Abstract MP59: Soluble Protein Oligomers Induce Endoplasmic Reticulum Stress In Mesenteric Resistance Arteries From Male And Female Mice

Amyloid β proteins, including toxic soluble oligomers (SPOs) are not only found in the brain duringAlzheimer’s, but also in the peripheral vascular system. The precise mechanism linking increasedcirculating levels of SPOs and vascular dysfunction remains unknown. We hypothesized that SPOslead to endoplasmic reticulum (ER) stress, further release of SPOs and vascular injury. Mesentericresistance arteries (MRAs) from 14 weeks old, male and female C57BL/6 mice were used forvascular function. Agonists were acetylcholine and phenylephrine (1nM-10mM). In acuteconditions, SPOs (0.1μM) caused pathologically exacerbated endothelium-dependent vasodilationcompared to vehicle (F12 media) [Male: EC50: SPOs: -7.0 ± 0.1 (n=4), vs. Vehicle -6.6 ± 0.1 (n=7)p=0.03; Female: EC50: SPOs: -7.3 ± 0.06 (n=5) vs. Vehicle -6.7 ± 0.1 (n=6), p=0.001]. Thisphenotype was similar to the positive control tunicamycin (5mg/ml) [Male: EC50: Tunicamycin: -7.3(n=4), vs. Vehicle -6.6 (n=7) p=0.2; Female: EC50: Tunicamycin: -7.7 (n=4) vs. Vehicle -6.8 (n=5)p=0.04]. To determine whether SPO’s cause ER stress, arteries were treated with ER stressinhibitor 4-Phenylbutyric acid (2mM). The ER stress inhibitor prevented the exacerbatedvasodilation induced by SPOs showing SPOs trigger ER stress in acute conditions independent ofsex. To determine whether SPOs are a consequence of ER stress, arteries were incubated withtunicamycin in the presence of the SPO inhibitor K01-162 (10mM). Interestingly, K01-162 did notprevent the tunicamycin-induced exacerbated vasodilation in arteries from male mice. However,this response was decreased in arteries from female mice showing that inducing ER stress leadsto the release of SPOs, escalating a feed-forward mechanism of further SPO release. There wereno changes in vascular contraction with tunicamycin or SPOs irrespective of sex. ER stress wasconfirmed with anti-KDEL antibody staining, specific for ER resident chaperones Grp78/94 andvisualized with multiphoton fluorescent confocal microscopy. These results demonstrate that SPO’sexacerbate endothelium-dependent vasodilation acutely and may contribute to brain and peripheralvascular edema and loss of autoregulation observed during cardiovascular and Alzheimer’sdisease.

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Soluble Protein Oligomers induce Endoplasmic Reticulum Stress in Acute Conditions in Mesenteric Resistance Arteries from Male and Female Mice

The FASEB Journal ◽

10.1096/fasebj.2021.35.s1.03855 ◽

2021 ◽

Vol 35 (S1) ◽

Author(s):

Emily Waigi ◽

Nicole Bearss ◽

Jonnelle Edwards ◽

Thaddaeus Castaneda ◽

Cameron McCarthy ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Soluble Protein ◽

Resistance Arteries ◽

Male And Female ◽

Female Mice ◽

Induce Endoplasmic Reticulum Stress ◽

Acute Conditions

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Abstract 33: Endoplasmic Reticulum Stress and Vascular Dysfunction in Hypertension

Circulation Research ◽

10.1161/res.111.suppl_1.a33 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Modar Kassan ◽

Maria Galan ◽

Megan Partyka ◽

Daniel Henrion ◽

Mohamed Trebak ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Vascular Function ◽

Vascular Dysfunction ◽

Ang Ii ◽

Resistance Arteries ◽

Cardiac Damage ◽

Stress Markers ◽

Enos Phosphorylation ◽

Dependent Mechanism

Objective: Cardiac damage and vascular dysfunction are major causes of morbidity and mortality in hypertension. In this study, we explored the beneficial therapeutic effect of endoplasmic reticulum (ER) stress inhibition on cardiac damage and vascular dysfunction in hypertension. Methods & results: Mice were infused with angiotensin II (Ang-II, 400 ng/kg/min) with or without ER stress inhibitors (Tudca and PBA) for two weeks. Mice infused with Ang-II displayed an increase in blood pressure, cardiac hypertrophy and fibrosis associated with enhanced collagen-I content, TGFβ1 activity, and ER stress markers, which were blunted after ER stress inhibition. Hypertension induced ER stress in aorta and mesenteric resistance arteries (MRA), enhanced TGFβ1 activity in aorta but not in MRA, and reduced eNOS phosphorylation and endothelium-dependent relaxation (EDR) in aorta and MRA. The inhibition of ER stress significantly reduced TGFβ1 activity, enhanced eNOS phosphorylation and improved EDR. The inhibition of TGFβ1 pathway improved EDR in aorta but not in MRA, while the reduction in ROS levels ameliorated EDR in MRA only. Infusion of tunicamycin in control mice induced ER stress in aorta and MRA, and reduced EDR by a TGFβ1-dependent mechanism in aorta and ROS-dependent mechanism in MRA. Conclusion: ER stress inhibition reduces cardiac damage and improves vascular function in hypertension. Therefore ER stress could be a potential target for cardiovascular diseases.

