High-throughput screening for agonists of ROS production in live human vascular endothelial cells.

Reactive oxygen species (ROS) are important physiological molecules, and identifying agonists for ROS production can yield useful tools for future research. Here we present an optimized protocol for high-throughput screening for agonists that induce ROS production. We describe the use of a fluorescent probe in human vascular endothelial cells, which can establish whether ROS production occurs in mitochondria or in the plasma membrane of live cells. For complete details on the use and execution of this profile, please refer to Sasahara et al. (2021).

Alzheimer's Aß assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells.

Amyloid ß-protein (Aß) may contribute to worsening of Alzheimer's disease (AD) through vascular dysfunction, but the molecular mechanism involved is unknown. Using ex vivo blood vessels and primary endothelial cells from human brain microvessels, we show that patient-derived Aß assemblies, termed amylospheroids (ASPD), exist on the microvascular surface in patients' brains and inhibit vasorelaxation through binding to the α3 subunit of sodium, potassium-ATPase (NAKα3) in caveolae on endothelial cells. Interestingly, NAKα3 is also the toxic target of ASPD in neurons. ASPD-NAKα3 interaction elicits neurodegeneration through calcium overload in neurons, while the same interaction suppresses vasorelaxation by increasing the inactive form of endothelial nitric oxide synthase (eNOS) in endothelial cells via mitochondrial ROS and protein kinase C, independently of the physiological relaxation system. Thus, ASPD may contribute to both neuronal and vascular pathologies through binding to NAKα3. Therefore, blocking the ASPD-NAKα3 interaction may be a useful target for AD therapy.

NMR-based site-resolved profiling of ß-amyloid misfolding reveals structural transitions from pathologically relevant spherical oligomer to fibril.

Increasing evidence highlights the central role of neurotoxic oligomers of the 42-residue-long ß-amyloid (Aß42) in Alzheimer's disease (AD). However, very limited information is available on the structural transition from oligomer to fibril, particularly for pathologically relevant amyloids. To the best of our knowledge, we present here the first site-specific structural characterization of Aß42 misfolding, from toxic oligomeric assembly yielding a similar conformation to an AD-associated Aß42 oligomer, into a fibril. Transmission EM (TEM) analysis revealed that a spherical amyloid assembly (SPA) of Aß42 with a 15.6 ± 2.1-nm diameter forms in a ∼30-µm Aß42 solution after a ∼10-h incubation at 4 °C, followed by a slow conversion into fibril at ∼180 h. Immunological analysis suggested that the SPA has a surface structure similar to that of amylospheroid (ASPD), a patient-derived toxic Aß oligomer, which had a diameter of 10-15 nm in negative-stain TEM. Solid-state NMR analyses indicated that the SPA structure involves a ß-loop-ß motif, which significantly differed from the triple-ß motif observed for the Aß42 fibril. The comparison of the 13C chemical shifts of SPA with those of the fibril prepared in the above conditions and interstrand distance measurements suggested a large conformational change involving rearrangements of intermolecular ß-sheet into in-register parallel ß-sheet during the misfolding. A comparison of the SPA and ASPD 13C chemical shifts indicated that SPA is structurally similar to the ASPD relevant to AD. These observations provide insights into the architecture and key structural transitions of amyloid oligomers relevant for AD pathology.

Alzheimer Aß Assemblies Accumulate in Excitatory Neurons upon Proteasome Inhibition and Kill Nearby NAKα3 Neurons by Secretion.

We identified ∼30-mer amyloid-ß protein (Aß) assemblies, termed amylospheroids, from brains of patients with Alzheimer disease (AD) as toxic entities responsible for neurodegeneration and showed that Na+,K+-ATPase α3 (NAKα3) is the sole target of amylospheroid-mediated neurodegeneration. However, it remains unclear where in neurons amylospheroids form and how they reach their targets to induce neurodegeneration. Here, we present an in vitro culture system designed to chronologically follow amylospheroid formation in mature neurons expressing amyloid precursor protein bearing early-onset AD mutations. Amylospheroids were found to accumulate mainly in the trans-Golgi network of excitatory neurons and were initially transported in axons. Proteasome inhibition dramatically increased amylospheroid amounts in trans-Golgi by increasing Aß levels and induced dendritic transport. Amylospheroids were secreted and caused the degeneration of adjacent NAKα3-expressing neurons. Interestingly, the ASPD-producing neurons later died non-apoptotically. Our findings demonstrate a link between ASPD levels and proteasome function, which may have important implications for AD pathophysiology.

