Improving the Classification of PCNSL and Brain Metastases by Developing a Machine Learning Model Based on 18F-FDG PET.

Background: The characteristic magnetic resonance imaging (MRI) and the positron emission tomography (PET) findings of PCNSL often overlap with other intracranial tumors, making definitive diagnosis challenging. PCNSL typically shows iso-hypointense to grey matter on T2-weighted imaging. However, a particular part of PCNSL can demonstrate T2-weighted hyperintensity as other intracranial tumors. Moreover, normal high uptake of FDG in the basal ganglia, thalamus, and grey matter can mask underlying PCNSL in 18F-FDG PET. In order to promote the efficiency of diagnosis, the MRI-based or PET/CT-based radiomics models combining histograms with texture features in diagnosing glioma and brain metastases have been widely established. However, the diagnosing model for PCNSL has not been widely reported. The study was designed to investigate a machine-learning (ML) model based on multiple parameters of 2-deoxy-2-[18F]-floor-D-glucose (18F-FDG) PET for differential diagnosis of PCNSL and metastases in the brain. Methods: Patients who underwent an 18F-FDG PET scan with untreated PCNSL or metastases in the brain were included between May 2016 and May 2022. A total of 126 lesions from 51 patients (43 patients with untreated brain metastases and eight patients with untreated PCNSL), including 14 lesions of PCNSL, and 112 metastatic lesions in the brain, met the inclusion criteria. PCNSL or brain metastasis was confirmed after pathology or clinical history. Principal component analysis (PCA) was used to decompose the datasets. Logistic regression (LR), support vector machine (SVM), and random forest classification (RFC) models were trained by two different groups of datasets, the group of multi-class features and the group of density features, respectively. The model with the highest mean precision score was selected. The testing sets and original data were used to examine the efficacy of models separately by using the weighted average F1 score and area under the curve (AUC) of the receiver operating characteristic curve (ROC). Results: The multi-class features-based RFC and SVM models reached identical weighted-average F1 scores in the testing set, and the score was 0.98. The AUCs of RFC and SVM models calculated from the testing set were 1.00 equally. Evaluated by the original dataset, the RFC model based on multi-class features performs better than the SVM model, whose weighted-average F1 scores of the RFC model calculated from the original data were 0.85 with an AUC of 0.93. Conclusions: The ML based on multi-class features of 18F-FDG PET exhibited the potential to distinguish PCNSL from brain metastases. The RFC models based on multi-class features provided comparatively high efficiency in our study.

PCAF Accelerates Vascular Senescence via the Hippo Signaling Pathway.

P300/CBP-Associated Factor (PCAF), one of the histone acetyltransferases (HATs), is known to be involved in cell growth and/or differentiation. PCAF is reported to be involved in atherosclerotic plaques and neointimal formation. However, its role in cellular senescence remains undefined. We investigated the potential mechanism for PCAF-mediated cellular senescence. Immunohistochemical (IHC) analysis showed PCAF was distinctly increased in the endothelia of aorta in aged mice. Palmitate acid (PA) or X radiation significantly induced the expression of senescence-associated markers and PCAF in human umbilical vein endothelial cells (HUVECs). PCAF silence in PA-treated HUVECs significantly rescued senescence-associated phenotypes, while PCAF overexpression accelerated it. Additionally, our results showed that Yes1 Associated Transcriptional Regulator (YAP) that acts as end effector of the Hippo signaling pathway is crucial in PCAF-mediated endothelial senescence. YAP activity declining was observed in aged vascular endothelia. Overexpression of YAP partially ameliorated PCAF-induced endothelial senescence. In vivo, endothelial-(EC-) specific PCAF downregulation in aged mice using adeno-associated virus revealed less vascular senescence-associated phenotypes. These results suggested that PCAF mediated endothelial senescence through the Hippo signaling pathway, implying that PCAF may become a potential target for the prevention and treatment of vascular aging.

