Glucose-induced regulation of novel iron transporters in vascular endothelial cell dysfunction

Increased iron indices have been associated with the development of diabetes and its complications. In the present study, we have investigated the glucose-induced alteration of iron transporters, divalent metal transporter-1 (DMT-1), iron regulated transporter protein-1 (IREG-1), and transferrin receptor (TfR), in endothelial cell iron accumulation and oxidative stress. Cells were exposed to high glucose levels and subjected to gene expression, protein expression, iron measurement and assessment of oxidative stress. Our results show, for the first time, expression of DMT-1 and IREG-1 in vascular endothelial cells. Our data further indicates upregulation of DMT-1 and IREG-1 mRNA and protein in response to high levels of glucose. TfR, however, exhibited a modest decrease in response to high levels of glucose. Increased expression of DMT-1 and IREG-1 was associated with iron accumulation and oxidative stress. Furthermore, our results show differential expression of iron transporters with treatment of high glucose-exposed cells with two different iron chelators. In conclusion, our study suggests that glucose-induced alteration of iron transporters may arbitrate iron accumulation and oxidative stress in endothelial cells.     

Nitrite supplementation reverses vascular endothelial dysfunction and large elastic artery stiffness with aging

We tested the hypothesis that short-term nitrite therapy reverses vascular endothelial dysfunction and large elastic artery stiffening with aging, and reduces arterial oxidative stress and inflammation. Nitrite concentrations were lower (P < 0.05) in arteries, heart, and plasma of old (26-28 month) male C57BL6 control mice, and 3 weeks of sodium nitrite (50 mg L(-1) in drinking water) restored nitrite levels to or above young (4-6 month) controls. Isolated carotid arteries of old control mice had lower acetylcholine (ACh)-induced endothelium-dependent dilation (EDD) (71.7 ± 6.1% vs. 93.0 ± 2.0%) mediated by reduced nitric oxide (NO) bioavailability (P < 0.05 vs. young), and sodium nitrite restored EDD (95.5 ± 1.6%) by increasing NO bioavailability. 4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), a superoxide dismutase (SOD) mimetic, apocynin, a nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, and sepiapterin (exogenous tetrahydrobiopterin) each restored EDD to ACh in old control, but had no effect in old nitrite-supplemented mice. Old control mice had increased aortic pulse wave velocity (478 ± 16 vs. 332 ± 12 AU, P < 0.05 vs. young), which nitrite supplementation lowered (384 ± 27 AU). Nitrotyrosine, superoxide production, and expression of NADPH oxidase were ～100-300% greater and SOD activity was ～50% lower in old control mice (all P < 0.05 vs. young), but were ameliorated by sodium nitrite treatment. Inflammatory cytokines were markedly increased in old control mice (P < 0.05), but reduced to levels of young controls with nitrite supplementation. Short-term nitrite therapy reverses age-associated vascular endothelial dysfunction, large elastic artery stiffness, oxidative stress, and inflammation. Sodium nitrite may be a novel therapy for treating arterial aging in humans.     

Myricetin ameliorates high glucose-induced endothelial dysfunction in human umbilical vein endothelial cells

Endothelial dysfunction is recognized as the initial detectable stage of cardiovascular disease, a serious complication of diabetes. In this study, we evaluated effects of myricetin on high glucose (HG)-elicited oxidative damage in human umbilical vein endothelial cells (HUVECs). The cells were pre-incubated with myricetin and then treated with HG to induce apoptosis. The effect of myricetin on viability was investigated by MTT assay. The levels of lipid peroxidation (LPO) were determined by thiobarbituric acid (TBA) method. The protein expression of Bax, Bcl-2 and caspase-3 was measured by western blot analysis. Moreover, the effect of myricetin on total antioxidant capacity (TAC) and total thiol molecules was also determined. Our results showed that myricetin was able to markedly restore the viability of endothelial cells under oxidative stress. Myricetin reduced HG-caused increase in LPO levels. Also, TAC and total thiol molecules were notably elevated by myricetin. Incubation with myricetin decreased the protein expression levels of Bax, whereas it increased the expression levels of the Bcl-2, compared with HG treatment alone. Furthermore, myricetin significantly decreased cleaved caspase-3 protein expression. It is concluded that myricetin may protect HUVECs from oxidative stress induced by HG via increasing cell TAC and reducing Bax/Bcl-2 protein ratio, and caspase-3 expression.     

