Oxidative Stress In Endothelial Cells Research Articles

Some Features of the Development of AMD and Other Diseases of the Posterior Pole Associated with the Virus Carrier and the Novel Coronavirus Disease COVID-19

This article highlights main aspects of pathogenesis of age-related macular degeneration (AMD) and the connection between SARS-CoV-2 and diseases of the posterior pole of the eyes. Background: A novel coronavirus disease COVID-19 is actual nowadays due to its multiple organ damage, including eye tissues. Retina is also a target organ. AMD is the most common cause of central vision loss and its development is connected not only with ageing, but also with some other factors. Last years ophthalmological community considers virus infection to be a predictor and trigger for developing of AMD. Objective: Several theories have been proposed for the development of AMD. The most common theories are vascular due to pathogenetically significant decrease in blood flow in the pool of the carotid arteries; metabolic disorders (systemic and local); oxidative stress in endothelial cells; hereditary predisposition. Inflammation as a pathogenetic mechanism appeared in a novel light of discoveries in developing of not only AMD, but also some other eye diseases. The connection between SARS-CoV-2 and diseases of the posterior pole known nowadays are conjunctivitis, central retinal vein occlusion, isolated inflammatory optic neuritis, acute bilateral demyelinating optic neuritis. Thus, the question is still open. Conclusion: no single theory of the development of AMD can absolutely explain the diversity of pathological changes in of the retina and choroid tissues. Further study of the role of each factor of pathogenesis– from molecular to tissue will allow the development of fundamentally novel and perspective directions of AMD therapy.

Hyperoxalemia Leads to Oxidative Stress in Endothelial Cells and Mice with Chronic Kidney Disease

Introduction: Cardiovascular disease is the most common cause of morbidity and mortality in patients with ESRD. In addition to phosphate overload, oxalate, a common uremic toxin, is also involved in vascular calcification in patients with ESRD. The present study investigated the role and mechanism of hyperoxalemia in vascular calcification in mice with uremia. Methods: A uremic atherosclerosis (UA) model was established by left renal excision and right renal electrocoagulation in apoE^−/− mice to investigate the relationship between oxalate loading and vascular calcification. After 12 weeks, serum and vascular levels of oxalate, vascular calcification, inflammatory factors (TNF-α and IL-6), oxidative stress markers (malondialdehyde [MDA], and advanced oxidation protein products [AOPP]) were assessed in UA mice. The oral oxalate-degrading microbe Oxalobacter formigenes (O. formigenes) was used to evaluate the effect of a reduction in oxalate levels on vascular calcification. The mechanism underlying the effect of oxalate loading on vascular calcification was assessed in cultured human aortic endothelial cells (HAECs) and human aortic smooth muscle cells (HASMCs). Results: Serum oxalate levels were significantly increased in UA mice. Compared to the control mice, UA mice developed more areas of aortic calcification and showed significant increases in aortic oxalate levels and serum levels of oxidative stress markers and inflammatory factors. The correlation analysis showed that serum oxalate levels were positively correlated with the vascular oxalate levels and serum MDA, AOPP, and TNF-α levels, and negatively correlated with superoxide dismutase activity. The O. formigenes intervention decreased serum and vascular oxalate levels, while did not improve vascular calcification significantly. In addition, systemic inflammation and oxidative stress were also improved in the O. formigenes group. In vitro, high concentrations of oxalate dose-dependently increased oxidative stress and inflammatory factor expression in HAECs, but not in HASMCs. Conclusions: Our results indicated that hyperoxalemia led to the systemic inflammation and the activation of oxidative stress. The reduction in oxalate levels by O. formigenes might be a promising treatment for the prevention of oxalate deposition in calcified areas of patients with ESRD.

The Providence Mutation (βK82D) in Human Hemoglobin Substantially Reduces βCysteine 93 Oxidation and Oxidative Stress in Endothelial Cells.

