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Leucine Rich Repeat Containing 8A (LRRC8A) is an essential component of volume‐regulated anion channels (VRACs), which regulate cell volume. These anion channels are activated by pro-inflammatory mediators such as angiotensin II and tumor necrosis factor-α (TNFα). We previously demonstrated that VRAC inhibition or LRRC8A knockdown reduced Nox1 activation and impaired the inflammatory response to TNFα in cultured vascular smooth muscle cells. We hypothesized that loss of LRRC8A would also modify the antioxidant status of VSMCs. Vascular smooth muscle-specific (Sm22alpha-Cre) LRRC8A KO mice were created. Primary VSMC cultures were prepared from aortae and mesenteric arteries from male mice. Western blotting of protein from WT and LRRC8A KO mesenteric artery VSMCs revealed significant upregulation of proteins that are known to be under the control of the Nrf-2 antioxidant response; heme oxygenase 1 (2.1 fold), thioredoxin 1 (1.8 fold), and superoxide dismutase 1 (1.3 fold) superoxide dismutase 3 (1.8 fold). There was no change in the abundance of catalase.To further explore the role of LRRC8A in intracellular signaling, LRRC8A was immunoprecipitated from cultured WT and LRRC8A KO VSMCs and proteins were analyzed by Mass Spectrometry (MS). Reads were sorted for proteins identified in wild type but not in KO cells. This approach identified the Trim21 E3 ubiquitin ligase, a known regulator of the antioxidant response, as an LRRC8A-associated protein. Trim21 ubiquitinates and thereby promotes degradation of p62 (Sequestosome-1, SQSTM1). p62 sequesters Kelch-like ECH-associated protein 1 (Keap1), preventing it from promoting ubiquitination and destruction of Nrf-2 by the proteosome. Consistent with this, TRIM21 KO mice have an augmented antioxidant status and are protected from myocardial inflammation in response to doxorubicin or ligation of the left coronary artery. We considered the possibility that association of TRIM21 with LRRC8A (directly or indirectly) regulates its activity. Co-immunoprecipitation and western blotting confirmed association of LRRC8A with TRIM21. Pulldown of ubiquitinated proteins from WT and KO VSMCs failed to provide evidence that LRRC8A is ubiquitinated, however, p62 ubiquitination was markedly reduced in KO VSMCs. Thus, reduced p62 turnover in LRRC8A KO cells may enhance Keap1 sequestration and increase Nrf2 activity. The ability of LRRC8A knockdown to both impair the inflammatory response to TNFα and to enhance the antioxidant status of VSMCs identify this protein as an appealing target for the treatment of vascular inflammation. This work was supported by our both grant from NIDDK (1R01DK132948) and NIH (1 R01 HL160975-01A1). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Volume-regulated anion channels (VRACs) are activated by tumor necrosis factor alpha (TNFα) and angiotensin II. VRACs are composed of Leucine Rich Repeat Containing 8 (LRRC8) family proteins (LRRC8A, B, C, D and E) and LRRC8A is required for all VRACs. LRRC8A associates with NADPH oxidase 1 (Nox1) and modulates production of extracellular superoxide in vascular smooth muscle cells (VSMCs). We investigated vascular reactivity in mesenteric arteries from wild type (WT) and smooth muscle-specific LRRC8A knockout (KO) mice. KO vessels displayed enhanced responsiveness to multiple vasodilator agents; acetylcholine, sodium nitroprusside, PKA (forskolin) or PKG (BAY60-2770) activators and a Rho kinase inhibitor (Y-27632), compared to WT. This was associated with decreased RhoA activity and reduced ROCK-dependent MYPT1 phosphorylation at T853. To explore the mechanism of enhanced relaxation we immunoprecipitated LRRC8A-associated proteins from WT and KO VSMCs and analyzed these by mass spectroscopy. Myosin Phosphatase Rho-Interacting protein (MPRIP) was identified only in WT lysates. MPRIP is a scaffolding protein that binds to RhoA, MYPT1, and actin and regulates activity of the myosin light chain phosphatase complex. Association of LRRC8A and MPRIP was confirmed using IP/western blot, proximity ligation assay (PLA) and immunohistochemistry. LRRC8A binds to the second plextrin homology domain of MPRIP. Knockdown of MPRIP (siRNA) increased protein expression of LRRC8A in cultured VSMCs. To explore the relationship between Nox1/LRRC8A, superoxide and intracellular redox signaling events sulfenylation of MPRIP cysteine residues (-SOH) was quantified using DCP-Bio1 (a biotin-labeled dimedone derivative) pulldown with streptavidin beads and western blotting. Sulfenylation was significantly increased 3 min following TNFα stimulation (2.3 fold, n = 3, p < 0.05). It remains to be determined how this impacts association of MPRIP with its four binding partners. Disruption of interaction between Nox1/LRRC8A and MPRIP/RhoA/MYPT1/actin may underly enhanced vasodilation in VSMC-specific LRRC8A null blood vessels. This multi-protein complex provides a potential mechanism by which oxidants produced by Nox1 impact vasomotor function, linking inflammation to increased vascular reactivity and hypertension. NIGMS (5R35GM138191-02), NIDDK (1R01DK132948-01), NHLBI (1R01HL160975-01A1) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BackgroundIn vascular smooth muscle cells (VSMCs), LRRC8A volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNFα), angiotensin II and stretch. LRRC8A physically associates with NADPH oxidase 1 (Nox1) and supports its production of extracellular superoxide (O2-•).Methods and ResultsMice lacking LRRC8A exclusively in VSMCs (Sm22α-Cre, KO) were used to assess the role of VRACs in TNFα signaling and vasomotor function. KO mesenteric vessels contracted normally to KCl and phenylephrine, but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). 48 hours ofex vivoexposure to TNFα (10ng/ml) markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 μM, 20 min) enhanced dilation of control rings and restored impaired dilation following TNFα exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 35 proteins that interacted with LRRC8A. Pathway analysis revealed actin cytoskeletal regulation as the most closely associated function of these proteins. Among these proteins, the Myosin Phosphatase Rho-Interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots which revealed LRRC8A binding at the second Pleckstrin Homology domain of MPRIP. siLRRC8A or CBX treatment decreased RhoA activity in cultured VSMCs, and MYPT1 phosphorylation at T853 was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNFα exposure.ConclusionsInteraction of Nox1/LRRC8A with MPRIP/RhoA/MYPT1/actin may allow redox regulation of the cytoskeleton and link Nox1 activation to both inflammation and vascular contractility.

