Vascular endothelium dysfunction: a conservative target in metabolic disorders

Aim

Vascular endothelium plays a role in capillary transport of nutrients and drugs and regulates angiogenesis, homeostasis, as well as vascular tone and permeability as a major regulator of local vascular homeostasis. The present study has been designed to investigate the role of endothelium in metabolic disorders.

Methods

The endothelium maintains the balance between vasodilatation and vasoconstriction, procoagulant and anticoagulant, prothrombotic and antithrombotic mechanisms.

Results

Diabetes mellitus causes the activation of aldose reductase, polyol pathway and advanced glycation-end-product formation that collectively affect the phosphorylation status and expression of endothelial nitric oxide synthatase (eNOS) and causes vascular endothelium dysfunction. Elevated homocysteine levels have been associated with increase in LDL oxidation, generation of hydrogen peroxides, superoxide anions that increased oxidative degradation of nitric oxide. Hyperhomocysteinemia has been reported to increase the endogenous competitive inhibitors of eNOS viz L-N-monomethyl arginine (L-NMMA) and asymmetric dimethyl arginine (ADMA) that may contribute to vascular endothelial dysfunction. Hypercholesterolemia stimulates oxidation of LDL cholesterol, release of endothelins, and generation of ROS. The increased cholesterol and triglyceride level and decreased protective HDL level, decreases the activity and expression of eNOS and disrupts the integrity of vascular endothelium, due to oxidative stress. Hypertension also stimulates release of endothelins, vasoconstrictor prostanoids, angiotensin II, inflammatory cytokines, xanthine oxidase and, thereby, reduces bioavailability of nitric oxide.

Conclusion

Thus, the cellular and molecular mechanisms underlying diabetes mellitus, hyperhomocysteinemia, hypercholesterolemia hypertension and hyperuricemia leads to an imbalance of phosphorylation and dephosphorylation status of lipid and protein kinase that cause modulation of vascular endothelial L-arginine/nitric oxide synthetase (eNOS), to produce vascular endothelium dysfunction.

     

Angiotensin 1–7, but not the thrombin-cleaved osteopontin C-terminal fragment,attenuates osteopontin-mediated macrophage-induced endothelial-cell inflammation

Objective and design

Evaluating the pro-/anti-inflammatory activity of the C-terminal cleavage product of osteopontin in comparison to angiotensin 1–7.

Material and subjects

Human coronary endothelial cells (hcEC) treated with conditioned media from human U937 macrophages.

Treatment

Macrophages were (pre)treated with C-terminal, full-length or N-terminal osteopontin (OPN-C, OPN-FL, OPN-N, respectively), angiotensin II, angiotensin 1–7 or TNF-α. OPN-C modulatory capacity was compared to that of Ang1–7 in inhibiting subsequent Ag II, OPN-FL or OPN-N-induced macrophage-mediated endothelial inflammation.

Methods

Protein expression of NFκB, IκB, vCAM-1 and iCAM-1 was assessed using western blot. Promotor activation by NFκB was also assessed by dual-luciferase reporter assay.

Results

Conditioned media of macrophages treated with OPN-C induced hcECs’ NfκB activation to a lower degree than OPN-FL or OPN-N. Priming of macrophages with angiotensin 1–7 attenuated the endothelial pro-inflammatory effect induced by subsequent exposure of the macrophages to angiotensin II, OPN-FL or OPN-N. This was evidenced by both NfκB activation and vCAM and iCAM expression. In contrast, priming macrophages with OPN-C did not significantly attenuate the subsequent response to the pro-inflammatory cytokines.

Conclusions

OPN-C induces lower macrophage-induced endothelial inflammation compared to OPN-FL or OPN-N, but unlike angiotensin 1–7, fails to prevent endothelial inflammation induced by subsequent pro-inflammatory macrophage stimulation.

     

Maoberry (<Emphasis Type="Italic">Antidesma bunius</Emphasis>) ameliorates oxidative stress and inflammation in cardiac tissues of rats fed a high-fat diet

Backgound

Chronic fat-rich diets consumption is increased risk associated with cardiovascular diseases (CVD). Prevention or reduction the progression of cardiac tissue deterioration could benefit in CVD. This study aimed to examine the effects of maoberry (Antidesma bunius), a antioxidant-rich tropical fruit, supplementation on oxidative stress and inflammation in cardiac tissues of rats fed a high-fat diet (HFD).

