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The analysis of chiral α-amino acids is of great significance in asymmetric synthesis, nutrition, food science, and microbiology. However, the ability of chiral recognition is difficult to achieve. Due to the demand for expensive equipment and skilled operators, traditional methods such as high-performance liquid chromatography are limited. The previously reported methods based on chemical sensor arrays usually cannot carry out the chiral analysis. Here, we developed a novel biosensor array based on the interaction between a suite of host-based luminescent bacteria and amino acids and used linear discriminant analysis to reflect their luminescence response patterns. This biosensor array could effectively discriminate chiral amino acids, including 19 L-amino acids, their corresponding D-enantiomers, and the achiral glycine. In addition, the determination of enantiomeric purity and quantitative ability has been proved. The successful identification of a complex system containing multiple chiral amino acids further demonstrates the superiority of the bioluminescent sensor array. Moreover, this sensor array could efficiently monitor the dynamic composition of free amino acids in the process of milk fermentation. Finally, the bioluminescence response mechanism of the luminescent bacteria for the recognition of chirality was clarified. This approach possessed the advantages of facile construction, high throughput, easy operation, high accuracy and fast response.

Accurate ecotoxicity assessment of contaminated soil is critical to public health, and the luminescent bacteria (Vibrio fischeri) method is the most commonly used. Hydrophobic compounds such as polycyclic aromatic hydrocarbons (PAHs) in soil cannot be in contact with luminescent bacteria due to their low water solubility so that the luminescence inhibitory effect cannot be observed. The underestimated biological toxicity makes the test unreliable and en-dangers public health and safety. The commonly adopted improved method of adding cosolvents has limited effect, it was only effective for low-hydrophobicity chemicals and could not be used for ecotoxicity evaluation of high-hydrophobicity chemicals. This is a problem that has always plagued researchers. Therefore, we constructed Pickering emulsions using luminescent bacteria modified with n-dodecanol in which PAHs were dissolved in the oil phase, n-tetradecane. Then the luminescent bacteria could tightly adhere to the oil-water interface and contact PAHs. As a result, their bioluminescence was suppressed to varying degrees depending on the chemical species and concentrations. With no solubility limitation, highly hydrophobic PAHs could even completely inhibit bacterial bioluminescence, hence the toxicity information was accurately displayed and the median effect concentration (EC

Background and purpose Inactivation of Cys674 (C674) in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) causes intracellular Ca2+ accumulation, which activates calcineurin-mediated nuclear factor of activated T-lymphocytes (NFAT)/NF-κB pathways, and results in the phenotypic modulation of smooth muscle cells (SMCs) to accelerate angiotensin II-induced aortic aneurysms. Our goal was to investigate the mechanism involved. Experimental approach We used heterozygous SERCA2 C674S knock-in (SKI) mice, where half of C674 was substituted by serine, to mimic partial irreversible oxidation of C674. The aortas of SKI mice and their littermate wild-type mice were collected for RNA sequencing, cell culture, protein expression, luciferase activity and aortic aneurysm analysis. Key results Inactivation of C674 inhibited the promoter activity and protein expression of PPARγ, which could be reversed by inhibitors of calcineurin or NF-κB. In SKI SMCs, inhibition of NF-κB by pyrrolidinedithiocarbamic acid (PDTC) or overexpression of PPARγ2 reversed the protein expression of SMC phenotypic modulation markers and inhibited cell proliferation, migration, and macrophage adhesion to SMCs. Pioglitazone, a PPARγ agonist, blocked the activation of NFAT/NF-κB, reversed the protein expression of SMC phenotypic modulation markers, and inhibited cell proliferation, migration, and macrophage adhesion to SMCs in SKI SMCs. Furthermore, pioglitazone also ameliorated angiotensin II-induced aortic aneurysms in SKI mice. Conclusions and implications The inactivation of SERCA2 C674 promotes the development of aortic aneurysms by disrupting the balance between PPARγ and NFAT/NF-κB. Our study highlights the importance of C674 redox status in regulating PPARγ to maintain aortic homeostasis.

Background and aims Our previous findings have demonstrated the protective effect of endothelial Nox4-based NADPH oxidase on atherosclerosis. One of the possible mechanisms is the inhibition of soluble epoxide hydrolase (sEH), a proinflammatory and atherogenic factor. Our goal was to investigate whether in vivo inhibition of sEH by 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) alleviates endothelial Nox4 dysfunction caused atherosclerosis and the regulatory mechanism of endothelial Nox4 on sEH. Methods & results: We used endothelial human Nox4 dominant-negative (EDN) transgenic mice in ApoE deficient background to mimic the dysfunction of endothelial Nox4 in atherosclerosis-prone conditions. In EDN aortic endothelium, sEH and the inflammatory marker vascular cell adhesion molecule 1 (VCAM1) were upregulated. TPPU reduced atherosclerotic lesions in EDN mice. In EDN endothelial cells (ECs), the endoplasmic reticulum (ER) stress markers (BIP, IRE1α, phosphorylation of PERK, ATF6) were upregulated, and they can be suppressed by ER stress inhibitor 4-phenyl butyric acid (4-PBA). In EDN ECs, 4-PBA downregulated the expression of sEH and VCAM1, suppressed inflammation, and its application in vivo reduced atherosclerotic lesions of EDN mice. Conclusions Endothelial Nox4 dysfunction upregulated sEH to enhance inflammation, probably by its induction of ER stress. Inhibition of ER stress or sEH is beneficial to alleviate atherosclerosis caused by endothelial Nox4 dysfunction.