Mitoferrin 2 deficiency prevents mitochondrial iron overload-induced endothelial injury and alleviates atherosclerosis.

Endothelial dysfunction is an early step in the development of atherosclerotic cardiovascular disease. Iron overload can lead to excessive mitochondrial reactive oxygen species (mtROS) production, resulting in mitochondrial dysfunction and vascular endothelial cell (EC) damage. Mitoferrin 2 (Mfrn2) is an iron transporter in the inner mitochondrial membrane. This study aimed to assess whether Mfrn2 and mitochondrial iron overload were involved in atherosclerosis progression and to explore the potential mechanism. We observed significant upregulation of Mfrn2 in the arteries of high-fat diet (HFD)-fed Apolipoprotein E-/- (ApoE-/-) mice and in TNF-α-induced mouse aortic endothelial cells (MAECs). Mfrn2 gene silencing inhibited mitochondrial iron overload, stabilized mitochondrial membrane potential and improved mitochondrial function in TNF-α-induced MAECs. Vascular EC-specific knockdown of Mfrn2 in ApoE-/- mice markedly decreased atherosclerotic lesion formation and the levels of ICAM-1 in aortas and reduced monocyte infiltration into the vascular wall. Furthermore, TNF-α increased the binding of 14-3-3 epsilon (ε) and Mfrn2, preventing Mfrn2 degradation and leading to mitochondrial iron overload in ECs, while 14-3-3ε overexpression increased Mfrn2 stability by inhibiting its ubiquitination. Together, our results reveal that Mfrn2 deficiency attenuates endothelial dysfunction by decreasing iron levels within the mitochondria and mitochondrial dysfunction. These findings may provide new insights into preventive and therapeutic strategies against vascular endothelial dysfunction in atherosclerotic disease.

CTRP13 Mitigates Abdominal Aortic Aneurysm Formation via NAMPT1.

Abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease characterized by localized dilation of the abdominal aorta. C1q/tumor necrosis factor (TNF)-related protein-13 (CTRP13) is a secreted adipokine that plays important roles in the cardiovascular system. However, the functional role of CTRP13 in the formation and development of AAA has yet to be explored. In this study, we determined that serum CTRP13 levels were significantly downregulated in blood samples from patients with AAA and in rodent AAA models induced by Angiotensin II (Ang II) in ApoE-/- mice or by CaCl2 in C57BL/6J mice. Using two distinct murine models of AAA, CTRP13 was shown to effectively reduce the incidence and severity of AAA in conjunction with reduced aortic macrophage infiltration, expression of proinflammatory cytokines (interleukin-6 [IL-6], TNF-α, and monocyte chemoattractant protein 1 [MCP-1]), and vascular smooth muscle cell (SMC) apoptosis. Mechanistically, nicotinamide phosphoribosyl-transferase 1 (NAMPT1) was identified as a new target of CTRP13. The decreased in vivo and in vitro expression of NAMPT1 was markedly reversed by CTRP13 supplementation in a ubiquitination-proteasome-dependent manner. NAMPT1 knockdown further blocked the beneficial effects of CTRP13 on vascular inflammation and SMC apoptosis. Overall, our study reveals that CTRP13 management may be an effective treatment for preventing AAA formation.

Akt phosphorylation regulated by IKKε in response to low shear stress leads to endothelial inflammation via activating IRF3.

Low shear stress (LSS) plays a critical role in the development of atherosclerotic plaques and vascular inflammation. Previous studies have reported Akt phosphorylation induced by LSS. However, the mechanism and role of Akt activation remains unclear in LSS-induced endothelial dysfunction. In this study, our results demonstrated the increased phosphorylation of IKKε, TBK1 and Akt in HUVECs exposed to LSS. Furthermore, IKKε silencing using small interfering RNAs significantly reduced LSS-induced Akt phosphorylation. In contrast, silencing of TBK1 or inhibition of PI3K and mTORC2 had no effect on LSS-induced Akt phosphorylation. Notably, Akt inhibition markedly diminished LSS-induced expression of ICAM-1, VCAM-1 and MCP-1, as well as LSS-induced IRF3 phosphorylation and nuclear translocation, without affecting the activation of NF-κB and STAT1. Moreover, endothelial cell specific Akt overexpression mediated by adeno-associated virus markedly increased intimal ICAM-1 and IRF3 expression at LSS area of partially ligated carotid artery in mice. In brief, our findings suggest that LSS-induced Akt phosphorylation is positively regulated by IKKε and promotes IRF3 activation, leading to endothelial inflammation.