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Abstract P217: Nox Compartmentalization, Protein Oxidation and ER Stress in Vascular Smooth Muscle Cells in Hypertension

Hypertension ◽

10.1161/hyp.70.suppl_1.p217 ◽

2017 ◽

Vol 70 (suppl_1) ◽

Author(s):

Livia L Camargo ◽

Augusto C Montezano ◽

Adam Harvey ◽

Sofia Tsiropoulou ◽

Katie Hood ◽

...

Keyword(s):

Er Stress ◽

Protein Oxidation ◽

Vascular Function ◽

Vascular Reactivity ◽

Stress Proteins ◽

Vascular Dysfunction ◽

Mesenteric Arteries ◽

Resistance Arteries ◽

Post Translational Modification ◽

Wistar Kyoto

In hypertension, activation of NADPH oxidases (Noxs) is associated with oxidative stress and vascular dysfunction. The exact role of each isoform in hypertension-associated vascular injury is still unclear. We investigated the compartmentalization of Noxs in VSMC from resistance arteries of Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Expression of Nox1 and Nox4 was increased in SHR cells (96.6±28.7% and 48.2±21.2% vs WKY, p<0.05), as well as basal ROS levels measured by chemiluminescence (110.2±26.4% vs WKY, p<0.05) and amplex red (105.2±33.2% vs WKY, p<0.05). Phosphorylation of unfolded protein response activators, PERK and IRE1α, and expression of ER chaperone BiP were elevated in SHR cells (p<0.05 vs WKY), indicating activation of ER stress response. Immunoblotting after organelle fractionation demonstrated that Noxs are expressed in an organelle-specific manner, with Nox1, 2 and 4 present in plasma membrane, ER and nucleus, but not in mitochondria. In SHR cells, NoxA1ds (Nox1 inhibitor, 10μM) and GKT136901 (Nox1/4 inhibitor, 10μM) decreased AngII-induced ROS levels (p<0.001 vs Ctl). Additionally, mito-tempol (mitochondrial-targeted antioxidant, 50nM) and 4-PBA (ER stress inhibitor, 1mM) decreased basal ROS levels in SHR cells (p<0.05 vs Ctl). Furthermore, oxidation of the antioxidant enzymes Peroxiredoxins (Prx) was increased in SHRSP compared to WKY (2.51±0.14 vs 0.56±0.07, p<0.001). One-dimensional isoelectric focusing revealed that cytosolic Prx2 and mitochondrial Prx3 were more oxidized in SHRSP than WKY cells. Using a biotin-tagged dimedone-based probe (DCP-Bio) we identified oxidation of ER stress proteins BiP and IRE1. To investigate the effect of protein oxidation in vascular function, vascular reactivity was evaluated in isolated mesenteric arteries. Inhibition of general oxidation (DTT 1mM; Emax: 111.7±33.1) and peroxiredoxin (Conoidin A 10nM; Emax: 116.0±7.3) reduces vascular contraction in response to noradrenalin in WKY rats (Emax: 166.6±30.2; p<0.05). These findings suggest an important role for Nox1/4 in redox-dependent organelle dysfunction and post-translational modification of proteins, processes that may play an important role in vascular dysfunction in hypertension.