Curcumin induces autophagy to protect vascular endothelial cell survival from oxidative stress damage

Our study first proposed that curcumin could protect human endothelial cells from the damage caused by oxidative stress via autophagy. Furthermore, our results revealed that curcumin causes some novel cellular mechanisms that promote autophagy as a protective effect. Pretreatment with curcumin remarkably improves the survival of human umbilical vein endothelial cells (HUVECs) from H₂O₂-induced viability loss, which specifically evokes an autophagic response. Exposed to H₂O₂, curcumin-treated HUVECs upregulate the level of microtubule-associated protein 1 light chain 3-II (LC3-II), the number of autophagosomes, and the degradation of p62. We show that this compound promotes BECN1 expression and inhibits the phosphatidylinositol 3-kinase (PtdIns3K)-AKT-mechanistic target of rapamycin (MTOR) signaling pathway. Curcumin can also reverse FOXO1 (a mediator of autophagy) nuclear localization along with causing an elevated level of cytoplasmic acetylation of FOXO1 and the interaction of acetylated FOXO1 and ATG7, under the circumstance of oxidative stress. Additionally, knockdown of FOXO1 by shRNA inhibits not only the protective effects that curcumin induced, but the autophagic process, from the quantity of LC3-II to the expression of RAB7. These results suggest that curcumin induces autophagy, indicating that curcumin has the potential for use as an autophagic-related antioxidant for prevention and treatment of oxidative stress. These data uncover a brand new protective mechanism involving FOXO1 as having a critical role in regulating autophagy in HUVECs, and suggest a novel role for curcumin in inducing a beneficial form of autophagy in HUVECs, which may be a potential multitargeted therapeutic avenue for the treatment of oxidative stress-related cardiovascular diseases.     

Mechanism of endoplasmic reticulum stress-induced vascular endothelial dysfunction

Background

We recently reported that ER stress plays a key role in vascular endothelial dysfunction during hypertension. In this study we aimed to elucidate the mechanisms by which ER stress induction and oxidative stress impair vascular endothelial function.

Methodology/principal findings

We conducted in vitro studies with primary endothelial cells from coronary arteries stimulated with tunicamycin, 1 μg/mL, in the presence or absence of two ER stress inhibitors: tauroursodeoxycholic acid (Tudca), 500 μg/mL, and 4-phenylbutyric acid (PBA), 5 mM. ER stress induction was assessed by enhanced phosphorylation of PERK and eIF2α, and increased expression of CHOP, ATF6 and Grp78/Bip. The ER stress induction increased p38 MAPK phosphorylation, Nox2/4 mRNA levels and NADPH oxidase activity, and decreased eNOS promoter activity, eNOS expression and phosphorylation, and nitrite levels. Interestingly, the inhibition of p38 MAPK pathway reduced CHOP and Bip expressions enhanced by tunicamycin and restored eNOS promoter activation as well as phosphorylation. To study the effects of ER stress induction in vivo, we used C57BL/6J mice and p47phox^−/− mice injected with tunicamycin or saline. The ER stress induction in mice significantly impaired vascular endothelium-dependent and independent relaxation in C57BL/6J mice compared with p47phox^−/− mice indicating NADPH oxidase activity as an intermediate for ER stress in vascular endothelial dysfunction.

Conclusion/significance

We conclude that chemically induced ER stress leads to a downstream enhancement of p38 MAPK and oxidative stress causing vascular endothelial dysfunction. Our results indicate that inhibition of ER stress could be a novel therapeutic strategy to attenuate vascular dysfunction during cardiovascular diseases.     