E3 Ligase FBXW2 Is a New Therapeutic Target in Obesity and Atherosclerosis.

Chronic low-grade inflammation orchestrated by macrophages plays a critical role in metabolic chronic diseases, like obesity and atherosclerosis. However, the underlying mechanism remains to be elucidated. Here, the E3 ubiquitin ligase F-box/WD Repeat-Containing Protein 2 (FBXW2), the substrate-binding subunit of E3 ubiquitin ligase SCF (a complex of FBXW2, SKP1, and cullin-1), as an inflammatory mediator in macrophages, is identified. Myeloid-specific FBXW2 gene deficiency improves both obesity-associated with insulin resistance and atherosclerosis in murine models. The beneficial effects by FBXW2 knockout are accompanied by decreased proinflammatory responses and macrophage infiltration in the microenvironment. Mechanistically, it is identified that KH-type splicing regulatory protein (KSRP) is a new bona fide ubiquitin substrate of SCFFBXW2. Inhibition of KSRP prevents FBXW2-deficient macrophages from exerting a protective effect on inflammatory reactions, insulin resistance and plaque formation. Furthermore, it is demonstrated that the C-terminus (P3) of FBXW2 competitively ablates the function of FBXW2 in KSRP degradation and serves as an effective inhibitor of obesity and atherogenesis progression. Thus, the data strongly suggest that SCFFBXW2 is an important mediator in the context of metabolic diseases. The development of FBXW2 (P3)-mimicking inhibitors and small-molecular drugs specifically abrogating KSRP ubiquitination-dependent inflammatory responses are viable approaches for obesity and atherosclerosis treatment.

Myeloid FBW7 deficiency disrupts redox homeostasis and aggravates dietary-induced insulin resistance.

The E3 ubiquitin ligase FBW7 plays critical roles in multiple pathological and physiological processes. Here, we report that after high-fat diet (HFD) feeding for 16 weeks, myeloid-specific FBW7-deficient mice demonstrate increased redox stress, inflammatory responses and insulin resistance. Macrophages activation under FBW7 deficiency decreases substrate flux through the pentose phosphate pathway (PPP) to produce less equivalents (NADPH and GSH) and aggravate the generation of intracellular reactive oxygen species (ROS) in macrophages, thereby over-activating proinflammatory reaction. Mechanistically, we identify that pyruvate kinase muscle isozyme M2 (PKM2) is a new bona fide ubiquitin substrate of SCFFBW7. While challenged with HFD stress, pharmacological inhibition of PKM2 protects FBW7-deficient macrophages against production of ROS, proinflammatory reaction and insulin resistance. Intriguingly, we further find an inverse correlation between FBW7 level and relative higher H2O2 level and the severity of obesity-related diabetes. Overall, the results suggest that FBW7 can play a crucial role in modulating inflammatory response through maintaining the intracellular redox homeostasis during HFD insults.

CTRP13 Preserves Endothelial Function by Targeting GTP Cyclohydrolase 1 in Diabetes.