Sepsis represents a life-threatening event often mediated by the host’s response to pathogens such as gram-negative organisms, which release the pro-inflammatory lipopolysaccharide (LPS). Within the endothelium, the mitogen-activated protein kinase (MAPK) pathway is an important driver of endothelial injury during sepsis, of which oxidant-sensitive apoptosis signal-regulating kinase 1 (ASK1) is postulated to be a critical upstream regulator. We hypothesized that ASK1 would play a key role in endothelial inflammation during bacterial challenge. Utilizing RNA sequencing data from patients and cultured human microvascular endothelial cells (HMVECs), ASK1 expression was increased in sepsis and after LPS challenge. Two ASK1 inhibitors, GS444217 and MSC2023964A, reduced cytokine production in HMVECs following LPS stimulation, but had no effect on permeability as measured by transendothelial electrical resistance (TEER) and intercellular space. MAPKs are known to interact with endothelial nitric oxide synthase (eNOS) and ASK1 expression levels correlated with eNOS expression in patients with septic shock. In addition, eNOS physically interacted with ASK1, though this interaction was not altered by ASK1 inhibition, nor did inhibition alter MAPK p38 activity. Instead, among MAPKs, ASK1 inhibition only impaired LPS-induced JNK phosphorylation. The reduction in JNK activation caused by ASK1 inhibition impaired JNK-mediated cytokine production without affecting permeability. Thus, LPS triggers JNK-dependent cytokine production that requires ASK1 activation, but both its effects on permeability and activation of p38 are ASK1-independent. These data demonstrate how distinct MAPK signaling pathways regulate endothelial inflammatory outputs during acute infectious challenge.