The highly toxic oxidative transformation of hemoglobin (Hb) to the ferryl state (HbFe4+) is known to occur in both in vitro and in vivo settings. We recently constructed oxidatively stable human Hbs, based on the Hb Providence (βK82D) mutation in sickle cell Hb (βE6V/βK82D) and in a recombinant crosslinked Hb (rHb0.1/βK82D). Using High Resolution Accurate Mass (HRAM) mass spectrometry, we first quantified the degree of irreversible oxidation of βCys93 in these proteins, induced by hydrogen peroxide (H2O2), and compared it to their respective controls (HbA and HbS). Both Hbs containing the βK82D mutation showed considerably less cysteic acid formation, a byproduct of cysteine irreversible oxidation. Next, we performed a novel study aimed at exploring the impact of introducing βK82D containing Hbs on vascular endothelial redox homeostasis and energy metabolism. Incubation of the mutants carrying βK82D with endothelial cells resulted in altered bioenergetic function, by improving basal cellular glycolysis and glycolytic capacity. Treatment of cells with Hb variants containing βK82D resulted in lower heme oxygenase-1 and ferritin expressions, compared to native Hbs. We conclude that the presence of βK82D confers oxidative stability to Hb and adds significant resistance to oxidative toxicity. Therefore, we propose that βK82D is a potential gene-editing target in the treatment of sickle cell disease and in the design of safe and effective oxygen therapeutics.

NOD2 induces VCAM-1 and ET-1 gene expression via NF-κB in human umbilical vein endothelial cells with muramyl dipeptide stimulation.

Endothelial dysfunction is involved in various aspects of vascular biology and different stages of cardiovascular diseases (CVDs). Nucleotide-binding oligomerization domain-containing protein (NOD)2, apivotal innate immune receptor for muramyl dipeptide (MDP), has been reported to be acentral regulator in CVDs. Previously, we reported that NOD2 played aleading role in MDP-triggered oxidative stress in endothelial cells (ECs). However, whether NOD2 participates in the regulatory mechanism of vascular cell adhesion molecule‑1 (VCAM-1) and endothelin‑1 (ET-1) expression was not elucidated. Human umbilical vein endothelial cells (HUVECs) were stimulated with MDP for 12 h. mRNA expression of VCAM‑1 and ET‑1 was detected using real time polymerase chain reaction (PCR). Scrambled control small interfering RNA (siRNA) and NOD2 siRNA were transfected into HUVECs using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA). Furthermore, pyrrolidine dithiocarbamate was adopted to investigate the effect of nuclear factor κB (NF-κB) on NOD2-mediated VCAM‑1 and ET‑1 gene expression in MDP-treated HUVECs. Data showed that MDP significantly increased VCAM‑1 and ET‑1 mRNA expression, which was dependent on NOD2. In addition, NF-κB inhibition suppressed NOD2-mediated gene expression of VCAM‑1 and ET‑1. Collectively, we confirmed NOD2 aggravated VCAM‑1 and ET‑1 gene expression through NF-κB in HUVECs treated with MDP.

Abstract 15092: The Yin-yang of Bmpr2 and Ces1 in the Pulmonary Endothelium Aad Its Role in Pulmonary Arterial Hypertension

Background/Hypothesis: Pulmonary Arterial Hypertension (PAH) is a life-threatening disease characterized by loss of pulmonary microvessels and vascular remodeling. Loss of function BMPR2 mutations contribute to pulmonary endothelial cell (EC) apoptosis and oxidative stress, but their reduced penetrance suggests need for additional modifiers. Carboxylesterase1 (CES1) is endoplasmic reticulum (ER) enzyme responsible for detoxification, proteostasis, and redox balance. Similar to BMPR2 mutations, we found that loss of CES1 is associated with oxidative stress and apoptosis. In this study, we explore a plausible link between BMPR2 pathway and CES1 regulation in promoting EC survival and angiogenesis. Methods: PECs & lung tissue from healthy donors and PAH patients were obtained from PHBI. To induce oxidative stress, we used H 2 O 2 (100 μM) & methamphetamine HCl (METH, 0.5-2mM). Both siRNA and pharmacological approaches were used to inhibit BMPR2, Nrf2, and CES1 expression. Caspase and Matrigel assays were used to assess PEC survival and tube formation, respectively. Results: RNAseq of BMPR2-mutant PECs showed significantly less CES1 expression, which correlated with reduced protein expression in PEC lysates and within lung vascular lesions. In healthy PECs, BMP9 stimulation led to increase in CES1 expression that was absent post BMPR2 knockdown. CES1 gene transcription was by BMPR2-dependent activation of Nrf2, a transcription factor responsible for antioxidant gene expression and mitochondrial biogenesis. Inhibition of Nrf2 activation by ML385 (5μM) abrogates BMP9 induced CES1 mRNA levels similar to BMPR2 knockdown. The connection between BMPR2 and CES1 was further strengthened by CES1 knockdown studies in PECs that demonstrated reduction in BMPR2 protein synthesis associated with ER stress and reduced autophagy. Finally, lung examination in CRISPR generated CES1 +/- mice demonstrated increased microvascular muscularization at normoxia compared to wild type mice. Conclusion: BMPR2 and CES1 are part of common signaling pathway that protects PECs against oxidative stress and mitochondrial damage through a positive feedback loop. Interventions that restore CES1 activity could rescue BMPR2 signaling and serve as novel PAH therapeutics.