Orthovanadate-Induced Vasoconstriction of Rat Mesenteric Arteries Is Mediated by Rho Kinase-Dependent Inhibition of Myosin Light Chain Phosphatase.

Orthovanadate (OVA), a protein tyrosine phosphatase inhibitor, induces vasoconstriction in a Rho kinase-dependent manner. The aim of this study was to determine the mechanism underlying OVA-induced vasoconstriction of rat mesenteric arteries. OVA-induced constriction of mesenteric arterial rings treated with N(G)-nitro-L-arginine methyl ester (L-NAME, 0.1 mM), a nitric oxide synthase inhibitor, was significantly blocked by the Rho kinase inhibitor Y-27632 (R-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 10 µM), extracellular signal-regulated kinase 1 and 2 (Erk1/2) inhibitor FR180204 (5-(2-phenyl-pyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-ylamine, 10 µM), Erk1/2 kinase (MEK) inhibitor PD98059 (2'-amino-3'-methoxyflavone, 10 µM), epidermal growth factor receptor (EGFR) inhibitor AG1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline, 10 µM), and Src inhibitor PP2 (4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 3 µM). However, the myosin light chain kinase inhibitor ML-7 (1-(5-iodonaphthalene-1-sulfonyl)-homopiperazine, 10 µM) did not affect OVA-induced constriction. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1, an index of Rho kinase activity) was abrogated by inhibitors of Src, EGFR MEK, Erk1/2, and Rho kinase. OVA-stimulated Erk1/2 phosphorylation was blocked by inhibitors of EGFR, Src, MEK, and Erk1/2 but not affected by an inhibitor of Rho kinase. OVA-induced Src phosphorylation was abrogated by an Src inhibitor but not affected by inhibitors of EGFR, MEK, Erk1/2, and Rho kinase. In addition, the metalloproteinase inhibitor TAPI-0 (N-(R)-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L-naphthylalanyl-L-alanine amide, 10 µM) and an inhibitor of heparin/epidermal growth factor binding (CRM 197, 10 µg/mL) did not affect OVA-induced contraction of rat mesenteric arterial rings. These results suggest that OVA induces vasoconstriction in rat mesenteric arteries via Src, EGFR, MEK, and Erk1/2 activation, leading to the inactivation of myosin light chain phosphatase through phosphorylation of MYPT1.

Na, K-ATPase α3 is a death target of Alzheimer patient amyloid-ß assembly.

Neurodegeneration correlates with Alzheimer's disease (AD) symptoms, but the molecular identities of pathogenic amyloid ß-protein (Aß) oligomers and their targets, leading to neurodegeneration, remain unclear. Amylospheroids (ASPD) are AD patient-derived 10- to 15-nm spherical Aß oligomers that cause selective degeneration of mature neurons. Here, we show that the ASPD target is neuron-specific Na(+)/K(+)-ATPase α3 subunit (NAKα3). ASPD-binding to NAKα3 impaired NAKα3-specific activity, activated N-type voltage-gated calcium channels, and caused mitochondrial calcium dyshomeostasis, tau abnormalities, and neurodegeneration. NMR and molecular modeling studies suggested that spherical ASPD contain N-terminal-Aß-derived "thorns" responsible for target binding, which are distinct from low molecular-weight oligomers and dodecamers. The fourth extracellular loop (Ex4) region of NAKα3 encompassing Asn(879) and Trp(880) is essential for ASPD-NAKα3 interaction, because tetrapeptides mimicking this Ex4 region bound to the ASPD surface and blocked ASPD neurotoxicity. Our findings open up new possibilities for knowledge-based design of peptidomimetics that inhibit neurodegeneration in AD by blocking aberrant ASPD-NAKα3 interaction.

Epidermal growth factor induces Ca(2+) sensitization through Rho-kinase-dependent phosphorylation of myosin phosphatase target subunit 1 in vascular smooth muscle.