Chloride channel-3 (ClC-3) Cl(−)/H(+) antiporters and leucine-rich repeat-containing 8 (LRRC8) family anion channels have both been associated with volume-regulated anion currents (VRACs). VRACs are often altered in ClC-3 null cells but are absent in LRRC8A null cells. To explore the relationship between ClC-3, LRRC8A and VRAC we localized tagged proteins in human epithelial kidney (HEK293) cells using multimodal microscopy. Expression of ClC-3-GFP induced large multivesicular bodies (MVBs) with ClC-3 in the delimiting membrane. LRRC8A-RFP localized to the plasma membrane and to small cytoplasmic vesicles. Co-expression demonstrated co-localization in small, highly mobile cytoplasmic vesicles that associated with the early endosomal marker Rab5A. However, most of the small LRRC8A-positive vesicles were constrained within large MVBs with abundant ClC-3 in the delimiting membrane. Dominant negative (S34A) Rab5A prevented ClC-3 overexpression from creating enlarged MVBs, while constitutively active (Q79L) Rab5A enhanced this phenotype. Thus, ClC-3 and LRRC8A are endocytosed together but independently sorted in Rab5A MVBs. Subsequently, LRRC8A-labelled vesicles were sorted to MVBs labelled by Rab27A and B exosomal compartment markers, but not to Rab11 recycling endosomes. VRAC currents were significantly larger in ClC-3 null HEK293 cells. This work demonstrates dependence of LRRC8A trafficking on ClC-3 which may explain the association between ClC-3 and VRACs.

KEY POINTS LRRC8A-containing anion channels associate with NADPH oxidase 1 (Nox1) and regulate superoxide production and tumour necrosis factor-α (TNFα) signalling. Here we show that LRRC8C and 8D also co-immunoprecipitate with Nox1 in vascular smooth muscle cells. LRRC8C knockdown inhibited TNFα-induced O2•- production, receptor endocytosis, nuclear factor-κB (NF-κB) activation and proliferation while LRRC8D knockdown enhanced NF-κB activation. Significant changes in LRRC8 isoform expression in human atherosclerosis and psoriasis suggest compensation for increased inflammation. The oxidant chloramine-T (ChlorT, 1 mM) weakly (∼25%) inhibited LRRC8C currents but potently (∼80%) inhibited LRRC8D currents. Substitution of the extracellular loop (EL1, EL2) domains of 8D into 8C conferred significantly stronger (69%) ChlorT-dependent inhibition. ChlorT exposure impaired subsequent current block by DCPIB, which occurs through interaction with EL1, further implicating external oxidation sites. LRRC8A/C channels most effectively sustain Nox1 activity at the plasma membrane. This may result from their ability to remain active in an oxidized microenvironment. ABSTRACT Tumour necrosis factor-α (TNFα) activates NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs), producing superoxide (O2•- ) required for subsequent signalling. LRRC8 family proteins A-E comprise volume-regulated anion channels (VRACs). The required subunit LRRC8A physically associates with Nox1, and VRAC activity is required for Nox activity and the inflammatory response to TNFα. VRAC currents are modulated by oxidants, suggesting that channel oxidant sensitivity and proximity to Nox1 may play a physiologically relevant role. In VSMCs, LRRC8C knockdown (siRNA) recapitulated the effects of siLRRC8A, inhibiting TNFα-induced extracellular and endosomal O2•- production, receptor endocytosis, nuclear factor-κB (NF-κB) activation and proliferation. In contrast, siLRRC8D potentiated NF-κB activation. Nox1 co-immunoprecipitated with 8C and 8D, and colocalized with 8D at the plasma membrane and in vesicles. We compared VRAC currents mediated by homomeric and heteromeric LRRC8C and LRRC8D channels expressed in HEK293 cells. The oxidant chloramine T (ChlorT, 1 mM) weakly inhibited 8C, but potently inhibited 8D currents. ChlorT exposure also impaired subsequent current block by the VRAC blocker DCPIB, implicating external sites of oxidation. Substitution of the 8D extracellular loop domains (EL1, EL2) into 8C conferred significantly stronger ChlorT-mediated inhibition of 8C currents. Our results suggest that LRRC8A/C channel activity can be effectively maintained in the oxidized microenvironment expected to result from Nox1 activation at the plasma membrane. Increased ratios of 8D:8C expression may potentially depress inflammatory responses to TNFα. LRRC8A/C channel downregulation represents a novel strategy to reduce TNFα-induced inflammation.