Abstract 14993: Dysfunctional Extracellular Microvesicles in Andean Highlanders With Excessive Erythrocytosis

Background: Chronic mountain sickness, a maladaptation to high altitude (>2,500 m) characterized by excessive erythrocytosis (EE) and often severe hypoxemia, is prevalent in Andean highlanders. EE increases the risk of cardiovascular events and contributes to vascular dysfunction. Circulating extracellular microvesicles (MVs) are key mediators of cardiovascular health and disease through their interaction with the vascular endothelium. The experimental aim of this study was to determine the effects of MVs isolated from adults with EE on endothelial cell inflammation, oxidative stress, apoptosis and nitric oxide (NO) production. Methods: Twenty-six male residents of Cerro de Pasco, Peru (4,340 m) were studied: 12 highlanders without EE (healthy; age: 40±4 yr; BMI: 26.4±1.7; Hb: 17.4±0.5 g/dL, SpO 2 : 86.9±1.0%) and 14 highlanders with EE (EE: 43±4 yr; 26.2±0.9; 24.4±0.4 g/dL; 79.7±1.6%). MVs were isolated from plasma by flow cytometry. Human umbilical vein endothelial cells were cultured and treated with MVs from either healthy or EE men. Results: MVs from highlanders with EE induced significantly higher release of interleukin (IL)-6 (89.8±2.7 vs 77.1±1.9 pg/mL) and IL-8 (62.0±2.7 vs 53.3±2.2 pg/mL) compared with MVs from healthy highlanders. Although intracellular expression of total NF-κB p65 (65.3±6.0 vs 74.9±7.8.9 AU) was not significantly affected, MVs from EE men resulted in ~25% higher (P<0.05) expression of p-NF-κB p65 (Ser536; active NF-κB) (173.6±14.3 vs 132.8±12.2 AU). Additionally, cell expression of the anti-inflammatory miR-146a and miR-181b were significantly suppressed by EE MVs. Cell oxidative stress and apoptotic susceptibility were not significantly affected by MVs from EE men. However, eNOS activation (231.3±15.5 vs 286.6±23.0 AU) and NO production (8.3±0.6 vs 10.7±0.7 μM/L) were significantly lower in cells treated with MVs from EE vs healthy men. Conclusion: Increased inflammation and decreased eNOS activity and NO production renders the vascular endothelium highly susceptible to atherosclerosis and thrombosis. Andean highlanders with EE exhibit dysfunctional circulating extracellular MVs that induce a proatherogenic endothelial phenotype contributing to their increased cardiovascular risk.

Sortilin evokes endothelial dysfunction and arterial hypertension through the dysregulation of sphingolipid metabolism and oxidative stress

Abstract Background Sortilin, a member of vacuolar protein sorting domain family Vps10, has been positively correlated with vascular and metabolic disorders in humans. Previous study has shown that, in response to Fas receptor stimulation, sortilin together with acid sphingomyelinase (ASMase) promote the clustering of lipid rafts and subsequent activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in coronary endothelial cells. However, whether sortilin plays a role in endothelial cells function is currently unknown. Purpose To assess whether sortilin per se was able to influence vascular function, thereby contributing to the pathogenesis of cardiovascular diseases. Methods Pressure myography was used to study vascular reactivity of mesenteric arteries. To investigate the involvement of acid sphingomyelinase (ASM), we performed gene silencing approach and fluorometric activity assay. NADPH oxidase lucigenin assay was used to evaluate oxidative stress in endothelial cells and resistance vessels. The effects of circulating sortilin on cardiovascular system was evaluated by systemic delivery of recombinant sortilin protein to wild-type (WT), sphingosine-1-phosphate receptor 3 (S1P3) and NADPH oxidase 2 (gp91phox/NOX2) deficient mice. Systolic arterial blood pressure (SBP) was noninvasively registered in conscious mice by tail-cuff blood monitoring. Finally, to explore the translational relevance of sortilin, we measured sortilin and NOX2 soluble derived peptide levels using ELISA and quantified sphingosine-1-phosphate (S1P) by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in plasma of hypertensive patients. Results Here we demonstrated that sortilin evoked endothelial dysfunction in mesenteric arteries due to increased NADPH oxidase-derived oxidative stress. Knockdown of ASM successfully prevented impairment of endothelial function. Using the inhibitor of sphingosine kinase type 1 (SphK1), sortilin failed to evoke endothelial impairment as well as NADPH oxidase activation. In endothelial cells, sortilin induced S1P-dependent activation of Rac1/NOX2 signaling axis, which was prevented by TY-52156, an antagonist of lysosphingolipid receptor S1P3. In vivo sortilin administration induced arterial hypertension in WT mice. In contrast, genetic deletion of S1P3 and gp91phox/NOX2 resulted in preservation of endothelial function and SBP unchanged levels after 14 days of systemic sortilin administration. Finally, to translate these research findings into a clinical setting, we found that hypertensive patients have higher plasma levels of sortilin, ASMase, S1P and soluble NOX2 derived peptide than normotensive subjects. Conclusions These results demonstrate the pathologic role of sortilin in the modulation of endothelial function and arterial blood pressure, suggesting that sortilin and its mediators might represent novel therapeutic targets in vascular diseases and hypertension. Funding Acknowledgement Type of funding source: None

