-cellular localization of eNOS toward the anterior progressive end of the tip cells, and eNOS became phosphorylated at serine 1177. Loss of eNOS suppressed tip cell formation. Live cell NO imaging demonstrated approximately 35% more NO in tip cells compared with stalk cells. Tip cells showed increased level of cGMP relative to stalk cells. Further, the dissection of NO downstream signaling using pharmacological inhibitors and inducers indicates that NO uses the sGC/cGMP pathway in tip cells to lead angiogenesis. Taken together, the present study confirms that eNOS/NO/cGMP signaling defines the direction of tip cell migration and thereby initiates new blood vessel formation.

Sinapic acid protects heart against ischemia/reperfusion injury and H9c2 cardiomyoblast cells against oxidative stress.

The present study was designed to evaluate antioxidant and cardioprotective potential of sinapic acid (SA) against ischemia/reperfusion (I/R) injury. Cardiac functional recovery after I/R was evaluated by percentage rate pressure product (%RPP) and percentage coronary flow (%CF). Myocardial injury was evaluated by 2,3,5-triphenyltetrazolium chloride (TTC) staining and LDH enzyme leakage. Oxidative stress was estimated by lipid peroxidation level. eNOS protein expression in reperfused heart was assessed using Western blot method. Finally, in order to support the antioxidant effect of SA, in vitro protective potential of SA was assessed on H2O2-induced oxidative stress in H9c2 cardiomyoblast cells. The overall results demonstrated that I/R induced cardiac dysfunction, injury and oxidative stress was attenuated by SA treatment. Moreover, in vitro results also shown that, SA protects H9c2 cells from oxidative stress and modulates mitochondrial membrane permeability transition (MPT). In conclusion, coupled results from both in vivo and in vitro experiments have confirmed that SA with antioxidant role protects cardiac cells and its functions from I/R induced oxidative stress.

A comparative study of NONOate based NO donors: spermine NONOate is the best suited NO donor for angiogenesis.

Nitric 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.

Breast cancer drugs dampen vascular functions by interfering with nitric oxide signaling in endothelium.

Widely used chemotherapeutic breast cancer drugs such as Tamoxifen citrate (TC), Capecitabine (CP) and Epirubicin (EP) are known to cause various cardiovascular side-effects among long term cancer survivors. Vascular modulation warrants nitric oxide (NO) signal transduction, which targets the vascular endothelium. We hypothesize that TC, CP and EP interference with the nitric oxide downstream signaling specifically, could lead to cardiovascular dysfunctions. The results demonstrate that while all three drugs attenuate NO and cyclic guanosine mono-phosphate (cGMP) production in endothelial cells, they caused elevated levels of NO in the plasma and RBC. However, PBMC and platelets did not show any significant changes under treatment. This implies that the drug effects are specific to the endothelium. Altered eNOS and phosphorylated eNOS (Ser-1177) localization patterns in endothelial cells were observed following drug treatments. Similarly, the expression of phosphorylated eNOS (Ser-1177) protein was decreased under the treatment of drugs. Altered actin polymerization was also observed following drug treatment, while addition of SpNO and 8Br-cGMP reversed this effect. Incubation with the drugs decreased endothelial cell migration whereas addition of YC-1, SC and 8Br-cGMP recovered the effect. Additionally molecular docking studies showed that all three drugs exhibited a strong binding affinity with the catalytic domain of human sGC. In conclusion, results indicate that TC, CP and EP cause endothelial dysfunctions via the NO-sGC-cGMP pathway and these effects could be recovered using pharmaceutical agonists of NO signaling pathway. Further, the study proposes a combination therapy of chemotherapeutic drugs and cGMP analogs, which would confer protection against chemotherapy mediated vascular dysfunctions in cancer patients.

Early and long-term outcomes of conventional and valve-sparing aortic root replacement.