Background: SERCA [sarco(endo)plasmic reticulum calcium ATPase] is regulated by oxidative posttranslational modifications at cysteine 674 (C674). Because sarcoplasmic reticulum (SR) calcium has been shown to play a critical role in mediating mitochondrial dysfunction in response to reactive oxygen species, we hypothesized that SERCA oxidation at C674 would modulate the effects of reactive oxygen species on mitochondrial calcium and mitochondria-dependent apoptosis in cardiac myocytes. Methods: Adult rat ventricular myocytes expressing wild-type SERCA2b or a redox-insensitive mutant in which C674 is replaced by serine (C674S) were exposed to H 2 O 2 (100 µmol/Lμ). Free mitochondrial calcium concentration was measured in adult rat ventricular myocytes with a genetically targeted fluorescent probe, and SR calcium content was assessed by measuring caffeine-stimulated release. Mice with heterozygous knock-in of the SERCA C674S mutation were subjected to chronic ascending aortic constriction. Results: In adult rat ventricular myocytes expressing wild-type SERCA, H 2 O 2 caused a 25% increase in mitochondrial calcium concentration that was associated with a 50% decrease in SR calcium content, both of which were prevented by the ryanodine receptor inhibitor tetracaine. In cells expressing the C674S mutant, basal SR calcium content was decreased by 31% and the H 2 O 2 -stimulated rise in mitochondrial calcium concentration was attenuated by 40%. In wild-type cells, H 2 O 2 caused cytochrome c release and apoptosis, both of which were prevented in C674S-expressing cells. In myocytes from SERCA knock-in mice, basal SERCA activity and SR calcium content were decreased. To test the effect of C674 oxidation on apoptosis in vivo, SERCA knock-in mice were subjected to chronic ascending aortic constriction. In wild-type mice, ascending aortic constriction caused myocyte apoptosis, LV dilation, and systolic failure, all of which were inhibited in SERCA knock-in mice. Conclusions: Redox activation of SERCA C674 regulates basal SR calcium content, thereby mediating the pathologic reactive oxygen species–stimulated rise in mitochondrial calcium required for myocyte apoptosis and myocardial failure.

Sarcoplasmic/endoplasmic reticulum Ca

The aberrant proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) are critical contributors to the pulmonary vascular remodeling that occurs during the development of Pulmonary arterial hypertension (PAH). Krüppel-like Factor 7 (KLF7) has been reported to be involved in the development of certain cardiovascular diseases. However, the role of KLF7 in PAH remains unknown. Here, we aimed to explore whether KLF7 mediates the proliferation and migration of PASMCs and its underlying mechanism. In this study, Sprague Dawley rats were exposed to 60 mg/kg monocrotaline (MCT) for 3 weeks to induce PAH and human PASMCs were stimulated with 20 ng/ml platelet-derived growth factor-BB (PDGF-BB) for 24 h to induce proliferation and migration. The mRNA and protein expression of KLF7 were significantly down-regulated in MCT-induced PAH rats and PDGF-BB-treated PASMCs. Under normal conditions, KLF7 knockdown obviously promoted PASMCs proliferation and migration, whereas KLF7 overexpression exhibited the opposite effects. Furthermore, PDGF-BB promoted the PASMCs proliferation and migration, increased the cell proportion in S phase, which was significantly attenuated by overexpression of KLF7. Mechanistic investigation indicated that KLF7 through activation its target protein, the cell cycle inhibitor p21, which finally leading to the inhibition of PASMCs growth. Consistently, UC2288, a specific inhibitor of p21, partially reversed the PASMCs proliferation inhibited by KLF7 overexpression. Taken collectively, the data suggested that KLF7 inhibits PASMCs proliferation and migration via p21 pathway and it may be used as a new therapeutic target for the PAH.

Pulmonary hypertension (PH) is a life-threatening disease characterized by pulmonary vascular remodeling, in which hyperproliferation of pulmonary artery smooth muscle cells (PASMCs) plays an important role. The cysteine 674 (C674) in the sarcoplasmic/endoplasmic reticulum Ca

Mechanosensitive ion channels are widely expressed in the cardiovascular system. They translate mechanical forces including shear stress and stretch into biological signals. The most prominent biological signal through which the cardiovascular physiological activity is initiated or maintained are intracellular calcium ions (Ca2+). Growing evidence show that the Ca2+ entry mediated by mechanosensitive ion channels is also precisely regulated by a variety of key proteins which are distributed in the cell membrane or endoplasmic reticulum. Recent studies have revealed that mechanosensitive ion channels can even physically interact with Ca2+ regulatory proteins and these interactions have wide implications for physiology and pathophysiology. Therefore, this paper reviews the cross-talk between mechanosensitive ion channels and some key Ca2+ regulatory proteins in the maintenance of calcium homeostasis and its relevance to cardiovascular health and disease.

BACKGROUND AND PURPOSE The cysteine674 (C674) thiol of Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is easily and irreversibly oxidized under atherosclerotic conditions. However, contribution of the C674 thiol redox status in the development of atherosclerosis remains unclear. Our goal was to elucidate the possible mechanism involved. EXPERIMENTAL APPROACH Heterozygous SERCA2 C674S knock-in (SKI) mice in which half of the C674 was substituted by serine674 were used to mimic removal of the reactive C674 thiol which occurs under patholog-ical conditions. The whole aorta and aortic root were isolated for histological analysis. Bone marrow derived macrophages (BMDMs) and a cardiac endothelial cell line were used for intra-cellular Ca2+, macrophage adhesion and protein expression analysis. KEY RESULTS SKI mice developed more severe atherosclerotic plaque and macrophage accumulation. Cell cul-ture studies suggest the partial substitution of SERCA2 C674 increased intracellular calcium lev-els and ER stress in both BMDMs and ECs. The release of pro-inflammatory factors and macro-phage adhesion increased in SKI BMDMs. In normal ECs, the overexpression of C674S mutant induced endothelial inflammation and promoted macrophage recruitment. Additionally, 4-phenyl butyric acid (4-PBA), an ER stress inhibitor, prevented the increased atherosclerosis observed in SKI mice, and alleviated ER stress and inflammatory responses in BMDMs and ECs exposed to 4-PBA. CONCLUSIONS AND IMPLICATIONS The substitution of SERCA2 C674 thiol accelerates the development of atherosclerosis by in-ducing ER stress and inflammation. Our findings highlight the importance of SERCA2 C674 redox status in the context of atherosclerosis, and open up a novel therapeutic strategy to combat atherosclerosis.