Stratified Treatment of Heart Failure with preserved Ejection Fraction: rationale and design of the STADIA-HFpEF trial.

AimsHigh myocardial stiffness in heart failure with preserved ejection fraction (HFpEF) is attributed to comorbidity‐induced structural and functional remodelling through inflammation and oxidative stress affecting coronary microvascular endothelial cells and cardiomyocytes, which augments interstitial fibrosis and cardiomyocyte stiffness. In murine and human HFpEF myocardium, sodium glucose co‐transporter 2 (SGLT2) inhibition ameliorates cardiac microvascular endothelial cell and cardiomyocyte oxidative stress, while enhancing myocardial protein kinase G activity and lowering titin‐based cardiomyocyte stiffness. Failure of previous HFpEF outcome trials refocuses attention to improving pathophysiological insight and trial design with better phenotyping of patients and matching of therapeutic targets to prevailing pathogenetic mechanisms. SGLT2 inhibition could represent a viable therapeutic option especially in HFpEF patients in whom high diastolic left ventricular (LV) stiffness is predominantly caused by elevated cardiomyocyte stiffness and associated endothelial dysfunction, whereas HFpEF patients with extensive myocardial fibrosis might be less responsive. This study aims to investigate a stratified treatment approach, using dapagliflozin in heart failure patients with preserved ejection fraction without evidence of significant myocardial fibrosis.Methods and resultsThe Stratified Treatment to Ameliorate DIAstolic left ventricular stiffness in early Heart Failure with preserved Ejection Fraction (STADIA‐HFpEF) is a Phase II, randomized, 2 × 2 crossover trial, evaluating the efficacy of 13 weeks of treatment with dapagliflozin 10 mg od in 26 patients with HFpEF, with normal cardiac magnetic resonance imaging‐derived extracellular volume. The co‐primary endpoint is echocardiographically derived change in E/e'/LV end‐diastolic volume index and change in mean LV e'.ConclusionsThe STADIA‐HFpEF trial will be the first study to evaluate the direct effects of dapagliflozin on amelioration of LV stiffness, using histological phenotyping to discern early HFpEF.

Protective effect of Shengmai injection on myocardial endothelial cell glycoprotein detachment after myocardial ischemia-reperfusion injury in isolated rat hearts.

To investigate effects of Shengmai injection (SMI) postconditioning on myocardial ischemia-reperfusion injury (MIRI) in isolated rat hearts. A total of thirty isolated hearts were randomly divided into three groups: Sham group, I/R group and SMI group. Sham group was continuously perfused with K-H solution for 120 minutes. I/R group and SMI group were given balanced perfusion for 30 min followed by reperfusion for 60 min, with an interval of 30 min, and those in the SMI group were given postconditioning with 1% SMI during the first 10 min of reperfusion. The left ventricular function, markers of myocardial injury, endothelial cell injury and oxidative stress injury were measured at 30 minutes after equilibration (t0), 30 minutes after ischemia (t2) and 60 minutes after reperfusion (t3). The results showed that there was no significant difference for all observation indexes at t0. Compared with the Sham group, real portfolio project and coronary arterial flow rate and the activity of superoxide dismutase were significantly decreased in the I/R group, whereas those in the SMI group were significantly higher. Left ventricular end-diastolic pressure, the concentrate of malondialdehyde, lactate dehydrogenase, cTn-I, hyaluronic acid, heparin sulphate, syndecan-1 in the I/R group were markedly higher than those in the Sham group, whereas those in the SMI group were significantly lower. In summary, the present study indicated that 1% SMI postconditioning can alleviate the detachment of endothelial cell glycoprotein envelope induced by myocardial ischemia-reperfusion injury, and its mechanism is probably related to the inhibition of the oxidative stress injury.