Endothelial dysfunction plays a crucial role in the progress of diabetic vasculopathy. C1q/tumor necrosis factor-related protein 13 (CTRP13) is a secreted adipokine that can ameliorate atherosclerosis and vascular calcification. However, the role of CTRP13 in regulating endothelial function in diabetes has yet to be explored. In this study, CTRP13 treatment improved endothelium-dependent relaxation in the aortae and mesenteric arteries of both db/db mice and streptozotocin-injected mice. CTRP13 supplement also rescued the impaired endothelium-dependent relaxation ex vivo in the db/db mouse aortae and in high glucose (HG)-treated mouse aortae. Additionally, CTRP13 treatment reduced reactive oxygen species overproduction and improved nitric oxide (NO) production and endothelial NO synthase (eNOS) coupling in the aortae of diabetic mice and in HG-treated human umbilical vein endothelial cells. Mechanistically, CTRP13 could increase GTP cyclohydrolase 1 (GCH1) expression and tetrahydrobiopterin (BH4) levels to ameliorate eNOS coupling. More importantly, CTRP13 rescued HG-induced inhibition of protein kinase A (PKA) activity. Increased PKA activity enhanced phosphorylation of the peroxisome proliferator-activated receptor α and its recruitment to the GCH1 promoter, thus activating GCH1 transcription and, ultimately, endothelial relaxation. Together, these results suggest that CTRP13 preserves endothelial function in diabetic mice by regulating GCH1/BH4 axis-dependent eNOS coupling, suggesting the therapeutic potential of CTRP13 against diabetic vasculopathy.

CTRP13 attenuates vascular calcification by regulating Runx2.

Vascular calcification is strongly associated with increased cardiovascular mortality and morbidity. C1q/TNF-related protein-13 (CTRP13) is a secreted adipokine that plays important roles in the cardiovascular system. However, the functional role of CTRP13 in the development of vascular calcification has yet to be explored. In this study, we collected blood samples from patients with chronic renal failure (CRF) and from rats with adenine-induced CRF. We found that the serum CTRP13 levels were decreased in patients and rats with CRF and were negatively associated with calcium deposition in the abdominal aorta. Compared to those of the controls, ectopic CTRP13 treatment significantly attenuated the calcium accumulation and alkaline phosphatase activity in the abdominal aorta of CRF rats, and ß-glycerophosphate induced the formation of arterial rings and of vascular smooth muscle cells (VSMCs) and decreased the number of VSMCs that transitioned from a contractile to an osteogenic phenotype. The overexpression of Runx2 blocked CTRP13-reduced VSMC calcification. Mechanistically, CTRP13 repressed the phosphorylation of tristetraprolin (TTP), thereby activating TTP and increasing the TTP binding to the 3'untranslated region of the Runx2 mRNA, accelerating the Runx2 mRNA destabilization and degradation. In summary, these findings reveal that CTRP13 regulation is a novel method for the prevention of vascular calcification, representing a novel mechanism of the regulation of Runx2 expression in VSMCs.-Li, Y., Wang, W., Chao, Y., Zhang, F., Wang, C. CTRP13 attenuates vascular calcification by regulating Runx2.

Activation of the PP2A catalytic subunit by ivabradine attenuates the development of diabetic cardiomyopathy.

Hyperglycemia-induced apoptosis plays a critical role in the pathogenesis of diabetic cardiomyopathy (DCM). Our previous study demonstrated that ivabradine, a selective If current antagonist, significantly attenuated myocardial apoptosis in diabetic mice, but the underlying mechanisms remained unknown. This study investigated the underlying mechanisms by which ivabradine exerts anti-apoptotic effects in experimental DCM. Pretreatment with ivabradine, but not ZD7288 (an established If current blocker), profoundly inhibited high glucose-induced apoptosis via inactivation of nuclear factor (NF)-κB signaling in neonatal rat cardiomyocytes. The effect was abolished by transfection of an siRNA targeting protein phosphatase 2A catalytic subunit (PP2Ac). In streptozotocin-induced diabetic mice, ivabradine treatment significantly inhibited left ventricular hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) and HCN4 (major components of the If current), activated PP2Ac, and attenuated NF-κB signaling activation and apoptosis, in line with improved histological abnormalities, fibrosis, and cardiac dysfunction without affecting hyperglycemia. These effects were not observed in diabetic mice with virus-mediated knockdown of HCN2 or HCN4 after myocardial injection, but were alleviated by knockdown of PP2Acα. Molecular docking and phosphatase activity assay confirmed direct binding of ivabradine to, and activation of, PP2Ac. In conclusion, ivabradine may directly activate PP2Ac, leading to inhibition of NF-κB signaling activation, myocardial apoptosis, and fibrosis, and eventually improving cardiac function in experimental DCM. Taken together, the present findings suggest that ivabradine may be a promising drug for treatment of DCM.