Tumor necrosis factor-α (TNFα) activates NADPH Oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs), producing superoxide (O2•-) required for subsequent signaling. LRRC8 family proteins A-E comprise volume-regulated anion channels (VRACs). The required subunit LRRC8A physically associates with Nox1, and VRAC activity is required for Nox activity and the inflammatory response to TNFα. LRRC8 channel currents are modulated by oxidants, suggesting that oxidant sensitivity and proximity to Nox1 may play a physiologically relevant role. In VSMCs, LRRC8C knockdown (siRNA) recapitulated the effects of siLRRC8A, inhibiting TNFα-induced extracellular and endosomal O2•- production, receptor endocytosis, NF-κB activation, and proliferation. In contrast, siLRRC8D potentiated NF-κB activation. Nox1 co-immunoprecipitated with 8C and 8D, and co-localized with 8D at the plasma membrane and in vesicles. We compared VRAC currents mediated by homomeric and heteromeric 8C and 8D channels expressed in HEK293 cells. The oxidant chloramine T (ChlorT, 1 mM) weakly inhibited LRRC8C, but potently inhibited 8D currents. ChlorT exposure also greatly reduced subsequent current block by DCPIB, implicating external sites of oxidation. Substitution of the extracellular loop domains (EL1, EL2) of 8D onto 8C conferred significantly stronger ChlorT-dependent inhibition. 8A/C channel activity is thus more effectively maintained in the oxidized microenvironment expected to result from Nox1 activation at the plasma membrane. Increased ratios of 8D:8C expression may potentially depress inflammatory responses to TNFα. LRRC8A/C channel downregulation represents a novel strategy to reduce TNFα-induced inflammation.Key PointsLRRC8A-containing anion channels associate with Nox1 and regulate superoxide production and TNFα signaling. Here we show that .LRRC8C and 8D also co-immunoprecipitate with Nox1 in vascular smooth muscle cells.LRRC8C knockdown inhibited TNFα-induced O2•- production, receptor endocytosis, NF-κB activation, and proliferation while LRRC8D knockdown enhanced NF-κB activation. Significant changes in LRRC8 isoform expression in human atherosclerosis and psoriasis suggest compensation for increased inflammation.The oxidant chloramine-T (ChlorT, 1 mM) weakly (∼25%) inhibited 8C currents but potently (∼80%) inhibited 8D currents. Substitution of the two extracellular loop (EL) domains of 8D onto 8C conferred significantly stronger ChlorT-dependent inhibition.ChlorT also impaired current block by DCPIB, which occurs through interaction with EL1, further implicating external sites of oxidation.8A/C channels most effectively maintain activity in an oxidized microenvironment, as is expected to result from Nox1 activity at the plasma membrane.

Hypertension and atherosclerosis, the predecessors of stroke and myocardial infarction, are chronic vascular inflammatory reactions. Tumor necrosis factor alpha (TNFα), the “master” proinflammatory cytokine, contributes to both the initiation and maintenance of vascular inflammation. TNFα induces reactive oxygen species (ROS) production which drives the redox reactions that constitute “ROS signaling.” However, these ROS may also cause oxidative stress which contributes to vascular dysfunction. Mice lacking TNFα or its receptors are protected against both acute and chronic cardiovascular injury. Humans suffering from TNFα-driven inflammatory conditions such as rheumatoid arthritis and psoriasis are at increased cardiovascular risk. When treated with highly specific biologic agents that target TNFα signaling (Etanercept, etc.) they display marked reductions in that risk. The ability of TNFα to induce endothelial dysfunction, often the first step in a progression toward serious vasculopathy, is well recognized and has been reviewed elsewhere. However, TNFα also has profound effects on vascular smooth muscle cells (VSMCs) including a fundamental change from a contractile to a secretory phenotype. This “phenotypic switching” promotes proliferation and production of extracellular matrix proteins which are associated with medial hypertrophy. Additionally, it promotes lipid storage and enhanced motility, changes that support the contribution of VSMCs to neointima and atherosclerotic plaque formation. This review focuses on the role of TNFα in driving the inflammatory changes in VSMC biology that contribute to cardiovascular disease. Special attention is given to the mechanisms by which TNFα promotes ROS production at specific subcellular locations, and the contribution of these ROS to TNFα signaling.