Decidual mesenchymal stem/stromal cell-derived extracellular vesicles ameliorate endothelial cell proliferation, inflammation, and oxidative stress in a cell culture model of preeclampsia.

To determine whether DMSC-secreted, extracellular vesicles (DMSC_EV) restore endothelial cell function and reduce oxidative stress. DMSC were isolated from the placentae of uncomplicated term pregnancies and DMSC_EV prepared by ultracentrifugation. Human umbilical vein endothelial cells (HUVEC) were treated with bacterial lipopolysaccharide (LPS), or with serum from PE patients, to model the effects of PE. DMSC_EV were then added to treated HUVEC and their growth profiles, inflammatory state, and oxidative stress levels measured. DMSC_EV displayed characteristic features of extracellular vesicles. In both LPS- and PE serum-treatment models, addition of DMSC_EV significantly increased HUVEC cell attachment and proliferation, and significantly reduced production of pro-inflammatory cytokine IL-6. The addition of DMSC_EV to LPS-treated HUVEC had no significant effect on total antioxidant capacity, superoxide dismutase levels or on lipid peroxidation levels. In contrast, the addition of DMSC_EV to PE serum-treated HUVEC resulted in a significant reduction in levels of lipid peroxidation. Addition of DMSC_EV had beneficial effects in both LPS- and PE serum- treated HUVEC but the two treatment models to induce endothelial cell dysfunction showed differences. The LPS treatment of HUVEC model may not accurately model the endothelial cell dysfunction characteristic of PE. Human cell culture models of PE show that DMSC_EV improve endothelial cell dysfunction in PE, but testing in in vivo models of PE is required.

Sirtuins family as a target in endothelial cell dysfunction: implications for vascular ageing.

The vascular endothelium is a protective barrier between the bloodstream and the vasculature that may be disrupted by different factors such as the presence of diseased states. Diseases like diabetes and obesity pose a great risk toward endothelial cell inflammation and oxidative stress, leading to endothelial cell dysfunction and thereby cardiovascular complications such as atherosclerosis. Sirtuins are NAD+-dependent histone deacetylases that are implicated in the pathophysiology of cardiovascular diseases, and they have been identified to be important regulators of endothelial cell function. A handful ofrecent studies suggest that disbalance in the regulation of endothelial sirtuins, mainly sirtuin 1 (SIRT1), contributes to endothelial cell dysfunction. Herein, we summarize how SIRT1 and other sirtuins may contribute to endothelial cell function and how presence of diseased conditions may alter their expressions to cause endothelial dysfunction. Moreover, we discuss how the beneficial effects of exercise on the endothelium are dependent on SIRT1. These mainly include regulation of signalingpathways related to endothelial nitric oxide synthase phosphorylation and nitric oxide production, mitochondrial biogenesis and mitochondria-mediated apoptotic pathways, oxidative stress and inflammatory pathways. Sirtuins as modulators of the adverse conditions in the endothelium hold a promising therapeutic potential for health conditions related to endothelial dysfunctionand vascular ageing.

Effects of circulating extracellular microvesicles from spinal cord-injured adults on endothelial cell function.

People with spinal cord injury (SCI) have three- to four-fold greater risk of cardiovascular disease (CVD) compared with those without SCI. Although circulating extracellular microvesicles are key effectors of vascular health and disease, how their functional phenotype might be altered with SCI is unknown. The aim of the present study was to determine the effects of microvesicles isolated from SCI adults on endothelial cell inflammation and oxidative stress as well as endothelial nitric oxide (NO) synthase (eNOS) activation and tissue-type plasminogen activator (t-PA) expression. Eighteen young and middle-aged adults were studied: 10 uninjured (7M/3F; age: 39 ± 3 years) and 8 cervical level spinal cord injured (SCI; 7M/1F; 46 ± 4 years; cervical injury: C3: n=1; C5: n=4; C6: n=3). Circulating microvesicles were isolated, enumerated and collected from plasma by flow cytometry. Human umbilical vein endothelial cells (HUVECs) were cultured and treated with microvesicles from either the uninjured or SCI adults. Microvesicles from SCI adults did not affect cellular markers or mediators of inflammation and oxidative stress. However, microvesicles from the SCI adults significantly blunted eNOS activation, NO bioavailability and t-PA production. Intercellular expression of phosphorylated eNOS at Ser1177 and Thr495 sites, specifically, were ∼65% lower and ∼85% higher, respectively, in cells treated with microvesicles from SCI compared with uninjured adults. Decreased eNOS activity and NO production as well as impaired t-PA bioavailability renders the vascular endothelium highly susceptible to atherosclerosis and thrombosis. Thus, circulating microvesicles may contribute to the increased risk of vascular disease and thrombotic events associated with SCI.