Inactivation of CysWe used heterozygous SERCA2 C674S knock-in (SKI) mice, where half of C674 was substituted by serine, to mimic partial irreversible oxidation of C674. The aortas of SKI mice and their littermate wild-type mice were collected for RNA sequencing, cell culture, protein expression, luciferase activity and aortic aneurysm analysis.Inactivation of C674 inhibited the promoter activity and protein expression of PPARγ, which could be reversed by inhibitors of calcineurin or NF-κB. In SKI SMCs, inhibition of NF-κB by pyrrolidinedithiocarbamic acid (PDTC) or overexpression of PPARγ2 reversed the protein expression of SMC phenotypic modulation markers and inhibited cell proliferation, migration, and macrophage adhesion to SMCs. Pioglitazone, a PPARγ agonist, blocked the activation of NFAT/NF-κB, reversed the protein expression of SMC phenotypic modulation markers, and inhibited cell proliferation, migration, and macrophage adhesion to SMCs in SKI SMCs. Furthermore, pioglitazone also ameliorated angiotensin II-induced aortic aneurysms in SKI mice.The inactivation of SERCA2 C674 promotes the development of aortic aneurysms by disrupting the balance between PPARγ and NFAT/NF-κB. Our study highlights the importance of C674 redox status in regulating PPARγ to maintain aortic homeostasis.

Background and Purpose Inactivation of Cys674 (C674) in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) by causing the accumulation of intracellular Ca2+ to activate calcineurin-mediated nuclear factor of activated T-lymphocytes (NFAT)/NF-κB pathways, resulted in the phenotypic modulation of smooth muscle cells (SMCs) to accelerate angiotensin II-induced aortic aneurysm. Our goal was to investigate the mechanism involved. Experimental Approach We used heterozygous SERCA2 C674S knock-in (SKI) mice, where half of C674 was substituted by serine, to represent partial irreversible oxidation of C674. The aortas of SKI mice and their littermate wild-type mice were collected for RNA sequencing, cell culture, protein expression, luciferase activity and aortic aneurysm analysis. KEY RESULTS Inactivation of C674 inhibited the promoter activity and protein expression of PPARγ, which could be reversed by inhibitors of calcineurin or NF-κB. Overexpression of PPARγ2 inhibited the phenotypic modulation of SKI SMCs. Pioglitazone, the activator of PPARγ, blocked the activation of NFAT/NF-κB, inhibited SMC phenotypic modulation, and ameliorated angiotensin II-induced aortic aneurysms in SKI mice. CONCLUSIONS AND IMPLICATIONS The inactivation of SERCA2 C674 promotes the development of aortic aneurysm by disrupting the balance between PPARγ and NFAT/NF-κB. Our study highlights the importance of C674 redox status in regulating PPARγ to maintain aortic homeostasis.

The arteriovenous fistula is the preferred type of hemodialysis vascular access for patients with end-stage renal disease, but a high proportion of newly created fistulas fail to mature for use. Stenosis caused by neointimal hyperplasia often is present in fistulas with maturation failure, suggesting that local mechanisms controlling vascular smooth muscle cell (SMC) migration and proliferation are important contributors to maturation failure. SMCs cultured from explants of vein tissue obtained at the time of fistula creation from 19 patients with end-stage renal disease were studied to determine whether smooth muscle responsiveness to nitric oxide is associated with fistula maturation outcomes. Nitric oxide–induced inhibition of smooth muscle cell migration, but not proliferation, was greater in cells from patients with subsequent fistula maturation success than from patients with subsequent fistula maturation failure (mean inhibition percentage, 17 versus 5.7, respectively; P = 0.035). Impaired nitric oxide responsiveness was associated with oxidation of the calcium regulatory protein, sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and was reversed by overexpressing SERCA (1.8-fold increase in inhibition, P = 0.0128) or down-regulating Nox4-based NADPH oxidase (2.3-fold increase in inhibition; P = 0.005). Our data suggest that the nitric oxide responsiveness of SMC migration is associated with fistula maturation success and raises the possibility that therapeutic restoration of nitric oxide responsiveness through manipulation of local mediators may prevent fistula maturation failure.

BACKGROUND AND PURPOSE: The kidney is essential in regulating sodium homeostasis and BP. The irreversible oxidation of Cys(674) (C674) in the sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) is increased in the renal cortex of hypertensive mice. Whether inactivation of C674 promotes hypertension is unclear. Here we have investigated the effects on BP of the inactivation of C674, and its role in the kidney. EXPERIMENTAL APPROACH: We used heterozygous SERCA2 C674S knock‐in (SKI) mice, where half of C674 was substituted by serine, to represent partial irreversible oxidation of C674. The BP, urine volume, and urine composition of SKI mice and their littermate wild‐type (WT) mice were measured. The kidneys were collected for cell culture, Na(+)/K(+)‐ATPase activity, protein expression, and immunohistological analysis. KEY RESULTS: Compared with WT mice, SKI mice had higher BP, lower urine volume and sodium excretion, up‐regulated endoplasmic reticulum (ER) stress markers and soluble epoxide hydrolase (sEH), and down‐regulated dopamine D(1) receptors in renal cortex and cells from renal proximal tubule. ER stress and sEH were mutually regulated, and both upstream of D(1) receptors. Inhibition of ER stress or sEH up‐regulated expression of D(1) receptors, decreased the activity of Na(+)/K(+)‐ATPase, increased sodium excretion, and lowered BP in SKI mice. CONCLUSIONS AND IMPLICATIONS: The inactivation of SERCA2 C674 promotes the development of hypertension by inducing ER stress and sEH. Our study highlights the importance of C674 redox status in BP control and the contribution of SERCA2 to sodium homeostasis and BP in the kidney.

Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) is vital to maintain intracellular calcium homeostasis. SERCA2 cysteine 674 (C674) is highly conservative and its irreversible oxidation is upregulated in human and mouse aortic aneurysms, especially in smooth muscle cells (SMCs). The contribution of SERCA2 and its redox C674 in the development of aortic aneurysm remains enigmatic. Objective: Our goal was to investigate the contribution of inactivation of C674 to the development of aortic aneurysm and the mechanisms involved. Approach and results: Using SERCA2 C674S knock-in (SKI) mouse line, in which half of C674 was substituted by serine 674 (S674) to represent partial irreversible oxidation of C674 in aortic aneurysm, we found that in aortic SMCs the replacement of C674 by S674 resulted in SMC phenotypic modulation. In SKI SMCs, the increased intracellular calcium activated calcium-dependent calcineurin, which promoted the nuclear translocation of nuclear factor of activated T-lymphocytes (NFAT) and nuclear factor kappa-B (NFκB), while inhibition of calcineurin blocked SMC phenotypic modulation. Besides, the replacement of C674 by S674 accelerated angiotensin II-induced aortic aneurysm. Conclusions: Our results indicate that the inactivation of C674 by causing the accumulation of intracellular calcium to activate calcineurin-mediated NFAT/NFκB pathways, resulted in SMC phenotypic modulation to accelerate aortic aneurysm, which highlights the importance of C674 redox state in the development of aortic aneurysms.

Pulmonary arterial hypertension (PAH) is a clinical hemodynamic syndrome characterized by elevated pulmonary arterial pressure and pulmonary vascular resistance leading to right heart failure and death. Vascular remodeling is the most prominent histopathological feature of PAH, which is regulated by many factors. Endoplasmic reticulum stress, calcium disorder and mitochondrial dysfunction are involved in the vascular cell proliferation and apoptosis by regulating intracellular calcium homeostasis and cellular metabolism. Epigenetic phenomenon such as DNA damage and abnormal expression of miRNA are also involved in the regulation of abnormal proliferation of vascular cells. Vascular cell phenotype switching including endothelial-mesenchymal transition and smooth muscle cell phenotype switching play an important role in abnormal proliferation of vascular cells. Vascular remodeling is produced by a variety of cells and molecular pathways, and aiming at multiple targets which is expected to find a new breakthrough in the treatment of PAH,and to improve abnormal vascular remodeling, delay or even reverse the progression of PAH.

Diabetic retinopathy is a multifactorial microvascular complication, and its pathogenesis hasn't been fully elucidated. The irreversible oxidation of cysteine 674 (C674) in the sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) was increased in the type 1 diabetic retinal vasculature. SERCA2 C674S knock-in (SKI) mouse line that half of C674 was replaced by serine 674 (S674) was used to study the effect of C674 inactivation on retinopathy. Compared with wild type (WT) mice, SKI mice had increased number of acellular capillaries and pericyte loss similar to those in type 1 diabetic WT mice. In the retina of SKI mice, pro-apoptotic proteins and intracellular Ca2+-dependent signaling pathways increased, while anti-apoptotic proteins and vessel density decreased. In endothelial cells, C674 inactivation increased the expression of pro-apoptotic proteins, damaged mitochondria, and induced cell apoptosis. These results suggest that a possible mechanism of retinopathy induced by type 1 diabetes is the interruption of calcium homeostasis in the retina by oxidation of C674. C674 is a key to maintain retinal health. Its inactivation can cause retinopathy similar to type 1 diabetes by promoting apoptosis. SERCA2 might be a potential target for the prevention and treatment of diabetic retinopathy.

Background and aims Angiotensin II (Ang II) is commonly used to induce aortic aneurysm and atherosclerosis in animal models. Ang II upregulates NADPH oxidase isoform Nox4 in aortic smooth muscle cells (SMCs) in mice. However, whether smooth muscle Nox4 is directly involved in Ang II-induced aortic aneurysm and atherosclerosis is unclear. Methods & results To address this, we used smooth muscle-specific Nox4 dominant-negative (SDN) transgenic mice, in which Nox4 activity is constitutively inhibited. In non-transgenic (NTg) mice, Ang II increased the expression of proteins known to contribute to both aortic aneurysm and atherosclerosis, namely osteopontin (OPN), collagen type I&III (Col I&III), matrix metalloproteinase 2 (MMP2), and vascular cell adhesion molecule 1 (VCAM1), which were all significantly downregulated in SDN mice. The number and size of Ang II-induced aorta collateral aneurysms and atherosclerotic lesions in the renal artery and aortic root of SDN mice were significantly decreased compared to NTg mice, and directly correlated with a decrease in OPN expression. Replenishing OPN in SDN SMCs, increased the expression of Col I&III, MMP2, and VCAM1, and promoted SMC proliferation, migration, and inflammation. Conclusions Our data demonstrate that smooth muscle Nox4 directly promotes the development of Ang II-induced aortic aneurysm and atherosclerosis, at least in part, through regulating OPN expression.

Autophagy is a highly conserved degradation process by which intracellular components, including soluble macromolecules (e.g. nucleic acids, proteins, carbohydrates, and lipids) and dysfunctional organelles (e.g. mitochondria, ribosomes, peroxisomes, and endoplasmic reticulum) are degraded by the lysosome. Autophagy is orchestrated by the autophagy related protein (Atg) composed protein complexes to form autophagosomes, which fuse with lysosomes to generate autolysosomes where the contents are degraded to provide energy for cell survival in response to environmental and cellular stress. Autophagy is an important player in cardiovascular disease development such as atherosclerosis, cardiac ischemia/reperfusion, cardiomyopathy, heart failure and hypertension. Autophagy in particular contributes to cardiac ischemia, hypertension and diabetes by interaction with reactive oxygen species generated in endoplasmic reticulum and mitochondria. This review highlights the dual role of autophagy in cardiovascular disease development. Full recognition of autophagy as an adaptive or maladaptive response would provide potential new strategies for cardiovascular disease prevention and management. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

Nox4-based NADPH oxidase is a major reactive oxygen species-generating enzyme in the vasculature, but its role in atherosclerosis remains controversial. Objective Our goal was to investigate the mechanisms of endothelial Nox4 in regulating atherosclerosis. Approach and results Atherosclerosis-prone conditions (disturbed blood flow, type I diabetes, and Western diet) downregulated endothelial Nox4 mRNA in arteries. To address whether the downregulated endothelial Nox4 was directly involved in the development of atherosclerosis, we generated mice carrying a human Nox4 P437H dominant negative mutation (Nox4DN), driven by the endothelial specific promoter Tie-2, on atherosclerosis-prone genetic background (ApoE deficient mice) to mimic the effect of decreased endothelial Nox4. Nox4DN significantly increased type I diabetes-induced aortic stiffness and atherosclerotic lesions. Gene analysis indicated that soluble epoxide hydrolase 2 (sEH) was significantly upregulated in Nox4DN endothelial cells (EC). Inhibition of sEH activity in Nox4DN EC suppressed inflammation and macrophage adhesion to EC. On the contrary, overexpression of endothelial wild type Nox4 suppressed sEH, ameliorated Western diet-induced atherosclerosis and decreased aortic stiffness. Conclusions Atherosclerosis-prone conditions downregulated endothelial Nox4 to accelerate the progress of atherosclerosis, at least in part, by upregulating sEH to enhance inflammation.