The role of endothelial dysfunction and oxidative stress in cerebrovascular diseases

Cerebrovascular diseases (CBD) are one of the most dangerous complications of atherosclerosis. The clinical consequences of CBD deeply impact quality of life and the prognosis of patients. Atherosclerosis is the main cause of CBD development. Hypertension, dyslipidemia, diabetes, smoking, obesity, and other risk factors explain the higher CBD incidence in the general population, as they are able to anticipate the clinical expression of atherosclerosis. These risk factors are effectively able to promote endothelial dysfunction which is the premise for the early, clinical expression of atherosclerosis. The mechanisms by which risk factors can influence the occurrence of CBD are different and not fully understood. The inflammatory background of atherosclerosis can explain a great part of it. In particular, the oxidative stress may promote the development of vascular lesions by negatively influencing biochemical cellular processes of the endothelium, thus predisposing the vascular tree to morphological and functional damages. The aim of this narrative review is to evaluate the role of endothelial dysfunction and oxidative stress in CBD development.     

人源性激肽释放酶结合蛋白与内皮细胞功能

人源性激肽释放酶结合蛋白（Kallistatin,Kal）是一种负性急性期内源性蛋白,与多种内皮相关性生理和病理过程密切相关,如血管生成及损伤修复、炎症、心功能不全、肾损伤、糖尿病等。炎症和氧化应激可引起内皮功能障碍,而Kal可抑制肿瘤坏死因子α引起的内皮细胞活化,通过KLF4-eNOS、PI3K-AKT-eNOS和AKT-FOXO1等信号通路,增加内皮细胞NO合酶的表达和NO生成,抑制内皮细胞损伤和凋亡。动物实验显示,Kal表达增加可减弱氧化应激诱导的细胞凋亡和器官损伤。基于内皮细胞所处的状态或来源,如健康或损伤情况,成熟内皮细胞或内皮祖细胞,Kal的作用可能有所区别。内皮细胞是参与肿瘤生长与转移的关键因素已达成共识,但肿瘤新生血管形成的机制尚待确认。Kal可诱导肿瘤内皮细胞凋亡,抑制肿瘤新生血管生成和肿瘤生长的能力已被证实。临床前研究结果表明,Kal具有多种药理作用,对氧化应激相关性疾病,特别是肿瘤治疗具有应用前景,但其药理作用的分子机制仍需深入探讨。     

Mechanisms of endothelial dysfunction with development of type 1 diabetes mellitus: role of insulin and C-peptide

Complications associated with insulin-dependent diabetes mellitus (type-1diabetes) primarily represent vascular dysfunction that has its origin in the endothelium. While many of the vascular changes are more accountable in the late stages of type-1diabetes, changes that occur in the early or initial functional stages of this disease may precipitate these later complications. The early stages of type-1diabetes are characterized by a diminished production of both insulin and C-peptide with a significant hyperglycemia. During the last decade numerous speculations and theories have been developed to try to explain the mechanisms responsible for the selective changes in vascular reactivity and/or tone and the vascular permeability changes that characterize the development of type-1diabetes. Much of this research has suggested that hyperglycemia and/or the lack of insulin may mediate the observed functional changes in both endothelial cells and vascular smooth muscle. Recent studies suggest several possible mechanisms that might be involved in the observed decreases in vascular nitric oxide (NO) availability with the development of type-1 diabetes. In addition more recent studies have indicated a direct role for both endogenous insulin and C-peptide in the amelioration of the observed endothelial dysfunction. These results suggest a synergistic action between insulin and C-peptide that facilitates increase NO availability and may suggest new clinical treatment modalities for type-1 diabetes mellitus.     