Macrophage stimulating 1-induced inflammation response promotes aortic aneurysm formation through triggering endothelial cells death and activating the NF-κB signaling pathway

Aortic aneurysm formation is associated with endothelial cells dysfunction through an undefined mechanism. Macrophage stimulating 1 (Mst1) and NF-κB signaling pathway have been found to be related to inflammation response in endothelial cell damage. The goal of our study is to explore the role of Mst1 in regulating endothelial cell viability with a focus on NF-κB signaling pathway and inflammation response. Endothelial cell viability and death were determined via immunofluorescence and ELISA. Agonist of NF-κB signaling pathway and siRNA against Mst1 were used. The results in our study demonstrated that Mst1 transcription and expression were significantly elevated after exposure to oxidative stress in endothelial cells. Once loss of Mst1 through transfection of siRNA (si-Mst1), endothelial cell viability and survival rate were rapidly increased in response to oxidative stress. In addition, we also found that Mst1 controlled inflammation response and mitochondrial function in endothelial cells. Re-activation of NF-κB signaling pathway was followed by an activation of inflammation response and mitochondrial dysfunction, as evidenced by increased expression of inflammation factors and decreased ATP synthesis. Altogether, our results identify Mst1 as the primary factors responsible for endothelial cells dysfunction in aneurysms formation through inducing inflammation response, endothelial apoptosis, and NF-κB signaling pathway activation.

MicroRNA-103 Protects Coronary Artery Endothelial Cells against H2O2-Induced Oxidative Stress via BNIP3-Mediated End-Stage Autophagy and Antipyroptosis Pathways.

Endothelial cell damage caused by oxidative stress is widely considered to be a triggering event in atherosclerosis (AS). However, the specific effect elicited by autophagy in endothelial cells undergoing oxidative stress remains controversial, especially during end-stage autophagy. The inhibition of end-stage autophagy has been reported to increase cell pyroptosis and contribute to endothelial damage. Several studies have shown that microRNA-103 is involved in end-stage autophagy; however, its specific mechanism of action is not yet characterized. In this study, we addressed the regulatory role of miR-103 in autophagy during oxidative stress of endothelial cells. Hydrogen peroxide (H2O2) treatment was used as an in vitro model of oxidative stress. MTS and ROS levels were measured to evaluate cell activity. qRT-PCR was used to detect the expression of miR-103. Autophagy was examined using western blot, immunofluorescence staining, and electron microscopy, while western blot analysis detected pyroptosis-related proteins. Results show that miR-103 expression decreased under oxidative stress. Further, miR-103 repressed transcription of Bcl-2/adenovirus E1B 19 kDa interacting protein (BNIP3). The oxidative stress caused by H2O2 caused cell damage from 2 hours (P < 0.05) and increased the level of intracellular reactive oxygen species (P < 0.05); at the same time, the damage could be further aggravated by the stimulation of bafA1 (P < 0.05). Under the stimulation of H2O2, the expression of miR-103 decreased (P < 0.05). However, high expression of miR-103 could reduce the accumulation of LC3II and P62 (P < 0.05) by inhibiting the downstream target gene Bcl-2/adenovirus E1B 19 kDa interacting protein (BNIP3), thus reducing the occurrence of cell pyroptosis (P < 0.05). This process could be blocked by end-stage autophagy inhibitor bafA1 (P < 0.05), which further indicated that miR-103 affected cell injury by autophagy. On the contrary, the low expression of miR-103 promoted the accumulation of autophagy protein and increased the occurrence of pyroptosis (P < 0.05). In conclusion, inhibition of miR-103 restrained end-stage of autophagy by regulating BNIP3, thus changing the occurrence of cell pyroptosis.