Ischemia is a complex phenomenon modulated by the concerted action of several cell types. We have identified that sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase 2 (SERCA 2) cysteine 674 (C674) S-glutathiolation is essential for ischemic angiogenesis, vascular endothelial growth factor (VEGF)-mediated endothelial cell (EC) migration and network formation. A heterozygote SERCA 2 C674S knockin (SKI) mouse shows impaired ischemic blood flow recovery after femoral artery ligation, and its ECs show depleted endoplasmic reticulum (ER) Ca(2+) stores and impaired angiogenic behavior. Here we studied the role of SERCA 2 C674 in the interaction between ECs and macrophages in the context of ischemia and discovered the involvement of the ER stress response protein, ER oxidoreductin-1α (ERO1). In wild type (WT) mice, expression of ERO1 was increased in the ischemic hind limb in vivo, as well as in ECs and macrophages exposed to hypoxia in vitro. The increase in ERO1 to ischemia/hypoxia was less in SKI mice. In WT ECs, both vascular cell adhesion molecule 1 (VCAM1) expression and bone marrow-derived macrophage adhesion to ECs were increased by hypoxia, and both were attenuated in SKI ECs. In WT ECs, ERO1 siRNA blocked hypoxia-induced VCAM1 expression and macrophage adhesion. In WT macrophages, hypoxia also stimulated both ERO1 and VEGF expression, and both were less in SKI macrophages. Compared with conditioned media of hypoxic SKI macrophages, conditioned media from WT macrophages had a greater effect on EC angiogenic behavior, and were blocked by VEGF neutralizing antibody. Taken together, under hypoxic conditions, SERCA 2 C674 and ERO1 enable increased VCAM1 expression and macrophage adhesion to ECs, as well as macrophage VEGF production that, in turn, promote angiogenesis. This study highlights the hitherto unrecognized interaction of two ER proteins, SERCA 2 C674 and ERO1, which mediate the EC and macrophage angiogenic response to ischemia/hypoxia.

The endothelium produces and responds to reactive oxygen and nitrogen species (RONS), providing important redox regulation to the cardiovascular system in physiology and disease. In no other situation are RONS more critical than in the response to tissue ischemia. Here, tissue healing requires growth factor-mediated angiogenesis that is in part dependent on low levels of RONS, which paradoxically must overcome the damaging effects of high levels of RONS generated as a result of ischemia. While generation of endothelial cell RONS in hypoxia/reoxygenation is acknowledged, the mechanism for their role in angiogenesis is still poorly understood. During ischemia, the major low molecular weight thiol glutathione (GSH) reacts with RONS and protein cysteines, producing GSH-protein adducts. Recent data indicate that GSH adducts on certain proteins are essential to growth factor responses in endothelial cells. Genetic deletion of the enzyme glutaredoxin-1, which selectively removes GSH protein adducts, improves, while its overexpression impairs, revascularization of the ischemic hindlimb of mice. Ischemia-induced GSH adducts on specific cysteine residues of several proteins, including p65 NFkB and the sarcoplasmic reticulum calcium ATPase-2 (SERCA2), appear to promote ischemic angiogenesis. Identifying the specific proteins in the redox response to ischemia has provided therapeutic opportunities to improve clinical outcomes of ischemia.

Endothelial cell (EC) migration in response to vascular endothelial growth factor (VEGF) is a critical step in both physiological and pathological angiogenesis. Although VEGF signaling has been extensively studied, the mechanisms by which VEGF-dependent reactive oxygen species (ROS) production affects EC signaling are not well understood. The aim of this study was to elucidate the involvement of Nox2- and Nox4-dependent ROS in VEGF-mediated EC Ca(2+) regulation and migration. VEGF induced migration of human aortic ECs into a scratch wound over 6 h, which was inhibited by overexpression of either catalase or superoxide dismutase (SOD). EC stimulation by micromolar concentrations of H2O2 was inhibited by catalase, but also unexpectedly by SOD. Both VEGF and H2O2 increased S-glutathiolation of SERCA2b and increased Ca(2+) influx into EC, and these events could be blocked by overexpression of catalase or overexpression of SERCA2b in which the reactive cysteine-674 was mutated to a serine. In determining the source of VEGF-mediated ROS production, our studies show that specific knockdown of either Nox2 or Nox4 inhibited VEGF-induced S-glutathiolation of SERCA, Ca(2+) influx, and EC migration. Treatment with H2O2 induced S-glutathiolation of SERCA and EC Ca(2+) influx, overcoming the knockdown of Nox4, but not Nox2, and Amplex red measurements indicated that Nox4 is the source of H2O2. These results demonstrate that VEGF stimulates EC migration through increased S-glutathiolation of SERCA and Ca(2+) influx in a Nox4- and H2O2-dependent manner, requiring Nox2 downstream.