OBJECTIVE: To determine the early and long-term outcomes of conventional aortic root (ARR) and valve-sparing root replacement (VSRR) using a standard perioperative and operative approach. METHODS: We present prospectively collected data of 609 consecutive patients undergoing elective and urgent aortic root surgery (470 ARR, 139 VSRR) between 2006 and 2020. Primary outcomes were operative mortality and incidence of postoperative complications. Secondary outcomes were long-term survival and requirement for reintervention. Median follow-up was 7.6 years (range 0.5-14.5). RESULTS: 189 patients (31%) had bicuspid aortic valves and 17 (6.9%) underwent redo procedures. Median cross-clamp time was 88 (range 54-208) min with cardiopulmonary bypass of 108 (range 75-296) min. In-hospital mortality was 10 (1.6%), with transient ischaemic attacks/strokes occurring in 1.1%. In-hospital mortality for VSRR was 0.7%. 12 patients (2.0%) required a resternotomy for bleeding and 14 (2.3%) received haemofiltration. Intensive care unit and hospital stay were 1.7 and 7.0 days, respectively. During follow-up, redo surgery for native aortic valve replacement was required in 1.4% of the VSRR group. Overall survival was 95.1% at 3 years, 93.1% at 5 years, 91.2% at 7 years and 88.6% at 10 years. CONCLUSIONS: ARR and VSRR can be performed with low mortality and morbidity as well as a low rate of reintervention during the period of long-term follow-up, if performed by an experienced team with a consistent perioperative approach. This series provides contemporary evidence to balance the risks of aortic aneurysms and their rupture at diameters of <5.5 cm against the risks and benefits of surgery.

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.

Surgical aortic valve replacement in the era of transcatheter aortic valve implantation: a review of the UK national database.

OBJECTIVES: To date the reported outcomes of surgical aortic valve replacement (SAVR) are mainly in the settings of trials comparing it with evolving transcatheter aortic valve implantation. We set out to examine characteristics and outcomes in people who underwent SAVR reflecting a national cohort and therefore 'real-world' practice. DESIGN: Retrospective analysis of prospectively collected data of consecutive people who underwent SAVR with or without coronary artery bypass graft (CABG) surgery between April 2013 and March 2018 in the UK. This included elective, urgent and emergency operations. Participants' demographics, preoperative risk factors, operative data, in-hospital mortality, postoperative complications and effect of the addition of CABG to SAVR were analysed. SETTING: 27 (90%) tertiary cardiac surgical centres in the UK submitted their data for analysis. PARTICIPANTS: 31 277 people with AVR were identified. 19 670 (62.9%) had only SAVR and 11 607 (37.1%) had AVR+CABG. RESULTS: In-hospital mortality for isolated SAVR was 1.9% (95% CI 1.6% to 2.1%) and was 2.4% for AVR+CABG. Mortality by age category for SAVR only were: <60 years=2.0%, 60-75 years=1.5%, >75 years=2.2%. For SAVR+CABG these were; 2.2%, 1.8% and 3.1%. For different categories of EuroSCORE, mortality for SAVR in low risk people was 1.3%, in intermediate risk 1% and for high risk 3.9%. 74.3% of the operations were elective, 24% urgent and 1.7% emergency/salvage. The incidences of resternotomy for bleeding and stroke were 3.9% and 1.1%, respectively. Multivariable analyses provided no evidence that concomitant CABG influenced outcome. However, urgency of the operation, poor ventricular function, higher EuroSCORE and longer cross clamp and cardiopulmonary bypass times adversely affected outcomes. CONCLUSIONS: Surgical SAVR±CABG has low mortality risk and a low level of complications in the UK in people of all ages and risk factors. These results should inform consideration of treatment options in people with aortic valve disease.

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.

The EXCEL Trial: The Surgeons' Perspective.

There have been several investigations comparing the efficacy of percutaneous coronary intervention and coronary artery bypass grafting surgery for treatment of left main stem disease. This includes the Evaluation of XIENCE versus Coronary Artery Bypass Graft Surgery for Effectiveness of Left Main Revascularizaton (EXCEL) trial, which has garnered significant controversy surrounding its experimental design and reporting of its results. The authors review the methodology, results, caveats and statements on the EXCEL trial. They also review the other trials in the management of left main stem disease comparing percutaneous coronary intervention with coronary artery bypass grafting, as well as the SYNTAX score and its role in future guidelines for revascularisation. These findings have significant implications for current practice, influencing the growing role for multidisciplinary team meeting and allowing clinicians and patients to make the right choice.

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.

Ulcerative Squamous Eyelid Papilloma: A Rare Presentation.

PURPOSE: To highlight the importance of histopathological evaluation of a lid mass to prognosticate the disease. We report a case of ulcerative squamous cell papilloma with clinical features suggesting malignancy. CASE REPORT: A 65-year-old man presented with a rapidly enlarging mass in the left upper eyelid, with clinical features suggesting a squamous cell carcinoma. However, a repeat histopathological examination showed no malignant cells. The patient was diagnosed with squamous cell papilloma. He was followed-up for 30 months and no recurrence was observed. No such case has previously been reported in the literature. CONCLUSION: This report highlights the need for histopathological examination of all eyelid lesions to enable surgeons to prognosticate the disease.

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.].