A cathelicidin-related antimicrobial peptide suppresses cardiac hypertrophy induced by pressure overload by regulating IGFR1/PI3K/AKT and TLR9/AMPK\u03b1

Cathelicidin-related antimicrobial peptide (CRAMP), an antimicrobial peptide, was reported to protect against myocardial ischemia/reperfusion injury. However, the effect of CRAMP on pressure overload-induced cardiac hypertrophy was unknown. This study explored the role of CRAMP on cardiac hypertrophy. A cardiac hypertrophy mouse model was induced by aortic banding surgery. Seven days after surgery, mice were given mCRAMP by intraperitoneal injection (8 mg/kg/d) for 7 weeks. Cardiac hypertrophy was evaluated by the hypertrophic response and fibrosis level as well as cardiac function. Mice were also injected with AAV9-shCRAMP to knockdown CRAMP in the mouse heart. CRAMP levels first increased and then reduced in the remodeling heart, as well as in angiotensin II-stimulated endothelial cells but not in cardiomyocytes and fibroblasts. mCRAMP protected against the pressure overload-induced cardiac remodeling process, while CRAMP knockdown accelerated this process. mCRAMP reduced the inflammatory response and oxidative stress in the hypertrophic heart, while mCRAMP deficiency deteriorated the pressure overload-induced inflammatory response and oxidative stress. mCRAMP inhibited the angiotensin II-stimulated hypertrophic response and oxidative stress in neonatal rat cardiomyocytes, but mCRAMP did not help the angiotensin II-induced inflammatory response and oxidative stress in endothelial cells. Mechanistically, we found that mCRAMP suppressed the cardiac hypertrophic response by activating the IGFR1/PI3K/AKT pathway via directly binding to IGFR1. AKT knockout mice completely reversed the anti-hypertrophic effect of mCRAMP but not its anti-oxidative effect. We also found that mCRAMP ameliorated cardiac oxidative stress by activating the TLR9/AMPKa pathway. This was confirmed by a TLR9 knockout mouse experiment, in which a TLR9 knockout partly reversed the anti-hypertrophic effect of mCRAMP and completely counteracted the anti-oxidative effect of mCRAMP. In summary, mCRAMP protected against pressure overload-induced cardiac hypertrophy by activating both the IGFR1/PI3K/AKT and TLR9/AMPKa pathways in cardiomyocytes.

Protective effects of beet (Beta vulgaris) leaves extract against oxidative stress in endothelial cells in vitro.

Beetroot is an herb used worldwide as a food product, raw material for food industry, ethanol production and source of food coloring. Beet leaves are an unconventional food with antioxidant properties, which might neutralize reactive oxygen species (ROS) induced by oxidized Low-Density Lipoprotein (LDL) present in dyslipidemias. This study aimed to elucidate the effects of beet leaves on the suppression of LDL oxidative processes. Beet leaves extract was produced, characterized, and tested for its antioxidant capacity using endothelial cells in vitro. A model of human umbilical vein endothelial cells was used in various tests, including viability assay, molecular analysis of antioxidant genes, ROS labeling, and macrophage adhesion assay. The extract improved the antioxidative protection of endothelial cells against different agents including oxidized LDL-cholesterol and H2 O2 . It acted on ROS directly due to its high content of natural antioxidants, but also due to the activation and improvement of cellular defenses such as Superoxide dismutase 1, Superoxide dismutase 2, and catalase. The inhibition of LDL-mediated oxidative effects on endothelial cells may turn this unconventional food a functional food with great potential for phytotherapy of atherosclerosis as an adjuvant for medicinal treatments.

Formation of a bovine serum albumin diligand complex with rutin for the suppression of heme toxicity

Serum albumin binds avidly to heme to form heme–serum albumin complex and can protect against the potentially toxic effects of heme. Rutin is a glycoside of the bioflavonoid quercetin with various protective effects due to its antioxidant ability. Clarification of the interaction mechanisms between serum albumin and bioactive components (such as heme and flavonoid) is important to develop effective carriers for encapsulation of heme and suppression of its toxicity. In this study, bindings of bovine serum albumin (BSA) to heme and/or rutin were investigated by experimental and molecular docking techniques. The fluorescence of BSA was quenched by both heme and rutin in static mode (i.e. formation of BSA-monoligand complexes), which was confirmed by Stern-Volmer calculations. Although heme showed higher affinity to BSA than rutin, the interactions of both components with BSA did locate within subdomain IIA (site I). BSA-diligand complexes were successfully formed after the simultaneous addition of heme and rutin. Bioactive rutin in the BSA-diligand complex still kept strong free radical scavenging activity compared to free rutin or BSA-monoligand complex. Hydrogen peroxide (H2O2)-induced heme degradation and free iron release was inhibited upon BSA binding and further decreased in BSA-diligand complexes. Consistently, the cytotoxicity of heme and oxidative stress in endothelial cells was decreased in the BSA-diligand complexes relative to those of heme or BSA-heme complex, where the co-presence of rutin played an important role. These results suggest the possibility and advantage of developing BSA-based carriers for the suppression of heme toxicity in their biomedical applications.