AMP-activated protein kinase regulates glycocalyx impairment and macrophage recruitment in response to low shear stress.

Low shear stress (LSS) increases degradation of the endothelial glycocalyx, leading to production of endothelial inflammation and atherosclerosis. However, the underlying mechanisms of how LSS diminishes the endothelial glycocalyx remain unclear. We showed that LSS inactivated AMPK, enhanced Na+-H+ exchanger (NHE)1 activity, and induced glycocalyx degradation. Activation of AMPK prevented LSS-induced NHE1 activity and endothelial glycocalyx impairment. We further identified hyaluronidase 2 (HYAL2) as a mediator of endothelial glycocalyx impairment in HUVECs exposed to LSS. Inactivation of AMPK by LSS up-regulates the activity of HYAL2, which acts downstream of NHE1. We characterized a left common carotid artery partial ligation (PL) model of LSS in C57BL/6 mice. The results showed decreased expression of hyaluronan (HA) in the endothelial glycocalyx and decreased thickness of the endothelial glycocalyx in PL mice. Pharmacological activation of AMPK by ampkinone not only attenuated glycocalyx impairment due to HA degradation but also blocked vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 expression increase and macrophage recruitment in the endothelia of PL mice. Our results revealed that AMPK dephosphorylation induced by LSS activates NHE1 and HYAL2 to promote HA degradation and glycocalyx injury, which may contribute to endothelial inflammatory reaction and macrophage recruitment.-Zhang, J., Kong, X., Wang, Z., Gao, X., Ge, Z., Gu, Y., Ye, P., Chao, Y., Zhu, L., Li, X., Chen, S. AMP-activated protein kinase regulates glycocalyx impairment and macrophage recruitment in response to low shear stress.

Norepinephrine stimulation downregulates the ß2 -adrenergic receptor-nitric oxide pathway in human pulmonary artery endothelial cells.

BACKGROUND: Norepinephrine (NE)-mediated vasoconstriction plays an important role in pulmonary hypertension associated with left heart disease (PH-LHD). However, the role of NE-mediated endothelial cell dysfunction in the pathogenesis of PH-LHD remains to be elucidated. METHODS AND RESULTS: An enzyme-linked immunosorbent assay showed that the NE concentration in the plasma of patients with PH-LHD was higher and the nitrate-nitrite concentration was lower than those in the control group. NE treatment decreased phospho-Ser633-eNOS and ß2 -adrenergic receptor (ß2 -AR) levels in the membrane of human pulmonary artery endothelial cells (HPAECs) analysed by western blot analysis. Consistently, fluorescence microscopy and flow cytometry showed that nitric oxide (NO) production was also decreased in HPAECs. Coimmunoprecipitation confirmed a direct interaction between ß2 -AR and endothelial NO synthase (eNOS). Overexpression of ß2 -AR attenuated the decline in phospho-Ser633-eNOS and NO production. Additionally, the expression of phospho-Ser633-eNOS and ß2 -AR was decreased in human pulmonary artery endothelium. Finally, our results indicate that NE stimulated HPAEC proliferation, which was blocked by protein kinase A inhibitor or protein kinase B (PKB-AKT) inhibitor. CONCLUSIONS: These data provide a novel mechanism for NE-decreased endothelium-derived NO and NE-induced HPAEC proliferation that leads to PH-LHD, suggesting a potential therapeutic target for PH-LHD.