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Upregulation of the Sarco-Endoplasmic Reticulum Calcium ATPase 1 Truncated Isoform Plays a Pathogenic Role in Alzheimer’s Disease

Cells ◽

10.3390/cells8121539 ◽

2019 ◽

Vol 8 (12) ◽

pp. 1539 ◽

Cited By ~ 4

Author(s):

Bussiere ◽

Oulès ◽

Mary ◽

Vaillant-Beuchot ◽

Martin ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Neuroblastoma Cells ◽

Amyloid Β ◽

Aβ Oligomers ◽

Human Neuroblastoma ◽

Endoplasmic Reticulum Calcium ◽

Protein Levels ◽

Functional Consequences ◽

Β Amyloid

Dysregulation of the Endoplasmic Reticulum (ER) Ca2+ homeostasis and subsequent ER stress activation occur in Alzheimer Disease (AD). We studied the contribution of the human truncated isoform of the sarco-endoplasmic reticulum Ca2+ ATPase 1 (S1T) to AD. We examined S1T expression in human AD-affected brains and its functional consequences in cellular and transgenic mice AD models. S1T expression is increased in sporadic AD brains and correlates with amyloid β (Aβ) and ER stress chaperone protein levels. Increased S1T expression was also observed in human neuroblastoma cells expressing Swedish-mutated β-amyloid precursor protein (βAPP) or treated with Aβ oligomers. Lentiviral overexpression of S1T enhances in return the production of APP C-terminal fragments and Aβ through specific increases of β-secretase expression and activity, and triggers neuroinflammation. We describe a molecular interplay between S1T-dependent ER Ca2+ leak, ER stress and βAPP-derived fragments that could contribute to AD setting and/or progression.

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Neuroprotective effects of verbascoside against Alzheimer’s disease via the relief of endoplasmic reticulum stress in Aβ-exposed U251 cells and APP/PS1 mice

Journal of Neuroinflammation ◽

10.1186/s12974-020-01976-1 ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Chunyue Wang ◽

Xueying Cai ◽

Ruochen Wang ◽

Siyu Zhai ◽

Yongfeng Zhang ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Endoplasmic Reticulum ◽

Er Stress ◽

Amyloid Β ◽

Terminal Deoxynucleotidyl Transferase ◽

Morris Water Maze Test ◽

Proteomics Analysis ◽

Neuroprotective Effects ◽

U251 Cells

Abstract Background Endoplasmic reticulum (ER) stress is involved in the progression of Alzheimer’s disease (AD). Verbascoside (VB), an active phenylethanoid glycoside that was first isolated from Verbascum sinuatum (the wavyleaf mullein), possesses anti-inflammatory, antioxidative, and anti-apoptotic effects. The purpose of this study was to elucidate the beneficial effects of VB in amyloid β (Aβ)1–42-damaged human glioma (U251) cells and in APPswe/PSEN1dE9 transgenic (APP/PS1) mice. Methods U251 cells were co-incubated with 10 μM of Aβ1-42 and treated with VB. The protective effects of VB were investigated by using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay, flow cytometry, fluorescence staining, and transmission electron microscopy. APP/PS1 transgenic mice were treated for 6 weeks with VB. Learning and memory were evaluated using a Morris water maze test. Immunohistochemistry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, thioflavin-S staining, and proteomics analysis were performed to study the potential neuroprotective mechanism. Enzyme-linked immunosorbent assays and western blot were performed to analyze altered protein levels of brain lysates in APP/PS1 mice and/or Aβ1-42-damaged U251 cells. Results In Aβ1-42-damaged U251 cells, VB significantly improved cell viability, inhibited apoptosis, reduced calcium accumulation and the intracellular concentrations of reactive oxygen species, and improved the morphology of mitochondria and ER. In APP/PS1 mice, 6-week administration of VB significantly improved memory and cognition. VB inhibited apoptosis, reduced the deposition of Aβ, reduced the formation of neurofibrillary tangles formed by hyperphosphorylated tau protein, and downregulated the expression levels of 4-hydroxynonenal and mesencephalic astrocyte-derived neurotrophic factor in the brains of APP/PS1 mice. Proteomics analysis of mouse hippocampus suggested that the neuroprotective effect of VB may be related to the reduction of ER stress. This was indicated by the fact that VB inhibited the three branches of the unfolded protein response, thereby attenuating ER stress and preventing apoptosis. Conclusions The results confirmed that VB possesses significant neuroprotective effects, which are related to the reduction of ER stress. These findings support the status of VB as a potentially effective treatment for AD and warrant further research.