The Effect of Hydro-alcoholic Extract of Rheum Turkestanicum Roots against Oxidative Stress in Endothelial Cells.

Introduction:Cardiovascular disorders (CVD) are a common cause of mortality worldwide. Oxidative stress is thought to be a major factor leading to CVD. Anti-oxidants such as medicinal plants may have a role in the mitigation of vascular problems through free radicals scavenging. In this study, we evaluated the protective effects of Rheum turkestanicum against hydrogen peroxide (H2O2)-induced toxicity in endothelial cells (BAE-1).Methods:To evaluate the protective effect of R. turkestanicum against H2O2 toxicity, four groups comprised of control group (the cells without any treatment), H2O2 group (the cells incubated with H2O2 (200 μM)), and treatment groups (the cells treated with R. turkestanicum (12200 μg/ml) alone or 24h before exposure to H2O2). Quercetin (30.23 μg/ml) was used as a bioactive ingredient of the extract. Then the cell viability, reactive oxygen species, lipid peroxidation, and apoptosis were evaluated.Results:H2O2 exposure reduced cell viability to 13.6 ± 1.6%, enhanced ROS generation to 1445 ± 80.7%, lipid peroxidation (LPO, 290 ± 13% of control), and apoptotic cells (P < 0.001). In contrast, compared with H2O2 group, R. turkestanicum and quercetin significantly restored the cell viability to 80.3 ± 1.6 and 87.2 ± 2.1%, ROS formation to 186 ± 10 and 129 ± 1%, as well as LPO to 130.7 ± 7.7 and 116 ± 2.5 of control, respectively (P < 0.001). Therefore, the extract reduced H2O2-induced toxicity in BAE-1 cells by scavenging of free radicals.Conclusion:Our findings demonstrated that the extract might reduce toxicity of endothelial cells by attenuation of oxidative stress, which can be related to the presence of active ingredients including quercetin.

Activation of AMPK/Sirt3 pathway by phloretin reduces mitochondrial ROS in vascular endothelium by increasing the activity of MnSOD via deacetylation.

As a dihydrochalcone, phloretin was reported to effectively attenuate palmitic acid (PA)-induced oxidative stress in endothelial cells. In the present study, we further investigated the antioxidant capacity of phloretin via restoring the activity of MnSOD through deacetylation in vitro and in vivo. The results revealed that phloretin (50 μM) treatment significantly increased the activity of MnSOD in the HUVECs and mouse aortas, and then obviously reduced the accumulation of mitochondrial ROS. Immunoprecipitation assay and Western blot analysis indicated that phloretin could decrease the lysine acetylation of MnSOD and restore its activity by promoting the expression of Sirt3 by increasing the phosphorylation of AMPK (Thr172). These findings provide a novel profile to explain the antioxidant activity of phloretin by reducing the acetylation level of MnSOD via an AMPK/Sirt3 signaling pathway.

Inhibition of miR-383 suppresses oxidative stress and improves endothelial function by increasing sirtuin 1.

Previous research has shown that suppression of miR-383 can prevent inflammation of the endothelium, as well as postpone the development of atherosclerosis. However, the role of miR-383 in endothelial cell apoptosis in diabetes remains unclear. The aim of this study was to investigate the function of miR-383 in high glucose-induced apoptosis and oxidative stress in endothelial cells. A series of experiments involving qualitative polymerase chain reaction, cell transfection, luciferase assay, assessment of cell death, detection of catalase and superoxide dismutase concentrations, detection of intracellular reactive oxygen species (ROS), and western blot analysis were performed in this study. We found that miR-383 expression was promoted, while NAD+-dependent deacetylase and sirtuin 1 (SIRT1) expressions were suppressed in the endothelium of the aorta in db/db mice as well as in human umbilical vein endothelial cells, which were treated with high glucose (HG). Increased expression of miR-383 decreased expression of SIRT1, while suppression of miR-383 promoted expression of SIRT1 in human umbilical vein endothelial cells (HUVECs). Furthermore, suppression of miR-383 following transfection with miR-383 suppressor repressed cell death and generation of ROS in HUVECs. SIRT1 knockdown by siRNA-SIRT1 reversed the suppressive effect of miR-383 inhibition on ROS production and cell apoptosis induced by HG treatment. Overall, the findings of our research suggested that suppression of miR-383 repressed oxidative stress and reinforced the activity of endothelial cells by upregulation of SIRT1 in db/db mice, and targeting miR-383 might be promising for effective treatment of diabetes.