RATIONALE: Precise regulation of cerebral blood flow (CBF) is critical for normal brain function. Insufficient CBF contributes to brain dysfunction and neurodegeneration. Carbon dioxide (CO(2)), via effects on local acidosis, is one of the most potent regulators of CBF. Although a role for nitric oxide (NO) in intermediate signaling has been implicated, mechanisms that initiate CO(2) induced-vasodilation remain unclear. OBJECTIVE: Acid-sensing ion channel-1A (ASIC1A) is a proton-gated cation channel that is activated by extracellular acidosis. Based on work which implicated ASIC1A in the amygdala and bed nucleus of the stria terminalis in CO(2)-evoked and acid-evoked behaviors, we hypothesized that ASIC1A might also mediate microvascular responses to CO(2). METHODS AND RESULTS: To test this hypothesis we genetically and pharmacologically manipulated ASIC1A and assessed effects on CO(2)-induced dilation of cerebral arterioles in vivo. Effects of inhalation of 5 or 10% CO(2) on arteriolar diameter were greatly attenuated in mice with global deficiency in ASIC1A (Asic1a(−/−)) or by local treatment with the ASIC inhibitor, psalmotoxin. Vasodilator effects of acetylcholine, which acts via endothelial NO synthase were unaffected, suggesting a non-vascular source of NO may be key for CO(2) responses. Thus, we tested whether neurons may be the cell type through which ASIC1A influences microvessels. Using mice in which Asic1a was specifically disrupted in neurons, we found effects of CO(2) on arteriolar diameter were also attenuated. CONCLUSIONS: Together, these data are consistent with a model wherein activation of ASIC1A, particularly in neurons, is critical for CO(2)-induced NO production and vasodilation. With these findings, ASIC1A emerges as major regulator of microvascular tone.

Tumor necrosis factor-α (TNFα) is a proinflammatory cytokine that is closely linked to the development of cardiovascular disease. TNFα activates NADPH oxidase 1 (Nox1) and reactive oxygen species (ROS), including superoxide (O2·−), production extracellularly is required for subsequent signaling in vascular smooth muscle cells (VSMCs). Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that is activated by oxidation of associated thioredoxin. The role of ASK1 in Nox1-mediated signaling by TNFα is poorly defined. We hypothesized that ASK1 is required for TNFα receptor endocytosis and subsequent inflammatory TNFα signaling. We employed a knockdown strategy to explore the role of ASK1 in TNFα signaling in VSMCs. siRNA targeting ASK1 had no effect on TNFα-induced extracellular O2·− production. However, siASK1 inhibited receptor endocytosis as well as phosphorylation of two endocytosis-related proteins, dynamin1 and caveolin1. Intracellular O2·− production was subsequently reduced, as were other inflammatory signaling steps including NF-κB activation, IL-6 production, inducible nitric oxide synthase and VCAM expression, and VSMC proliferation. Prolonged exposure to TNFα (24 h) increased tumor necrosis factor receptor (TNFR) subtype 1 and 2 expression, and these effects were also attenuated by siASK1. ASK1 coimmunoprecipitated with both Nox1 and the leucine rich repeat containing 8A anion channel, two essential components of the TNFR1 signaling complex. Activation of ASK1 by autophosphorylation at Thr845 occurs following thioredoxin dissociation, and this requires the presence of Nox1. Thus, Nox1 is part of the multiprotein ASK1 signaling complex. In response to TNFα, ASK1 is activated by Nox1-derived oxidants, and this plays a critical role in translating these ROS into a physiologic response in VSMCs. NEW & NOTEWORTHY Apoptosis signal-regulating kinase 1 (ASK1) drives dynamin1 and caveolin1 phosphorylation and TNFα receptor endocytosis. ASK1 modulates TNFα-induced NF-κB activation, survival, and proliferation. ASK1 and NADPH oxidase 1 (Nox1) physically associate in a multiprotein signaling complex. Nox1 is required for TNFα-induced ASK1 activation.