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Endoplasmic reticulum stress inhibition blunts the development of essential hypertension in the spontaneously hypertensive rat

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00523.2018 ◽

2019 ◽

Vol 316 (5) ◽

pp. H1214-H1223 ◽

Cited By ~ 6

Author(s):

Safaa Naiel ◽

Rachel E. Carlisle ◽

Chao Lu ◽

Victor Tat ◽

Jeffrey G. Dickhout

Keyword(s):

Blood Pressure ◽

Endoplasmic Reticulum ◽

Essential Hypertension ◽

Er Stress ◽

Spontaneously Hypertensive Rat ◽

Resistance Arteries ◽

Spontaneously Hypertensive ◽

Functional Changes ◽

Hypertensive Rat ◽

Hypertension Development

Essential hypertension is the leading cause of premature death worldwide. However, hypertension’s cause remains uncertain. endoplasmic reticulum (ER) stress has recently been associated with hypertension, but it is unclear whether ER stress causes hypertension. To clarify this question, we examined if ER stress occurs in blood vessels before the development of hypertension and if ER stress inhibition would prevent hypertension development. We used the spontaneously hypertensive rat (SHR) as a model of human essential hypertension and the Wistar-Kyoto (WKY) rat as its normotensive control. Resistance arteries collected from young rats determined that ER stress was present in SHR vessels before the onset of hypertension. To assess the effect of ER stress inhibition on hypertension development, another subset of rats were treated with 4-phenylbutyric acid (4-PBA; 1 g·kg−1·day−1) for 8 wk from 5 wk of age. Blood pressure was measured via radiotelemetry and compared with untreated SHR and WKY rats. Mesenteric resistance arteries were collected and assessed for structural and functional changes associated with hypertension. Systolic and diastolic blood pressures were significantly lower in the 4-PBA-treated SHR groups than in untreated SHRs. Additionally, 4-PBA significantly decreased the media-to-lumen ratio and ER stress marker expression, improved vasodilatory response, and reduced contractile responses in resistance arteries from SHRs. Overall, ER stress inhibition blunted the development of hypertension in the SHR. These data add evidence to the hypothesis that a component of hypertension in the SHR is caused by ER stress. NEW & NOTEWORTHY In this study, 4-phenylbutyric acid’s (4-PBA’s) molecular chaperone capability was used to inhibit endoplasmic reticulum (ER) stress in the small arteries of young spontaneously hypertensive rats (SHRs) and reduce their hypertension. These effects are likely mediated through 4-PBA's effects to reduce resistant artery contractility and increase nitric oxide-mediated endothelial vasodilation through a process preventing endothelial dysfunction. Overall, ER stress inhibition blunted the development of hypertension in this young SHR model. This suggests that a component of the increase in blood pressure found in SHRs is due to ER stress. However, it is important to note that inhibition of ER stress was not able to fully restore the blood pressure to normal, suggesting that a component of hypertension may not be due to ER stress. This study points to the inhibition of ER stress as an important new physiological pathway to lower blood pressure, where other known approaches may not achieve blood pressure-lowering targets.

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Opioids Cause Sex-Specific Vascular Changes via Cofilin-Extracellular Signal-Regulated Kinase Signaling: Female Mice Present Higher Risk of Developing Morphine-Induced Vascular Dysfunction than Male Mice

Journal of Vascular Research ◽

10.1159/000517555 ◽

2021 ◽

pp. 1-11

Author(s):

Soyoung Cheon ◽

Jeremy C. Tomcho ◽

Jonnelle M. Edwards ◽

Nicole R. Bearss ◽

Emily Waigi ◽

...

Keyword(s):

Vascular Dysfunction ◽

Actin Dynamics ◽

Male Mice ◽

Resistance Arteries ◽

Opioid Use ◽

Female Mice ◽

Extracellular Signal ◽

Increased Risk ◽

Vascular Age ◽

Pulmonary Arterial

Recent studies have shown that chronic use of prescription or illicit opioids leads to an increased risk of cardiovascular events and pulmonary arterial hypertension. Indices of vascular age and arterial stiffness are also shown to be increased in opioid-dependent patients, with the effects being more marked in women. There are currently no studies investigating sex-specific vascular dysfunction in opioid use, and the mechanisms leading to opioid-induced vascular damage remain unknown. We hypothesized that exposure to exogenous opioids causes sex-specific vascular remodeling that will be more pronounced in female. Acknowledging the emerging roles of cofilins and extracellular signal-regulated kinases (ERKs) in mediating actin dynamics, we investigated the effects of morphine on these molecules. Twenty-four hour exposure to morphine increased inactivated cofilin and activated ERKs in resistance arteries from female mice, which may promote stress fiber over-assembly. We also performed continuous intraluminal infusion of morphine in pressurized resistance arteries from male and female mice using culture pressure myographs. We observed that morphine reduced the vascular diameter in resistance arteries from female, but not male mice. These results have significant implications for the previously unexplored role of exogenous opioids as a modifiable cardiovascular risk factor, especially in women.