We previously found that the protein tyrosine phosphatase inhibitor orthovanadate evoked a vasoconstrictor effect in rat aortas via Rho-kinase-dependent inactivation of myosin light chain phosphatase (MLCP) downstream of epidermal growth factor (EGF) receptor signaling. To determine whether the direct activation of EGF receptor by EGF also induces Rho-kinase-dependent vasoconstriction, isometric tension changes were measured in rat aortic rings without endothelium. Although EGF did not produce a contractile effect, the Ca(2+)-induced force in Ca(2+)-depleted rings significantly increased after treatment with 100nM EGF, suggesting that EGF induces Ca(2+) sensitization by MLCP inactivation. In addition, EGF induced the activation of Rho-kinase and phosphorylation of myosin phosphatase target subunit 1 (MYPT1) in rat aortic smooth muscle cells (VSMCs). The effects of EGF on Ca(2+) sensitivity in aortas and MYPT1 phosphorylation in VSMCs were blocked by inhibitors of EGF receptor (AG1478), Rho-kinase (Y27632), extracellular signal-regulated kinase 1/2 (Erk1/2; FR180204), and mitogen/extracellular signal-regulated kinase (MEK; PD98059), but not by inhibitors of p38 kinase (SB203580) and c-Jun amino-terminal kinase (AS601245). EGF-induced Erk1/2 phosphorylation was not abrogated by the Rho-kinase inhibitor, suggesting that Rho-kinase-dependent phosphorylation of MYPT1 is downstream of EGF receptor/MEK/Erk1/2 signaling. These results suggest that EGF induces Ca(2+) sensitization in vascular smooth muscle by Rho-kinase-dependent inactivation of MLCP mediated by the EGF receptor/MEK/Erk1/2 pathway.

Orthovanadate-induced vasocontraction is mediated by the activation of Rho-kinase through Src-dependent transactivation of epidermal growth factor receptor.

Orthovanadate (OVA), a protein tyrosine phosphatase (PTPase) inhibitor, exerts contractile effects on smooth muscle in a Rho-kinase-dependent manner, but the precise mechanisms are not elucidated. The aim of this study was to determine the potential roles of Src and epidermal growth factor receptor (EGFR) in the OVA-induced contraction of rat aortas and the phosphorylation of myosin phosphatase target subunit 1 (MYPT1; an index of Rho-kinase activity) in vascular smooth muscle cells (VSMCs). Aortic contraction by OVA was significantly blocked not only by Rho kinase inhibitors Y-27632 [R-[+]-trans-N-[4-pyridyl]-4-[1-aminoethyl]-cyclohexanecarboxamide] and hydroxyfasudil [1-(1-hydroxy-5-isoquinolinesulfonyl)homopiperazine] but also by Src inhibitors PP2 [4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine] and Src inhibitor No. 5 [4-(3'-methoxy-6'-chloro-anilino)-6-methoxy-7(morpholino-3-propoxy)-quinazoline], and the EGFR inhibitors AG1478 [4-(3-chloroanilino)-6,7-dimethoxyquinazoline] and EGFR inhibitor 1 [cyclopropanecarboxylic acid-(3-(6-(3-trifluoromethyl-phenylamino)-pyrimidin-4-ylamino)-phenyl)-amide]. OVA induced rapid increases in the phosphorylation of MYPT1 (Thr-853), Src (Tyr-416), and EGFR (Tyr-1173) in VSMCs, and Src inhibitors abolished these effects. OVA-induced Src phosphorylation was abrogated by Src inhibitors, but not affected by inhibitors of EGFR and Rho-kinase. Inhibitors of Src and EGFR, but not Rho-kinase, also blocked OVA-induced EGFR phosphorylation. Furthermore, a metalloproteinase inhibitor TAPI-0 [N-(R)-[2-(hydroxyaminocarbonyl) methyl]-4-methylpentanoyl-l-naphthylalanyl-l-alanine amide] and an inhibitor of heparin-binding EGF (CRM 197) not only abrogated the OVA-induced aortic contraction, but also OVA-induced EGFR and MYPT1 phosphorylation, suggesting the involvement of EGFR transactivation. OVA also induced EGFR phosphorylation at Tyr-845, one of residues phosphorylated by Src. These results suggest that OVA-induced vasocontraction is mediated by the Rho-kinase-dependent inactivation of myosin light-chain phosphatase via signaling downstream of Src-induced transactivation of EGFR.

p38 mitogen-activated protein kinase mediates hyperosmolarity-induced vasoconstriction through myosin light chain phosphorylation and actin polymerization in rat aorta.