Methods

The male rats orally received HFD with maoberry extract doses of 0.38, 0.76 or 1.52?g/kg or simvastatin (10?mg/kg) for 12?weeks. At the end of the experimental period, the rats were fasted, euthanized and harvested for the hearts.

Results

Significantly reduced oxidative stress (malondialdehyde levels) and enhanced antioxidant capacity (ferric-reducing activities) in cardiac tissues of the rats were found. Maoberry extract remarkably ameliorated the expressions of genes involved with pro-inflammatory such as the tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1) and endothelial nitric oxide synthase (eNOS).

Conclusions

Our findings suggest that maoberry extract has remarkable effects on preventing progression of cardiac tissue deterioration at least through lowering oxidative stress and inflammation.

     

Resistin and NGAL are associated with inflammatory response,endothelial activation and clinical outcomes in sepsis

Objective and design

Resistin and neutrophil gelatinase-associated lipocalin (NGAL) are upregulated in circulating leucocytes in sepsis, but the significance of this is uncertain. We evaluated associations between Resistin and NGAL with endothelial cell activation and clinical outcomes in a prospective observational study in the Emergency Department (ED).

Methods

Serum levels of Resistin, NGAL, inflammatory cytokines (IL-6, IL-10) and soluble endothelial adhesion molecules (VCAM-1, ICAM-1) were measured at defined time points up to 24 h. Patterns and relationships between markers were investigated using linear mixed regression models. Predictive values for clinical outcomes for markers at enrollment were assessed by logistic regression and receiver operator characteristic (ROC) curves.

Results

186 participants (89 septic-shock, 69 sepsis, 28 uncomplicated infection) were compared with 29 healthy controls. Median Resistin and NGAL were higher in uncomplicated infection compared to controls, and in septic shock compared to non-shock sepsis. Resistin and NGAL correlated with IL-6 and IL-10, with VCAM-1 and ICAM-1, and with organ failure. Resistin and NGAL were associated with septic shock but had limited predictive utility for mortality.

Conclusion

Resistin and NGAL correlate with expression of endothelial cell adhesion molecules in sepsis. Further evaluation of the role of Resistin and NGAL in sepsis pathogenesis is warranted.

     

Hypermethylation of MEG3 promoter correlates with inactivation of MEG3 and poor prognosis in patients with retinoblastoma

Background

In our previous study, we revealed that MEG3 was a tumor suppressor gene in retinoblastoma and inhibited proliferation of retinoblastoma cells by regulating the activity of the Wnt/β-catenin pathway. Here, we further explored the mechanism of MEG3 inactivation in retinoblastoma.

Methods

MSP and qRT-PCR were performed to detect the methylation status of MEG3 promoter and levels of MEG3 expression, respective. To further explore relationship between MEG3 expression and epigenetic modifications, 5-Aza-CdR was used to interfere with DNA methylation. In addition, we evaluated proliferation, apoptosis and the expression of β-catenin via CCK-8, flow cytometric analysis and western blot analysis, respective.

Results

Hypermethylation of MEG3 promoter was observed more frequently in retinoblastoma tissues and was highly associated with low MEG3 expression and poor survival of retinoblastoma patients. We also provided evidence demonstrating that hypermethylation of MEG3 promoter depressed MEG3 expression, promoted proliferation, inhibited apoptosis and increased β-catenin expression of retinoblastoma cells in vitro.

Conclusions

Our present study indicates that promoter silencing by hypermethylation may account for the loss of MEG3 expression and predict poor prognosis.

     

Multicolor Flow Cytometry for the Diagnosis of Primary Immunodeficiency Diseases

Purpose

Primary immunodeficiency diseases (PIDDs) are rare inherited diseases that impair the human immune system. We established a multicolor flow cytometric assay to comprehensively evaluate the immune status and immunological characteristics of patients with PIDDs.

Methods

Fifty-nine normal controls and 75 patients with PIDDs, including X-linked severe combined immunodeficiency (X-SCID), X-linked agammaglobulinemia (XLA), X-linked hyper IgM syndrome (X-HIGM), ataxia telangiectasia (AT), Wiskott-Aldrich syndrome (WAS), hyper IgE syndrome (HIES), and chronic mucocutaneous candidiasis disease (CMCD), were enrolled in this study. Immunophenotyes were evaluated by multicolor flow cytometry using seven different panels that allowed the detection of major leukocyte populations in peripheral blood.