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Abstract 18708: Vascular Dysfunction in Hyperhomocysteinemia: In vitro Evidence for Endoplasmic Reticulum Stress-mediated Calcium-activated Potassium Channel Dysfunction

Circulation ◽

10.1161/circ.132.suppl_3.18708 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Wen-Tao Sun ◽

Xiang-Chong Wang ◽

Cheuk-Man Yu ◽

Shun-Hay Pun ◽

Qin Yang

Keyword(s):

Endoplasmic Reticulum ◽

Smooth Muscle ◽

Cell Surface ◽

Er Stress ◽

Vascular Dysfunction ◽

Cell Protein ◽

Α Subunit ◽

Whole Cell

Objectives: KCa channels play an important role in the control of vascular tone. Opening of IKCa and SKCa in endothelial cells underlies the classic EDHF pathway and promotes NO production. Smooth muscle BKCa provides a negative feedback mechanism opposing vasoconstriction and is an effector of NO and EDHF. Previous studies demonstrated homocysteine (Hcy), a risk factor for atherosclerosis, compromises NO and EDHF function, however, whether KCa is involved is poorly studied and the underlying mechanisms remain unknown. We studied the effect of Hcy on vascular KCa with the role of endoplasmic reticulum (ER) stress explored. Methods: In vitro studies were performed in porcine coronary arteries and primary cultured porcine coronary endothelial (PCECs) and smooth muscle cells (PCSMCs). IKCa and SKCa-, and BKCa-mediated relaxations were studied in endothelium-intact and -denuded arteries in a myograph. IKCa and SKCa currents in PCECs and BKCa current in PCSMCs were analyzed by whole-cell patch clamp and channel expressions were examined by western blot. Results: Hcy impairs the role of IKCa and SKCa, and BKCa in vasorelaxation. Relaxant responses to channel activators NS309 and NS1619 were attenuated and EDHF-type response was inhibited. Hcy suppressed IKCa and SKCa currents in PCECs and BKCa currents in PCSMCs. Inhibition of ER stress enhanced KCa currents and improved EDHF-type and channel activators-induced responses. Whole-cell protein levels of IKCa and SKCa remained unchanged in Hcy-exposed PCECs whereas IKCa and SKCa at cell surface were significantly decreased. Hcy lowered protein of β1 but not α subunit of BKCa in PCSMCs. The decrease in cell surface IKCa and SKCa and reduction of BKCa β1 were restored by ER stress inhibition. Further, inhibition of PERK increased BKCa β1 protein and enhanced BKCa current. Conclusion: ER stress mediates Hcy-induced vascular dysfunction through inhibition of KCa. Suppression of cell surface expression underlies ER stress-mediated IKCa and SKCa inhibition. Downregulation of BKCa β1 by PERK-ER stress pathway plays a key role in the loss of BKCa function. This study provides new mechanistic insights into the role of ER stress in vascular dysfunction. Supported by RGC GRF CUHK4774/12M & CUHK14118414, and NSFC 81200123.

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Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Aβ-induced cell death

The Journal of Cell Biology ◽

10.1083/jcb.200310015 ◽

2004 ◽

Vol 165 (3) ◽

pp. 347-356 ◽

Cited By ~ 583

Author(s):

Junichi Hitomi ◽

Taiichi Katayama ◽

Yutaka Eguchi ◽

Takashi Kudo ◽

Manabu Taniguchi ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Endoplasmic Reticulum ◽

Er Stress ◽

Amyloid Β ◽

Small Interfering Rnas ◽

Caspase 12 ◽

Induced Apoptosis ◽

Interfering Rna ◽

Human Caspase

Recent studies have suggested that neuronal death in Alzheimer's disease or ischemia could arise from dysfunction of the endoplasmic reticulum (ER). Although caspase-12 has been implicated in ER stress-induced apoptosis and amyloid-β (Aβ)–induced apoptosis in rodents, it is controversial whether similar mechanisms operate in humans. We found that human caspase-4, a member of caspase-1 subfamily that includes caspase-12, is localized to the ER membrane, and is cleaved when cells are treated with ER stress-inducing reagents, but not with other apoptotic reagents. Cleavage of caspase-4 is not affected by overexpression of Bcl-2, which prevents signal transduction on the mitochondria, suggesting that caspase-4 is primarily activated in ER stress-induced apoptosis. Furthermore, a reduction of caspase-4 expression by small interfering RNA decreases ER stress-induced apoptosis in some cell lines, but not other ER stress-independent apoptosis. Caspase-4 is also cleaved by administration of Aβ, and Aβ-induced apoptosis is reduced by small interfering RNAs to caspase-4. Thus, caspase-4 can function as an ER stress-specific caspase in humans, and may be involved in pathogenesis of Alzheimer's disease.

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