Leucine Rich Repeat Containing 8A (LRRC8A) is a required component of volume-regulated anion channels (VRACs). In vascular smooth muscle cells, tumor necrosis factor-α (TNFα) activates VRAC via type 1 TNFα receptors (TNFR1), and this requires superoxide (O2•−) production by NADPH oxidase 1 (Nox1). VRAC inhibitors suppress the inflammatory response to TNFα by an unknown mechanism. We hypothesized that LRRC8A directly supports Nox1 activity, providing a link between VRAC current and inflammatory signaling. VRAC inhibition by 4-(2-butyl-6,7-dichlor-2-cyclopentylindan-1-on-5-yl) oxobutyric acid (DCPIB) impaired NF-κB activation by TNFα. LRRC8A siRNA reduced the magnitude of VRAC and inhibited TNFα-induced NF-κB activation, iNOS and VCAM expression, and proliferation of VSMCs. Signaling steps disrupted by both siLRRC8A and DCPIB included; extracellular O2•− production by Nox1, c-Jun N-terminal kinase (JNK) phosphorylation and endocytosis of TNFR1. Extracellular superoxide dismutase, but not catalase, selectively inhibited TNFR1 endocytosis and JNK phosphorylation. Thus, O2•− is the critical extracellular oxidant for TNFR signal transduction. Reducing JNK expression (siJNK) increased extracellular O2•− suggesting that JNK provides important negative feedback regulation to Nox1 at the plasma membrane. LRRC8A co-localized by immunostaining, and co-immunoprecipitated with, both Nox1 and its p22phox subunit. LRRC8A is a component of the Nox1 signaling complex. It is required for extracellular O2•− production, which is in turn essential for TNFR1 endocytosis. These data are the first to provide a molecular mechanism for the potent anti-proliferative and anti-inflammatory effects of VRAC inhibition.

Prostaglandin E2 (PGE2), a cyclooxygenase metabolite that generally acts as a systemic vasodepressor, has been shown to have vasopressor effects under certain physiologic conditions. Previous studies have demonstrated that PGE2 receptor signaling modulates angiotensin II (Ang II)-induced hypertension, but the interaction of these two systems in the regulation of vascular reactivity is incompletely characterized. We hypothesized that Ang II, a principal effector of the renin-angiotensin-aldosterone system, potentiates PGE2-mediated vasoconstriction. Here we demonstrate that pre-treatment of arterial rings with 1 nM Ang II potentiated PGE2-evoked constriction in a concentration dependent manner (AUC−Ang II 2.778 ± 2.091, AUC+Ang II 22.830 ± 8.560, ***P

Figure S1. Lack of iNOS induction in human endothelial cells: HMVECs (left) were exposed to Poly I:C (10 μg/mL) for 90 minutes, 6 hours or 16 hours, lysates were collected and examined for iNOS expression by western blot. No bands were detectable at the estimated molecular weight of ~135 kD. For a positive control for the antibody, murine vascular smooth muscle cells (right) were exposed to TNFα (10 ng/mL) and examined for iNOS induction via western blot. Representative images are shown for a single experiment (n=4 replicates per agonist group). Figure S2. Temporal change in phospho-eNOS and total eNOS after Poly I:C: HMVECs were exposed to Poly I:C (10 μg/mL) for 90 minutes, 6 hours or 16 hours, lysates were collected and examined via western blot for phospho-eNOS at residue serine 1177 compared to total eNOS (left) or total eNOS compared to α-tubulin (right) at the respective time points. Normalized protein ratios are shown above representative images of a single comparison (n=4 replicates per time point). * = p < 0.05 between compared groups, † = p