Hyaluronidase2 (Hyal2) modulates low shear stress-induced glycocalyx impairment via the LKB1/AMPK/NADPH oxidase-dependent pathway.

The endothelium glycocalyx layer (ECL), presents on the apical surface of endothelial cells, creates a barrier between circulating blood and the vessel wall. Low shear stress (LSS) may accelerate the degradation of the glycocalyx via hyaluronidase2 (Hyal2) and then alter the cell polarity. Yet the liver kinase B1 (LKB1) signaling pathway plays an important role in regulating cell polarity. However, the relationship between LKB1 and glycocalyx during LSS is not clear. In the current study, we demonstrate that LSS attenuates LKB1 and AMP-activated protein kinase activation as well as activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (p47phox ) and Hyal2 in the human umbilical vein endothelial cell (HUVEC). Pretreatment with 5-Aminoimidazole-4-carboxamide1-ß-D-ribofuranoside (AICAR), or diphenyleneiodonium (DPI chloride) and transfection with LKB1 overexpression vector and p47phox small interfering RNA downregulated LSS-induced Hyal2 activation. By coimmunoprecipitation, we discovered the existence of p47phox /Hyal2 complex. LSS induced the dissociation of p47phox /Hyal2 complex, which was inhibited by LKB1 overexpression and AICAR. Furthermore, knockdown of Hyal2 performed a positive feedback on LKB1 activity. In addition, we also show that LSS enhanced LKB1 translocation from the cytosol to the nucleus. Taken together, these data indicate that Hyal2 regulates LSS-induced injury of the glycocalyx via LKB1/AMPK/NADPH oxidase signaling cascades.

Exogenous hydrogen sulfide attenuates the development of diabetic cardiomyopathy via the FoxO1 pathway.

BACKGROUND: Previous studies have suggested that exogenous hydrogen sulfide can alleviate the development of diabetic cardiomyopathy (DCM) by inhibiting oxidative stress, inflammation, and apoptosis. However, the underlying mechanism is not fully understood. Nuclear expression and function of the transcription factor Forkhead box protein O (FoxO1) have been associated with cardiovascular diseases, and thus, the importance of FoxO1 in DCM has gained increasing attention. This study was designed to investigate the interactions between hydrogen sulfide (H2 S) and nuclear FoxO1 in DCM. METHODS: Diabetes was induced in adult male C57BL/6J mice by intraperitoneal injection of streptozotocin and was treated with H2 S donor sodium hydrosulfide for 12 weeks. The H9C2 cardiomyoblast cell line and neonatal rat cardiomyocytes (NRCMs) were treated with the slow-releasing H2 S donor GYY4137 before high-glucose (HG) exposure with or without pretreatment with the Akt inhibitor MK-2206 2HCl. Changes in FoxO1 protein phosphorylation and subcellular localization were determined in H9C2 cells, NRCMs, and cardiac tissues from normal and diabetic mice. Cardiac structure and function in the diabetic mice were evaluated by echocardiography and histological analysis and compared with those in control animals. RESULTS: The echocardiographic and histopathological data indicated that exogenous H2 S improved cardiac function and attenuated cardiac hypertrophy and myocardial fibrosis in diabetic mice. H2 S also improved HG-induced oxidative stress and apoptosis in cardiac tissue and NRCMs. In addition, H2 S induced FoxO1 phosphorylation and nuclear exclusion in vitro and in vivo, and this function was not inhibited by MK-2206 2HCl. Alanine substitution mutation of three sites in FoxO1-enhanced FoxO1 transcriptional activity, and subsequent treatment with exogenous H2 S could not prevent HG-induced nuclear retention. CONCLUSIONS: Our data indicate that H2 S is a novel regulator of FoxO1 in cardiac cells and provide evidence supporting the potential of H2 S in inhibiting the progression of DCM.

Berberine attenuates pulmonary arterial hypertension via protein phosphatase 2A signaling pathway both in vivo and in vitro.