Hyperosmotic stress induces the contractile response of vascular smooth muscle cells (VSMCs). Previous studies have demonstrated that cytoskeleton reorganization and Rho/Rho-kinase-mediated inactivation of myosin light chain phosphatase (MLCP) play an important role in hyperosmotic vasoconstriction, but the precise mechanism is unknown. This study aimed to investigate the contractile response of endothelium-denuded rings of rat aortas to hyperosmolar sucrose (160 mM) in the presence or absence of inhibitors for various protein kinases. We found that the hyperosmotic constriction of aortic rings was attenuated not only by ML-7 or hydroxyfasudil, specific inhibitor for myosin light chain kinase (MLCK) or Rho-kinase, respectively, but also by SB203580, a specific inhibitor for p38 mitogen-activated kinase (p38 MAPK). Hyperosmolar sucrose evoked a transient increase in cytosolic free Ca(2+) in rat VSMCs, and this response was not affected by SB203580. Western blot analysis of proteins extracted from rings showed that the hyperosmolar sucrose stimulated phosphorylation of the Rho-kinase-mediated myosin phosphatase target subunit 1, myosin light chain (MLC), and p38 MAPK. The experiments performed using a combination of the kinase inhibitors showed that hyperosmolarity-induced MLC phosphorylation is partially mediated via the SB203580-sensitive pathway and is independent of both MLCK and Rho-kinase-mediated inactivation of MLCP. Furthermore, the hyperosmolarity-induced increase in the F-actin/G-actin ratio in rings was attenuated not only by hydroxyfasudil but also by SB203580. These results suggest that p38 MAPK is involved in hyperosmotic vasoconstriction via stimulation of MLC phosphorylation and cytoskeleton reorganization through pathways independent of activation of MLCK and/or Rho-kinase-mediated mechanisms.

Na+/H+ exchanger inhibitor augments hyperosmolarity-induced vasoconstriction by enhancing actin polymerization.

Vascular smooth muscle cells (VSMCs) exhibit shrinkage-induced activation of Na(+)/H(+) exchanger isoform 1 (NHE-1) and Na(+), K(+), 2Cl(-) cotransporter (NKCC) under hyperosmotic conditions. To investigate the roles of these ion transporters in vascular smooth muscle force induced by hyperosmotic stress, we tested the effects of 5-(N, N-dimethyl)-amiloride (DMA; NHE inhibitor), cariporide (a selective NHE-1 inhibitor), and bumetanide (NKCC inhibitor) on the contractile response of rat aortic rings to hyperosmolar solutions. NHE inhibitors significantly augmented the maximum force response and contractile sensitivity to hyperosmolar sucrose, NaCl, and glucose in endothelium-denuded rings. Bumetanide elicited a comparatively modest increase in sensitivity. NHE inhibitors blocked the increase in intracellular pH and enhanced the cell volume decrease of cultured VSMCs after exposure to hyperosmolar sucrose. However, DMA had no effect on the increase in cytosolic free Ca(2+) concentration ([Ca(2+)]i) in rat VSMCs and on the increases in phosphorylation of myosin phosphatase target subunit 1 and myosin light chain (MLC) in aortic rings in response to hyperosmolar sucrose. Hyperosmolar sucrose-induced force was significantly attenuated by cytochalasin B in the presence or absence of DMA. Exposure to hyperosmolar sucrose increased the ratio of F- to G-actin; the ratio was further elevated by DMA. These results suggest that the potentiation of hyperosmotic shrinkage by NHE inhibition promotes actin polymerization in VSMCs and augments force production independent of changes in [Ca(2+)]i and MLC phosphorylation.

Na(+)/H(+) exchanger inhibitor induces vasorelaxation through nitric oxide production in endothelial cells via intracellular acidification-associated Ca2(+) mobilization.

The objective of this study was to determine the mechanism by which Na(+)/H(+) exchanger (NHE) inhibitors induce vasodilatation. The NHE inhibitors, 5-(N,N-dimethyl)-amiloride (DMA), cariporide, and amiloride, evoked endothelium-dependent relaxation in rat aortas with ED50 values of 16, 89, and 148µM, respectively, and these effects were abolished by treatment with N(G)-nitro-l-arginine methyl ester (L-NAME). The relaxation effects induced by DMA and cariporide were strongly attenuated in aortas of the endothelial NO synthase (eNOS)-deficient mice, as compared to the effects in wild-type mice. The DMA-induced relaxation in rat aorta was attenuated by a calmodulin (CaM) inhibitor, calmidazolium, and a soluble guanylyl cyclase inhibitor, [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, but was not affected by a phosphoinositide 3-kinase inhibitor, wortmannin. Immunoblots for endothelial eNOS on immunoprecipitated CaM complexes showed that DMA enhanced the association of eNOS with CaM in rat aortas. Both DMA and cariporide induced the reduction of intracellular pH (pHi) in bovine aortic endothelial cells (BAECs), which was accompanied by a sustained elevation of cytosolic Ca(2+) ([Ca(2+)]i). This DMA-induced rise of [Ca(2+)]i was not affected by removing external Ca(2+) from the buffer, but was abolished in thapsigargin-pretreated BAECs. These results suggest that lowering of pHi by NHE inhibitors in endothelial cells induces the mobilization of Ca(2+) from the thapsigargin-sensitive stores of endoplasmic reticulum, which in turn stimulates NO production via the CaM-dependent activation of eNOS.