Prior exposure to lipopolysaccharide (LPS) produces a reduced or “tolerant” inflammatory response to subsequent challenges with LPS, however the potent pro-inflammatory effects of LPS limit its clinical benefit. The adjuvant monophosphoryl lipid A (MPLA) is a weak toll-like receptor 4 (TLR4) agonist that induces negligible inflammation but retains potent immunomodulatory properties. We postulated that pre-treatment with MPLA would inhibit the inflammatory response of endothelial cells to secondary LPS challenge. Human umbilical vein endothelial cells (HUVECs), were exposed to MPLA (10 μg/ml), LPS (100 ng/ml) or vehicle control. HUVECs were then washed and maintained in culture for 24 h before being challenged with LPS (100 ng/ml). Supernatants were collected and examined for cytokine production in the presence or absence of siRNA inhibitors of critical TLR4 signalling proteins. Pre-treatment with MPLA attenuated interleukin (IL)-6 production to secondary LPS challenge to a similar degree as LPS. The application of myeloid differentiation primary response gene 88 (MyD88) siRNA dramatically reduced MPLA-induced tolerance while TIR-domain-containing adapter-inducing interferon-β (TRIF) siRNA had no effect. The tolerant phenotype in endothelial cells was associated with reduced IκB kinase (IKK), p38 and c-Jun N-terminal kinase (JNK) phosphorylation and enhanced IL-1 receptor associated kinase-M (IRAK-M) expression for LPS-primed HUVECs, but less so in MPLA primed cells. Instead, MPLA-primed HUVECs demonstrated enhanced p-extracellular-signal-regulated kinase (ERK) phosphorylation. In contrast with leucocytes in which tolerance is largely TRIF-dependent, MyD88 signalling mediated endotoxin tolerance in endothelial cells. Most importantly, MPLA, a vaccine adjuvant with a wide therapeutic window, induced tolerance to LPS in endothelial cells.

Tumor necrosis factor-α (TNFα), a proinflammatory cytokine, causes vascular smooth muscle cell (VSMC) proliferation and migration and promotes inflammatory vascular lesions. Nuclear factor-kappa B (NF-κB) activation by TNFα requires endosomal superoxide production by Nox1. In endothelial cells, TNFα stimulates c-Jun N-terminal kinase (JNK), which inhibits NF-κB signaling. The mechanism by which JNK negatively regulates TNFα-induced NF-κB activation has not been defined. We hypothesized that JNK modulates NF-κB activation in VSMC, and does so via a Nox1-dependent mechanism. TNFα-induced NF-κB activation was TNFR1- and endocytosis-dependent. Inhibition of endocytosis with dominant-negative dynamin (DynK44A) potentiated TNFα-induced JNK activation, but decreased ERK activation, while p38 kinase phosphorylation was not altered. DynK44A attenuated intracellular, endosomal superoxide production in wild-type (WT) VSMC, but not in NADPH oxidase 1 (Nox1) knockout (KO) cells. siRNA targeting JNK1 or JNK2 potentiated, while a JNK activator (anisomycin) inhibited, TNFα-induced NF-κB activation in WT, but not in Nox1 KO cells. TNFα-stimulated superoxide generation was enhanced by JNK1 inhibition in WT, but not in Nox1 KO VSMC. These data suggest that JNK suppresses the inflammatory response to TNFα by reducing Nox1-dependent endosomal ROS production. JNK and endosomal superoxide may represent novel targets for pharmacologic modulation of TNFα signaling and vascular inflammation.

Endothelial dysfunction, characterized by changes in eNOS, is a common finding in chronic inflammatory vascular diseases. These states are associated with increased infectious complications. We hypothesized that alterations in eNOS would enhance the response to LPS-mediated TLR4 inflammation. Human microvascular endothelial cells were treated with sepiapterin or N-nitro-L-arginine methylester (L-NAME) to alter endogenous NO production, and small interfering RNA to knockdown eNOS. Alterations of endogenous NO by sepiapterin, and L-NAME provided no significant changes to LPS inflammation. In contrast, eNOS knockdown greatly enhanced endothelial IL-6 production and permeability in response to LPS. Knockdown of eNOS enhanced LPS-induced p38. Inhibition of p38 with SB203580 prevented IL-6 production, without altering permeability. Knockdown of p38 impaired NF-κB activation. Physical interaction between p38 and eNOS was demonstrated by immunoprecipitation, suggesting a novel, NO-independent mechanism for eNOS regulation of TLR4. In correlation, biopsy samples in patients with systemic lupus erythematous showed reduced eNOS expression with associated elevations in TLR4 and p38, suggesting an in vivo link. Thus, reduced expression of eNOS, as seen in chronic inflammatory disease, was associated with enhanced TLR4 signaling through p38. This may enhance the response to infection in patients with chronic inflammatory conditions.-Stark, R. J., Koch, S. R., Choi, H., Mace, E. H., Dikalov, S. I., Sherwood, E. R., Lamb, F. S. Endothelial nitric oxide synthase modulates Toll-like receptor 4-mediated IL-6 production and permeability via nitric oxide-independent signaling.