Excessive proliferation, migration, and antiapoptosis of pulmonary artery (PA) smooth muscle cells (PASMCs) underlies the development of pulmonary vascular remodeling. The innervation of the PA is predominantly sympathetic, and increased levels of circulating catecholamines have been detected in pulmonary arterial hypertension (PAH), suggesting that neurotransmitters released by sympathetic overactivation may play an essential role in PAH. However, the responsible mechanism remains unclear. Here, to investigate the effects of norepinephrine (NE) on PASMCs and the related mechanism, we used 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, the proliferating cell nuclear antigen and the cell counting kit-8 assay to evaluate the proliferation of PASMCs, Boyden chamber migration, and wound-healing assays to assess migration and western blot analysis to investigate protein expression. We demonstrated that the phosphorylation level of the protein phosphatase 2A (PP2A) catalytic subunit (Y307) was higher in PAH patients and PAH models than in controls, both in vivo and in vitro. In addition, NE induced the proliferation and migration of PASMCs, which was attenuated by berberine (BBR), a Chinese herbal medicine, and/or PP2A overexpression. PP2A inhibition worsened NE-induced PAH and could not be reversed by BBR. Thus, PP2A is critical in driving PAH, and BBR may alleviate PAH via PP2A signaling pathways, thereby offering a potential therapeutic option for PAH.

Low shear stress induces vascular eNOS uncoupling via autophagy-mediated eNOS phosphorylation.

Uncoupled endothelial nitric oxide synthase (eNOS) produces O2- instead of nitric oxide (NO). Earlier, we reported rapamycin, an autophagy inducer and inhibitor of cellular proliferation, attenuated low shear stress (SS) induced O2- production. Nevertheless, it is unclear whether autophagy plays a critical role in the regulation of eNOS uncoupling. Therefore, this study aimed to investigate the modulation of autophagy on eNOS uncoupling induced by low SS exposure. We found that low SS induced endothelial O2- burst, which was accompanied by reduced NO release. Furthermore, inhibition of eNOS by L-NAME conspicuously attenuated low SS-induced O2- releasing, indicating eNOS uncoupling. Autophagy markers such as LC3 II/I ratio, amount of Beclin1, as well as ULK1/Atg1 were increased during low SS exposure, whereas autophagic degradation of p62/SQSTM1 was markedly reduced, implying impaired autophagic flux. Interestingly, low SS-induced NO reduction could be reversed by rapamycin, WYE-354 or ATG5 overexpression vector via restoration of autophagic flux, but not by N-acetylcysteine or apocynin. eNOS uncoupling might be ascribed to autophagic flux blockade because phosphorylation of eNOS Thr495 by low SS or PMA stimulation was also regulated by autophagy. In contrast, eNOS acetylation was not found to be regulated by low SS and autophagy. Notably, although low SS had no influence on eNOS Ser1177 phosphorylation, whereas boosted eNOS Ser1177 phosphorylation by rapamycin were in favor of the eNOS recoupling through restoration of autophagic flux. Taken together, we reported a novel mechanism for regulation of eNOS uncoupling by low SS via autophagy-mediated eNOS phosphorylation, which is implicated in geometrical nature of atherogenesis.

Low shear stress induces endothelial reactive oxygen species via the AT1R/eNOS/NO pathway.