Background : Myocyte contractile dysfunction occurs in pathological remodeling in association with abnormalities in calcium regulation. Mice with cardiac myocyte–specific overexpression of Gαq develop progressive left ventricular failure associated with myocyte contractile dysfunction and calcium dysregulation. Objective : We tested the hypothesis that myocyte contractile dysfunction in the Gαq mouse heart is mediated by reactive oxygen species, and in particular, oxidative posttranslational modifications, which impair the function of sarcoplasmic reticulum Ca 2+ -ATPase (SERCA). Methods and Results : Freshly isolated ventricular myocytes from Gαq mice had marked abnormalities of myocyte contractile function and calcium transients. In Gαq myocardium, SERCA protein was not altered in quantity but displayed evidence of oxidative cysteine modifications reflected by decreased biotinylated iodoacetamide labeling and evidence of specific irreversible oxidative modifications consisting of sulfonylation at cysteine 674 and nitration at tyrosines 294/295. Maximal calcium-stimulated SERCA activity was decreased 47% in Gαq myocardium. Cross-breeding Gαq mice with transgenic mice that have cardiac myocyte-specific overexpression of catalase (a) decreased SERCA oxidative cysteine modifications, (b) decreased SERCA cysteine 674 sulfonylation and tyrosine 294/295 nitration, (c) restored SERCA activity, and (d) improved myocyte calcium transients and contractile function. Conclusions : In Gαq-induced cardiomyopathy, myocyte contractile dysfunction is mediated, at least in part, by 1 or more oxidative posttranslational modifications of SERCA. Protein oxidative posttranslational modifications contribute to the pathophysiology of myocardial dysfunction and thus may provide a target for therapeutic intervention.

Vascular disease in hypertension and diabetes is associated with increased oxidants. The oxidants arise from NADPH oxidase, xanthine oxidase, and mitochondria. Superoxide anion and hydrogen peroxide are produced by both leukocytes and vascular cells. Nitric oxide is produced in excess by inducible nitric oxide synthase, and the potent oxidant, peroxynitrite, is formed from superoxide and nitric oxide. The damage to proteins caused by oxidants is selective, affecting specific oxidant-sensitive amino acid residues. With some important vascular proteins, for example, endothelial nitric oxide synthase, prostacycline synthase, and superoxide dismutase, oxidation of a single susceptible amino acid inactivates the enzyme. The beneficial effects of antioxidants, at least in animal models of hypertension and diabetes, can in part be ascribed to protection of these and other proteins. Mutant proteins lacking their reactive constituent can recapitulate some disease phenotypes suggesting a pathogenic role of the oxidation. Thus, many of the shared functional abnormalities of hypertensive and diabetic blood vessels may be caused by oxidants. Although studies using antioxidants have failed in patients, the successful treatment of vascular disease with HMG-CoA reductase inhibitors, thromboxane A2 antagonists, and polyphenols may depend on their anti-inflammatory effects and ability to decrease production of damaging oxidants.

Nitric oxide (NO) causes S-glutathiolation of the reactive cysteine-674 in the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA), thus increasing SERCA activity, and inhibiting Ca(2+) influx and migration of vascular smooth muscle cells (VSMC). Because increased VSMC migration contributes to accelerated neointimal growth and atherosclerosis in diabetes, the effect of culture of VSMC in high glucose (HG) was determined. Rat aortic VSMC were exposed to normal (5.5 mmol/L) or high (25 mmol/L) glucose for 3 days, and serum-induced cell migration during 6 h into a wounded cell monolayer was measured 5 min after adding the NO donor S-nitroso-N-acetylpenicillamine (SNAP) or 24 h after interleukin-1beta (IL-1beta) to express inducible nitric oxide synthase (iNOS). In normal glucose, SNAP or IL-1beta significantly inhibited migration in cells infected with adenovirus to express GFP or SERCA wild type (WT), but not with a C674S SERCA mutant. After HG, NO failed to inhibit migration, nor did it decrease calcium-dependent association of calmodulin with calcineurin, indicating that NO failed to decrease intracellular calcium levels via SERCA. In contrast, overexpression of SERCA WT, but not the SERCA C674S mutant, preserved the ability for NO to inhibit migration despite exposing the cells to HG. The antioxidant, Tempol, or overexpression of superoxide dismutase also prevented the effects of HG. Further studies showed that both biotinylated-iodoacetamide and NO-induced biotinylated glutathione labeling of SERCA C674 were decreased by HG, and a sequence-specific sulfonic acid antibody detected oxidation of the C674 SERCA thiol. These results indicate that failure of NO to inhibit migration in VSMC exposed to HG is due to oxidation of the SERCA reactive cysteine-674.

Objectives— Nitric oxide inhibits smooth muscle cell migration after arterial injury, but the detailed mechanism is not fully understood. The sarco/endoplasmic reticulum calcium ATPase (SERCA) lowers cell Ca 2+ by increasing intracellular Ca 2+ uptake and inhibiting extracellular Ca 2+ influx. Our previous studies showed that NO causes cyclic GMP-independent arterial relaxation by increasing SERCA activity by inducing reversible S -glutathiolation at cysteine-674. Because Ca 2+ is an important second messenger for cell migration, we hypothesized that NO also inhibits cell migration through redox regulation of SERCA activity via cysteine-674. Methods and Results— To test our hypothesis, overexpression of either wild type (WT) or mutant SERCA in which cysteine-674 was mutated to serine was accomplished by stable transfection of HEK 293 or adenoviral expression in rat aortic smooth muscle cells (VSMCs). In the cell models expressing mutant SERCA, biotinylated-iodoacetamide (BIAM) and biotinylated-glutathione labeling of SERCA was decreased, and NO failed to increase SERCA activity or decrease Ca 2+ influx, thus validating that the expression of mutant SERCA prevents its redox-dependent activation. In the absence of NO, fetal bovine serum stimulated migration of both cell types expressing WT or C674S SERCA at similar rates. The NO donor S-nitrosopenicillamine inhibited migration of cells with WT SERCA, but had no effect on the migration of either HEK cells or VSMCs with C674S SERCA. The same result was obtained in VSMCs in which endogenous NO was produced by iNOS induced by interleukin (IL)-1β. Blocking cyclic GMP did not prevent the inhibition of migration by NO. Conclusions— In cells overexpressing SERCA, the cyclic GMP-independent, redox regulation of SERCA cysteine-674 is required for the inhibition of cell migration by both exogenous and endogenously generated NO.