The role of iron in Campylobacter gene regulation,metabolism and oxidative stress defense

Enteric Campylobacter species cause gastrointestinal diseases in humans. Like almost all organisms, campylobacters have an absolute requirement for iron, but are faced with variable availability of iron in the environment and host tissues. Campylobacters have developed mechanisms to scavenge sufficient iron for metabolism and growth. However, iron also participates in the formation of reactive oxygen species, and this forces pathogens to maintain intracellular iron homeostasis and to cope with oxidative stresses. The presence of two separate, but possibly overlapping iron-responsive regulatory systems, which regulate iron acquisition and oxidative stress defense, and the presence of genes encoding multiple iron acquisition and detoxification systems in Campylobacter indicate the central role that iron plays in Campylobacter gene regulation and virulence.     

Up‐regulation of HDACs,a harbinger of uraemic endothelial dysfunction,is prevented by defibrotide

Endothelial dysfunction is an earlier contributor to the development of atherosclerosis in chronic kidney disease (CKD), in which the role of epigenetic triggers cannot be ruled out. Endothelial protective strategies, such as defibrotide (DF), may be useful in this scenario. We evaluated changes induced by CKD on endothelial cell proteome and explored the effect of DF and the mechanisms involved. Human umbilical cord vein endothelial cells were exposed to sera from healthy donors (n = 20) and patients with end‐stage renal disease on haemodialysis (n = 20). Differential protein expression was investigated by using a proteomic approach, Western blot and immunofluorescence. HDAC1 and HDAC2 overexpression was detected. Increased HDAC1 expression occurred at both cytoplasm and nucleus. These effects were dose‐dependently inhibited by DF. Both the HDACs inhibitor trichostatin A and DF prevented the up‐regulation of the endothelial dysfunction markers induced by the uraemic milieu: intercellular adhesion molecule‐1, surface Toll‐like receptor‐4, von Willebrand Factor and reactive oxygen species. Moreover, DF down‐regulated HDACs expression through the PI3/AKT signalling pathway. HDACs appear as key modulators of the CKD‐induced endothelial dysfunction as specific blockade by trichostatin A or by DF prevents endothelial dysfunction responses to the CKD insult. Moreover, DF exerts its endothelial protective effect by inhibiting HDAC up‐regulation likely   through PI3K/AKT.     

S-adenosyl methionine prevents endothelial dysfunction by inducing heme oxygenase-1 in vascular endothelial cells

S-adenosyl methionine (SAM) is a key intermediate in the metabolism of sulfur amino acids and is a major methyl donor in the cell. Although the low plasma level of SAM has been associated with atherosclerosis, the effect of SAM administration on atherosclerosis is not known. Endothelial dysfunction is an early prerequisite for atherosclerosis. This study was undertaken to investigate the possible preventive effect of SAM on endothelial dysfunction and the molecular mechanism of its action. SAM treatment prevented endothelial dysfunction in high fat diet (HFD)-fed rats. In cultured human aortic endothelial cells, linoleic acid (LA) increased and SAM decreased cell apoptosis and endoplasmic reticulum stress. Both LA and SAM increased heme oxygenase-1 (HO-1) expression in an NF-E2-related factor 2-dependent manner. However, knockdown of HO-1 reversed only the SAM-induced preventive effect of cell apoptosis. The LA-induced HO-1 expression was dependent on PPARα, whereas SAM induced HO-1 in a PPAR-independent manner. These data demonstrate that SAM treatment prevents endothelial dysfunction in HFDfed animals by inducing HO-1 in vascular endothelial cells. In cultured endothelial cells, SAM-induced HO-1 was responsible for the observed prevention of cell apoptosis. We propose that SAM treatment may represent a new therapeutic strategy for atherosclerosis.     