Reactive oxygen species (ROS) contribute to many aspects of physiological and pathological cardiovascular processes. However, the underlying mechanism of ROS induction by low shear stress (LSS) remains unclear. Accumulating evidence has shown that the angiotensin II type 1 receptor (AT1R) is involved in inflammation, apoptosis, and ROS production. Our aim was to explore the role of AT1R in LSS-mediated ROS induction. We exposed human umbilical vein endothelial cells (HUVECs) to LSS (3 dyn/cm2 ) for different periods of time. Western blotting and immunofluorescence showed that LSS significantly induced AT1R expression in a time-dependent manner. Using immunohistochemistry, we also noted a similar increase in AT1R expression in the inner curvature of the aortic arch compared to the descending aorta in C57BL/6 mice. Additionally, HUVECs were cultured with a fluorescent probe, either DCFH, DHE or DAF, after being subjected to LSS. Cell chemiluminescence and flow cytometry results revealed that LSS stimulated ROS levels and suppressed nitric oxide (NO) generation in a time-dependent manner, which was reversed by the AT1R antagonist Losartan. We also found that Losartan markedly increased endothelial NO synthase (eNOS) phosphorylation at Ser(633,1177) and dephosphorylation at Thr(495), which involved AKT and ERK. Moreover, the ROS level was significantly reduced by endogenous and exogenous NO donors (L-arginine, SNP) and increased by the eNOS inhibitor L-NAME. Overall, we conclude that LSS induces ROS via AT1R/eNOS/NO.

Inhibition of angiotension II type 1 receptor reduced human endothelial inflammation induced by low shear stress.

Low shear stress (LSS)-induced endothelial inflammation is the basis for the development of atherosclerosis. However, the mechanism underlying LSS-induced inflammation is not well understood. The angiotensin II type 1 receptor (AT1R), a component of the renin-angiotensin system, participates in atherosclerotic plaque progression. The aim of this study was to investigate the role of AT1R in LSS-induced endothelial activation. Using immunohistochemistry, we noted significant increases in AT1R, vascular endothelial adhesion cell-1 (VCAM1), and intercellular adhesion molecule-1 (ICAM1) expression in the inner curvature of the aortic arch in C57BL/6 mice compared to the descending aorta in these mice. Moreover, western blotting revealed that these LSS-induced increases in AT1R, ICAM1 and VCAM1 expression were time dependent. However, the expression of these proteins was significantly abolished by treatment with the AT1R antagonist Losartan (1µM) or AT1R small interfering RNA (siRNA). AT1R inhibition significantly suppressed extracellular signal-regulated kinase 1/2 (ERK) upregulation, which also resulted in decreases in ICAM1 and VCAM1 protein expression. These findings demonstrate that LSS induces endothelial inflammation via AT1R/ERK signaling and that Losartan has beneficial effects on endothelial inflammation.

Modulation of low shear stress­induced eNOS multi­site phosphorylation and nitric oxide production via protein kinase and ERK1/2 signaling.

Physiological shear stress has been demonstrated to serve an atheroprotective function by stimulating endothelial nitric oxide synthase (eNOS) multi­site phosphorylation. Low shear stress (LSS) serves an atheroprone role by increasing endothelial cell apoptosis and inflammation. The present study assessed whether LSS inhibited nitric oxide (NO) production in human umbilical vein endothelial cells by modulating eNOS phosphorylation and potential signaling pathways. A parallel flow chamber imposed with 2 dyn/cm2 shear stress on endothelial cells was used. Western blotting and 4,5­diaminofluorescein diacetate were used to analyze the protein expression levels and NO production. LSS activated eNOS­Ser1177 and eNOS­Thr495, but inhibited eNOS­Ser633. NO production was decreased after a transient increase at 5 min. LSS­stimulated phosphorylation of eNOS­Ser1177 and ­Thr495 were suppressed by the Akt inhibitor, perifosine, and extracellular signal regulated kinases1/2 (ERK1/2) inhibitor, PD98059, respectively. Additionally, the phosphorylation of eNOS­Ser633 inhibited by LSS was restored by the protein kinase A activator, 8­Bromo­cAMP. PD98059 completely inhibited the LSS­induced downregulation of NO production. NO downregulation in response to LSS was intensified by perifosine and was partly inhibited by 8­Bromo­cAMP. These results indicated that LSS­induced activation of ERK1/2/eNOS­Thr495 serves a major role in inhibiting endothelial NO synthase, which may explain the proinflammatory and proatherosclerotic properties of LSS.