Results

Multicolor flow cytometry revealed distinct leukocyte populations and immunological features of patients with X-SCID, XLA, X-HIGM, AT, WAS, HIES, and CMCD.

Conclusions

Immunophenotyping by multicolor flow cytometry is useful to evaluate immune status and contributes to the diagnosis and management of patients with PIDDs.

     

Deficiencies in elements involved in TLR4-receptor signalling in RBL-2H3 cells

Objective

RBL-2H3 cells express Toll-like receptors, including TLR4. This study aims to assess various aspects of the TLR4 pathway.

Methods

RBL-2H3 cells were indirectly stained for cell surface TLR4, 25 CD14 and intracellular MyD88 proteins and analysed through flow cytometry for single-colour staining.

Results

While TLR4-receptors are expressed in RBL-2H3 cells, associated elements involved in the signaling pathway, CD14 and MyD88, are not.

Conclusion

Care should be taken if RBL-2H3 cells are used to study aspects of the innate immune system in mast cells.

     

NETosis provides the link between activation of neutrophils on hemodialysis membrane and comorbidities in dialyzed patients

Introduction

Neutrophil extracellular traps (NETs) are formed by activated neutrophils during the process of NETosis in which the nuclear material is released into extracellular space, including DNA molecules, citrullinated histones, and neutrophil granule enzymes, such as elastase. This material forms networks that are able not only to physically entrap bacteria but also to provide elevated concentration of bactericidal components. Over the last years, it has become clear that NETs can also be formed under numerous sterile inflammatory conditions, i.e., thrombosis, cancer, SLE, atherosclerosis, and diabetes.

Method

We reviewed studies published until July 2016 to find possible associations between elevated cell-free DNA levels in dialyzed patients and the process of NETosis and its consequences.

Results

The process of NETosis, its elevated activation, or impaired clearance provides the link between clinical conditions and elevated levels of cell-free DNA found in plasma after the hemodialytic procedure which itself is able to activate neutrophils via platelets and ROS formation. NETs stimulate thrombosis and endothelial damage, and their formation may contribute to the development of spectrum of comorbidities described in dialyzed patients.

Conclusion

The study of plasma cell-free DNA levels together with markers of NETosis could contribute to the evaluation of the influence of hemodialysis on the immune system of patients.

     

TLR4 regulates insulin-resistant proteins to increase apoptosis in the mouse retina

Objective and design

Work in multiple organs has suggested that toll-like receptor 4 (TLR4) may play a role in insulin resistance. Additional studies have shown a negative role for TLR4 on retinal health. We have previously reported that β-adrenergic receptors can regulate both TLR4 signal transduction, as well as insulin signaling in the retina and in retinal endothelial cells. Thus, we hypothesized that TLR4 would regulate retinal insulin signaling.

Materials and methods

We used endothelial cell-specific TLR4 knockout mice, as well as TLR4-overexpressing mice for these studies.

Methods

Western blotting and ELISA analyses were done for investigations of insulin receptor, insulin receptor substrate 1 (IRS-1) serine 307, and Akt phosphorylation, as well as cleaved caspase 3 levels in the mouse retina.

Results

We found that loss of TLR4 led to increased insulin receptor and Akt phosphorylation, as well as decreased IRS-1^Ser307 levels. In support of these results, TLR4 overexpression decreased insulin signaling and the cleavage of caspase 3.

Conclusions

Therefore, these results suggest that TLR4 plays a key role in insulin signaling in the retina. Reduction of TLR4 levels may be protective to the retina.

     

Hepatic mobilization of zinc after an experimental surgery,and its relationship with inflammatory cytokines release,and expression of metallothionein and Zip14 transporter

Objective

To study the relationship between the release of inflammatory cytokines and mobilization of zinc into liver, and the expression of metallothionein and Zip14 transporter after an abdominal surgery in rats.

Materials

Thirty-five male Wistar rats were subjected to experimental surgical stress, then the subgroups of five animals were killed at 3, 6, 9, 12, 16, 20 and 24 h. Matched groups without surgery were used as controls.

Methods

Zinc levels were determined by AAS, intracellular zinc by zinquin and dithizone staining. Hepatic metallothionein was assayed by a Cd-saturation method, and IL-1β, IL-6, and TNF-β by immunoassays. Zip14 expression was analyzed by real-time RT-PCR, and protein level by immunohistochemistry and Western blot.