K(ATP) channels are involved in regulating coronary function, but the contribution of endothelial K(ATP) channels remains largely uncharacterized. We generated a transgenic mouse model to specifically target endothelial K(ATP) channels by expressing a dominant negative Kir6.1 subunit only in the endothelium. These animals had no obvious overt phenotype and no early mortality. Histologically, the coronary endothelium in these animals was preserved. There was no evidence of increased susceptibility to ergonovine-induced coronary vasospasm. However, isolated hearts from these animals had a substantially elevated basal coronary perfusion pressure. The K(ATP) channel openers, adenosine and levcromakalim, decreased the perfusion pressure whereas the K(ATP) channel blocker glibenclamide failed to produce a vasoconstrictive response. The inducible endothelial nitric oxide pathway was intact, as evidenced by vasodilation caused by bradykinin. In contrast, basal endothelin-1 release was significantly elevated in the coronary effluent from these hearts. Treatment of mice with bosentan (endothelin-1 receptor antagonist) normalized the coronary perfusion pressure, demonstrating that the elevated endothelin-1 release was sufficient to account for the increased coronary perfusion pressure. Pharmacological blockade of K(ATP) channels led to elevated endothelin-1 levels in the coronary effluent of isolated mouse and rat hearts as well as enhanced endothelin-1 secretion from isolated human coronary endothelial cells. These data are consistent with a role for endothelial K(ATP) channels to control the coronary blood flow by modulating the release of the vasoconstrictor, endothelin-1.

Nox4-based NADPH oxidase is a major reactive oxygen species-generating enzyme in the vasculature, but its role in atherosclerosis remains controversial. Objective Our goal was to investigate the role of smooth muscle Nox4 in atherosclerosis. Approach and results Atherosclerosis-prone conditions (disturbed blood flow and Western diet) increased Nox4 mRNA level in smooth muscle of arteries. To address whether upregulated smooth muscle Nox4 under atherosclerosis-prone conditions was directly involved in the development of atherosclerosis, mice carrying a human Nox4 P437H dominant negative mutation (Nox4DN), specifically in smooth muscle, were generated on a FVB/N ApoE deficient genetic background to counter the effect of increased smooth muscle Nox4. Nox4DN significantly decreased aortic stiffness and atherosclerotic lesions, with no effect on blood pressure. Gene analysis indicated that soluble epoxide hydrolase 2 (sEH) was significantly downregulated in Nox4DN smooth muscle cells (SMC), at both mRNA and protein levels. Downregulation of sEH by siRNA decreased SMC proliferation and migration, and suppressed inflammation and macrophage adhesion to SMC. Conclusions Downregulation of smooth muscle Nox4 inhibits atherosclerosis by suppressing sEH, which, at least in part, accounts for inhibition of SMC proliferation, migration and inflammation.

Elevated levels of reactive oxygen species (ROS) in the vascular wall play a key role in the development of neointimal hyperplasia. Nox4-based NADPH oxidase is a major ROS generating enzyme in the vasculature, but its roles in neointimal hyperplasia remain unclear. Objective Our purpose was to investigate the role of smooth muscle cell (SMC) Nox4 in neointimal hyperplasia. Approach and results Mice overexpressing a human Nox4 mutant form, carrying a P437H dominant negative mutation (Nox4DN) and driven by SM22α promoter, to achieve specific expression in SMC, were generated in a FVB/N genetic background. After wire injury-induced endothelial denudation, Nox4DN had significantly decreased neointima formation compared with non-transgenic littermate controls (NTg). ROS production, serum-induced proliferation and migration, were significantly decreased in aortic SMCs isolated from Nox4DN compared with NTg. Both mRNA and protein levels of thrombospondin 1 (TSP1) were significantly downregulated in Nox4DN SMCs. Downregulation of TSP1 by siRNA decreased cell proliferation and migration in SMCs. Similar to Nox4DN, downregulation of Nox4 by siRNA significantly decreased TSP1 expression level, cell proliferation and migration in SMCs. Conclusions Downregulation of smooth muscle Nox4 inhibits neointimal hyperplasia by suppressing TSP1, which in part can account for inhibition of SMC proliferation and migration.

The sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) is key to Ca(2+) homeostasis and is redox-regulated by reversible glutathione (GSH) adducts on the cysteine (C) 674 thiol that stimulate Ca(2+) uptake activity and endothelial cell angiogenic responses in vitro. We found that mouse hind limb muscle ischemia induced S-glutathione adducts on SERCA in both whole muscle tissue and endothelial cells. To determine the role of S-glutathiolation, we used a SERCA 2 C674S heterozygote knock-in (SKI) mouse lacking half the key thiol. Following hind limb ischemia, SKI animals had decreased SERCA S-glutathione adducts and impaired blood flow recovery. We studied SKI microvascular endothelial cells in which total SERCA 2 expression was unchanged. Cultured SKI microvascular endothelial cells showed impaired migration and network formation compared with wild type (WT). Ca(2+) studies showed decreased nitric oxide (·NO)-induced (45)Ca(2+) uptake into the endoplasmic reticulum (ER) of SKI cells, while Fura-2 studies revealed lower Ca(2+) stores and decreased vascular endothelial growth factor (VEGF)- and ·NO-induced Ca(2+) influx. Adenoviral overexpression of calreticulin, an ER Ca(2+) binding protein, increased ionomycin-releasable stores, VEGF-induced Ca(2+) influx and endothelial cell migration. Taken together, these data indicate that the redox-sensitive Cys-674 thiol on SERCA 2 is required for normal endothelial cell Ca(2+) homeostasis and ischemia-induced angiogenic responses, revealing a novel redox control of angiogenesis via Ca(2+) stores.