Study of the association of adrenomedullin and basic-fibroblast growth factors with the peripheral arterial blood flow and endothelial dysfunction biomarkers in type 2 diabetic patients with peripheral vascular insufficiency

Background

Progressive micro-vascular vaso-degeneration is the major factor in progression of diabetic complications. Adrenomedullin (AM) and basic-Fibroblast growth factor (b-FGF) are strongly correlated with angiogenesis in vascular diseases. This study aims to provide base line data regarding the vascular effects and correlation of AM, and b-FGF with the peripheral blood flow in diabetic patients with peripheral vascular disease (PVD), and their effect on endothelial dysfunction markers. Ninety age- and sex matched females were enrolled in the study: 30 were controls, 30 had diabetes without complications (group II) and 30 had diabetes with PVD (group III) diagnosed by ankle/ brachial index (A/BI). Plasma levels of AM, b-FGF, intercellular adhesion molecule −1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were measured by indirect enzyme immunoassay (ELISA).

Results

There was a significant increase in plasma AM, VCAM-1and ICAM-1, while a significant decrease in plasma b-FGF in diabetic patients with PVD (p < 0.05). A positive correlation was observed between plasma AM, b-FGF and A/BI and a negative correlation with VCAM −1 and ICAM in diabetic PVD. AM was not a predictor, while b-FG, VCAM-1 and ICAM-1 could be predictors for peripheral blood flow in diabetic PVD.

Conclusion

This study elucidates for the first time that AM and b-FGF are correlated and have a direct impact on the peripheral blood flow, the rise of AM in diabetic PVD may be a consecutive and compensatory vasculo-protective effect as its angiogenic and anti-inflammatory properties act to relief the endothelial insult. Down expression of b-FGF may be a predisposing factor for micro-vascular derangement. It is not clear if the rise of AM and the decline of b- FGF levels may be consequences or predisposing factors for VCAM-1 and ICAM-1 elevation as these endothelial dysfunction biomarkers could reduce peripheral blood flow and vascular integrity. It is optimistic to believe that drug intervention through AM and b-FGF administration together with reversing the endothelial inflammatory process by targeting VCAM and ICAM could reduce the prevalence of diabetic vascular complications, reduce the risk of cerebrovascular and cardiovascular morbidity in diabetes through normalizing vascular endothelium function and peripheral blood flow.     

Nicotinamide mononucleotide supplementation reverses vascular dysfunction and oxidative stress with aging in mice

We tested the hypothesis that supplementation of nicotinamide mononucleotide (NMN), a key NAD⁺ intermediate, increases arterial SIRT1 activity and reverses age‐associated arterial dysfunction and oxidative stress. Old control mice (OC) had impaired carotid artery endothelium‐dependent dilation (EDD) (60 ± 5% vs. 84 ± 2%), a measure of endothelial function, and nitric oxide (NO)‐mediated EDD (37 ± 4% vs. 66 ± 6%), compared with young mice (YC). This age‐associated impairment in EDD was restored in OC by the superoxide () scavenger TEMPOL (82 ± 7%). OC also had increased aortic pulse wave velocity (aPWV, 464 ± 31 cm s^?1 vs. 337 ± 3 cm s^?1) and elastic modulus (EM, 6407 ± 876 kPa vs. 3119 ± 471 kPa), measures of large elastic artery stiffness, compared with YC. OC had greater aortic production (2.0 ± 0.1 vs. 1.0 ± 0.1 AU), nitrotyrosine abundance (a marker of oxidative stress), and collagen‐I, and reduced elastin and vascular SIRT1 activity, measured by the acetylation status of the p65 subunit of NFκB, compared with YC. Supplementation with NMN in old mice restored EDD (86 ± 2%) and NO‐mediated EDD (61 ± 5%), reduced aPWV (359 ± 14 cm s^?1) and EM (3694 ± 315 kPa), normalized production (0.9 ± 0.1 AU), decreased nitrotyrosine, reversed collagen‐I, increased elastin, and restored vascular SIRT1 activity. Acute NMN incubation in isolated aortas increased NAD⁺ threefold and manganese superoxide dismutase (MnSOD) by 50%. NMN supplementation may represent a novel therapy to restore SIRT1 activity and reverse age‐related arterial dysfunction by decreasing oxidative stress.