Results

Experimental surgery produced a hypozincemia, and the increase of hepatic zinc also produced the release of IL-1β, IL-6 in serum, and the increase of hepatic MT. Histochemistry showed a decrease of free zinc at 3–6 h, but an increase at 9 h (zinquin); meanwhile, total intracellular zinc increased after 9 h (dithizone). RNAm and protein levels of Zip14 were elevated between 6 and 20 h after surgery.

Conclusion

Biochemical changes described in this work could be part of the APR, and directed to respond to the damage produced during surgical trauma.

     

Lack of MMP-9 expression is a marker for poor prognosis in Dukes’ B colorectal cancer

Background

Matrix metalloproteinases (MMPs) play a role in cancer progression by degrading extracellular matrix and basement membranes, assisting in tumour neovascularization and in supporting immune response in cancer.

Methods

We studied the prognostic value of immunohistochemical expression of MMP-2, MMP-8, and MMP-9 in a series of 619 colorectal cancer patients using tissue microarray specimens.

Results

Of the samples, 56% were positive for MMP-2, 78% for MMP-8, and 60% for MMP-9. MMP-9 associated with low WHO grade (p?<?0.001). In univariate analysis of Dukes’ B tumours, MMP-9 negativity associated with poor survival (p?=?0.018), and MMP-9 positivity was an independent prognostic marker in multivariate analysis of these tumours (p?=?0.034).

Conclusion

Negative MMP-9 expression can predict poor prognosis in Dukes’ B colorectal tumours and may prove useful for identifying patients, who should be offered adjuvant treatment.

     

Tinnitus,Traumatic Brain Injury,and Posttraumatic Stress Disorder in the Military

Purpose

Acoustic trauma is more prevalent in military settings, especially among individuals with combat-related military occupational specialties. Gunfire, improvised explosive devices, and mortar explosions are a few examples that may cause hearing degradation and tinnitus. It is possible that the same events that are associated with auditory problems can cause traumatic brain injury (TBI) and posttraumatic stress disorder (PTSD).

Method

This paper reviews the distinct and overlapping symptoms of tinnitus, TBI, and PTSD, and how these disorders interact to synergistically promote negative outcomes.

Results

Tinnitus may serve as a significant contributor to symptoms of TBI and PTSD. Therefore, tinnitus subtypes could be identified as physiologically or psychologically based, or both.

Conclusions

Additional research is warranted to determine the common and unique symptoms and associated neurological pathways of tinnitus, TBI, and PTSD. Brief treatment recommendations are provided, including a multidisciplinary approach for the physical and psychological distress associated with tinnitus.

     

Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), an anticancer agent,exerts an anti-inflammatory effect in activated human mast cells

Objective

Inflammation has been closely associated with the development and progression of cancer. Previously, we reported that mast cells play a critical role in tumor growth. The purpose of this study is to investigate the anti-inflammatory effect of an anticancer agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), on an activated human mast cell line, in this case HMC-1 cells.

Methods

We evaluated the effect and specific molecular mechanism of Dp44mT on phorbol 12-myristate 13-acetate and calcium ionophore A23187 (PMACI) using HMC-1 cells.

Results

Here, we demonstrated that Dp44mT significantly decreased the protein levels of hypoxia-inducible factor-1α and vascular endothelial growth factor without exposing activated HMC-1 cells to any cytotoxicity. In activated mast cells, Dp44mT mitigated the strong production and mRNA expression of inflammatory cytokines, in this case, interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and thymic stromal lymphopoietin, through a blockade of caspase-1 and nuclear factor-κB activities. Furthermore, phosphorylations of the mitogen-activated protein kinase family included in inflammatory signaling cascades were significantly inhibited by a Dp44mT treatment.

Conclusions

Overall, our results indicate that the anticancer agent Dp44mT has an anti-inflammatory effect and may be of therapeutic importance for the treatment of mast cell-mediated inflammatory diseases.

     

Leptin stimulates tissue rat mast cell pro-inflammatory activity and migratory response

Objective

The aim of this study was to determine whether leptin, a member of the adipocytokines involved in immune and inflammatory response regulation, may influence some aspects of mast cell biology.