The endoplasmic reticulum (ER) plays a pivotal role in lipid and protein biosynthesis as well as calcium store regulation, which determines its essential role in cell function. Hypoxia, nutrient deprivation, perturbation of redox status and aberrant calcium regulation can all trigger the ER stress response, which is mediated through three main sensors, namely inositol requiring element-1 (IRE-1), protein kinase-like ER kinase (PERK) and activating transcription factor 6 (ATF6). This review explores the interaction of ER stress and ER stress-associated pathological processes, including inflammation, apoptosis, aberrant autophagy, mitochondrial dysfunction and hypoxic responses. In addition, the correlation of ER stress with lipid and calcium homeostasis and dysregulation, and its role in disease development is also presented. Improved understanding of ER stress and its cofactors in pathological processes may provide new perspective on disease development and control.

Diet-induced obesity and metabolic syndrome are important contributors to cardiovascular diseases. The decreased nitric oxide (NO) bioactivity in endothelium and the impaired response of smooth muscle cell (SMC) to NO significantly contribute to vascular pathologies, including atherosclerosis and arterial restenosis after angioplasty. Sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) is an important mediator of NO function in both endothelial cells and SMCs, and its irreversible oxidation impairs its stimulation by NO. We used C57BL/6J mice fed a high fat high sucrose diet (HFHSD) to study the role of SMC SERCA in diet-induced obesity and metabolic syndrome. We found that HFHSD upregulated Nox2 based NADPH oxidase, induced inflammation, increased irreversible SERCA oxidation, and suppressed the response of aortic SERCA to NO. Cultured aortic SMCs from mice fed HFHSD showed increased reactive oxygen species production, Nox2 upregulation, irreversible SERCA oxidation, inflammation, and a decreased ability of NO to inhibit SMC migration. Overexpression of wild type SERCA2b or downregulation of Nox2 restored NO-mediated inhibition of migration in SMCs isolated from HFHSD-fed mice. In addition, tumor necrosis factor alpha (TNFα) increased Nox2 which induced SERCA oxidation and inflammation. Taken together, Nox2 induced by HFHSD plays significant roles in controlling SMC responses to NO and TNFα-mediated inflammation, which may contribute to the development of cardiovascular diseases in diet-induced obesity and metabolic syndrome.

Background A hallmark of aging of the cardiac myocyte is impaired sarcoplasmic reticulum ( SR ) calcium uptake and relaxation due to decreased SR calcium ATP ase ( SERCA ) activity. We tested the hypothesis that H 2 O 2 ‐mediated oxidation of SERCA contributes to impaired myocyte relaxation in aging. Methods and Results Young (5‐month‐old) and senescent (21‐month‐old) FVB wild‐type (WT) or transgenic mice with myocyte‐specific overexpression of catalase were studied. In senescent mice, myocyte‐specific overexpression of catalase (1) prevented oxidative modification of SERCA as evidenced by sulfonation at Cys674, (2) preserved SERCA activity, (3) corrected impaired calcium handling and relaxation in isolated cardiac myocytes, and (4) prevented impaired left ventricular relaxation and diastolic dysfunction. Nitroxyl, which activates SERCA via S ‐glutathiolation at Cys674, failed to activate SERCA in freshly isolated ventricular myocytes from senescent mice. Finally, in adult rat ventricular myocytes in primary culture, adenoviral overexpression of SERCA in which Cys674 is mutated to serine partially preserved SERCA activity during exposure to H 2 O 2 . Conclusion Oxidative modification of SERCA at Cys674 contributes to decreased SERCA activity and impaired myocyte relaxation in the senescent heart. Strategies to decrease oxidant levels and/or protect target proteins such as SERCA may be of value to preserve diastolic function in the aging heart.

Endoplasmic Reticulum Stress and Related Pathological Processes The endoplasmic reticulum (ER) plays a pivotal role in lipid and protein biosynthesis as well as calcium store regulation, which determines its essential role in cell function. Hypoxia, nutrient deprivation, perturbation of redox status and aberrant calcium regulation can all trigger the ER stress response, which is mediated through three main sensors, namely inositol requiring element-1 (IRE-1), protein kinase-like ER kinase (PERK) and activating transcription factor 6 (ATF6). This review explores the interaction of ER stress and ER stress-associated pathological processes, including inflammation, apoptosis, aberrant autophagy, mitochondrial dysfunction and hypoxic responses. In addition, the correlation of ER stress with lipid and calcium homeostasis and dysregulation, and its role in disease development is also presented. Improved understanding of ER stress and its cofactors in pathological processes may provide new perspective on disease development and control.

Aims: Vascular endothelial growth factor (VEGF) increases angiogenesis by stimulating endothelial cell (EC) migration. VEGF-induced nitric oxide (•NO) release from •NO synthase plays a critical role, but the proteins and signaling pathways that may be redox-regulated are poorly understood. The aim of this work was to define the role of •NO-mediated redox regulation of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) in VEGF-induced signaling and EC migration. Results: VEGF-induced EC migration was prevented by the •NO synthase inhibitor, N (G)-nitro-L-arginine methyl ester (LNAME). Either VEGF or •NO stimulated endoplasmic reticulum (ER) 45Ca2+ uptake, a measure of SERCA activity, and knockdown of SERCA2 prevented VEGF-induced EC migration and 45Ca2+ uptake. S-glutathione adducts on SERCA2b, identified immunochemically, were increased by VEGF, and were prevented by LNAME or overexpression of glutaredoxin-1 (Glrx-1). Furthermore, VEGF failed to stimulate migration of ECs overexpressing Glrx-1. VEGF or •NO increased SERCA S-glutathiolation and stimulated migration of ECs in which wild-type (WT) SERCA2b was overexpressed with an adenovirus, but did neither in those overexpressing a C674S SERCA2b mutant, in which the reactive cysteine-674 was mutated to a serine. Increased EC Ca2+ influx caused by VEGF or •NO was abrogated by overexpression of Glrx-1 or the C674S SERCA2b mutant. ER store-emptying through the ryanodine receptor (RyR) and Ca2+ entry through Orai1 were also required for VEGF- and •NO-induced EC Ca2+ influx. Innovation and Conclusions: These results demonstrate that •NO-mediated activation of SERCA2b via S-glutathiolation of cysteine-674 is required for VEGF-induced EC Ca2+ influx and migration, and establish redox regulation of SERCA2b as a key component in angiogenic signaling. Antioxid. Redox Signal. 00, 000–000.