Materials and methods

Experiments were done in vitro on fully mature tissue rat mast cells isolated from the peritoneal cavity, and leptin was used at concentrations 0.001–100 ng/ml. The effect of leptin on mast cell degranulation (histamine release assay), intracellular Ca²⁺ level (fluorimetry), pro-inflammatory mediator release (ELISA technique), surface receptor expression (flow cytometry and confocal microscopy), and migration (Boyden microchamber assay) was estimated.

Results

Leptin was found to stimulate mast cells to degranulation and histamine release. It induced the intracellular Ca²⁺ increase, as well. In response to leptin stimulation, mast cells generated and released cysLTs and chemokine CCL3. Leptin-induced upregulation of CYSLTR1 and CYSLTR2 surface expression was observed. Moreover, this adipocytokine stimulated mast cells to migratory response, even in the absence of extracellular matrix (ECM) proteins.

Conclusions

Our observations clearly documented that leptin promotes the pro-inflammatory activity of mast cells, and it thereby engages these cells in the inflammatory processes.

     

Examining the Associations Between Overeating,Disinhibition, and Hunger in a Nonclinical Sample of College Women

Background

Binge eating (BE) has long been identified as a correlate of overweight and obesity. However, less empirical attention has been given to overeating with and without loss of control (LOC) in nonclinical samples.

Purpose

The goal of the present study was to examine the association of (1) established correlates of BE, namely, weight and shape concerns, dietary restraint, and negative affect, and (2) three additional correlates, disinhibition, hunger, and interoceptive awareness (IA), to overeating in a nonclinical sample of college women.

Method

Female students (n?=?1,447) aged 18 to 21 years recruited from colleges in three Canadian metropolitan areas completed self-report questionnaires in class to assess sociodemographic and anthropomorphic characteristics, overeating, LOC, dietary restraint, negative affect, weight and shape concerns, IA, disinhibition, and hunger.

Results

The established correlates of BE were significant correlates of all types of overeating and explained 33 % of the variance. Disinhibition was the most strongly associated correlate of overeating.

Conclusions

Findings suggest that established correlates of BE are associated with other types of overeating such as objective overeating (OOE), as are disinhibition and hunger.

     

Social Relationship Factors,Preoperative Depression,and Hospital Length of Stay in Surgical Patients

Purpose

The interrelated associations of social relationship factors, depression, and outcomes of surgical patients are yet unexplored. The purpose of this study was to investigate whether depression mediates effects of general social support, loneliness, and living alone on hospital length of stay (LOS) of 2487 patients from diverse surgical fields.

Method

Social relationship factors and depression were assessed prior to surgery. The PROCESS macro for SPSS was used to conduct three simple mediation models that tested the indirect effects of social relationship factors on LOS mediated through depression. The models were adjusted for age, gender, preoperative physical health, surgical field, severity of medical comorbidity, and extent of surgical procedure.

Results

Social support and loneliness had significant indirect effects on LOS that were statistically mediated by preoperative depression. Lower social support and the feeling of loneliness were considerably related to higher depression which predicted longer LOS. While social support and loneliness had no direct effects on LOS, there was a small significant direct association of living alone with shorter LOS.

Conclusion

Data suggest that social support and loneliness are indirectly related with surgical outcomes by an association with depression which in turn is related to worse outcomes.

Trial Registration

NCT01357694

     

Ashwagandha attenuates TNF-α- and LPS-induced NF-κB activation and CCL2 and CCL5 gene expression in NRK-52E cells

Background

The aging kidney is marked by a chronic inflammation, which may exacerbate the progression of renal dysfunction, as well as increase the susceptibility to acute injury. The identification of strategies to alleviate inflammation may have translational impact to attenuate kidney disease.

Methods

We tested the potential of ashwaganda, sutherlandia and elderberry on tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) induced chemokine (CCL2 and CCL5) expression in vitro.

Results

Elderberry water-soluble extract (WSE) was pro-inflammatory, while sutherlandia WSE only partially attenuated the TNF-α-induced changes in CCL5. However, ashwaganda WSE completely prevented TNF-α-induced increases in CCL5, while attenuating the increase in CCL2 expression and NF-κB activation. The same pattern of ashwagandha protection was seen using LPS as the pro-inflammatory stimuli.

Conclusions

Taken together, these results demonstrate the ashwaganda WSE as a valid candidate for evaluation of therapeutic potential for the treatment of chronic renal dysfunction.