Monophosphoryl lipid A (MPLA) is a TLR4 agonist that is used as an immunomodulator in human vaccines; additionally, it has been shown to be protective in models of sepsis. As endothelial cells regulate inflammation, we hypothesized that MPLA would decrease activation of human umbilical vein endothelial cells (HUVECs) to LPS. We studied HUVECs challenged with LPS (100 ng/ml), MPLA (0.001–100 µg/ml) or a combination. Secretion of IL-6, RANTES (CCL5) and IP-10 (CXCL10) were assessed by ELISA. Activation of MAPK phosphorylation and cytokine transcription were assessed by Western blot analysis and PCR, respectively. MPLA alone was a weak stimulator of myeloid differentiation primary response protein 88-dependent IL-6 and did not induce TIR-domain-containing adapter-inducing IFN-β (TRIF)-dependent chemokine responses. MPLA significantly reduced LPS-mediated IL-6 production. This inhibitory effect was also conferred for the TRIF-dependent chemokines RANTES and IP-10. Inhibition of LPS-mediated activation by MPLA was associated with reduced p38 phosphorylation and mRNAs encoding inflammatory cytokines. MPLA inhibition of LPS signaling appeared to be at the level of the TLR4 receptor, acting as a receptor antagonist with weak agonistic properties. This study provides evidence of a novel mechanism for the inhibitory effect of MPLA on LPS-induced endothelial activation.

Mitochondrial aldehyde dehydrogenase (ALDH2) is an enzyme that detoxifies aldehydes to carboxylic acids. ALDH2 deficiency is known to increase oxidative stress, which is the imbalance between reactive oxygen species (ROS) generation and antioxidant defense activity. Increased ROS contribute to vascular dysfunction and structural remodeling in hypertension. We hypothesized that ALDH2 plays a protective role to reduce vascular contraction in angiotensin-II (AngII) hypertensive mice. Endothelium-denuded aortic rings from C57BL6 mice, treated with AngII (3.6 μg/kg/min, 14 days), were used to measure isometric force development. Rings treated with daidzin (10 μmol/L), an ALDH2 inhibitor, potentiated contractile responses to phenylephrine (PE) in AngII mice. Tempol (1 mmol/L) and catalase (600 U/mL) attenuated the augmented contractile effect of daidzin. In normotensive mice, contraction to PE in the presence of the daidzin was not different from control, untreated values. AngII aortic rings transfected with ALDH2 recombinant protein decreased contractile responses to PE compared with control. These data suggest that ALDH2 reduces vascular contraction in AngII hypertensive mice. Because tempol and catalase blocked the contractile response of the ALDH2 inhibitor, ROS generation by AngII may be decreased by ALDH2, thereby preventing ROS-induced contraction.

The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10μM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility.

Tumor necrosis factor-α (TNFα), a pro-inflammatory cytokine, can cause either vascular smooth muscle cell (VSMC) apoptosis (JNK-dependent) or proliferation (NF-κB-dependent). We hypothesized that J...

Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that causes endothelial dysfunction. Endocytosis of TNF-α receptors (TNFR) precedes endosomal reactive oxygen species (ROS) production, which is required for NF-κB activation in vascular smooth muscle cells. It is unknown how endocytosis of TNFRs impacts signaling in endothelial cells. We hypothesized that TNF-α-induced endothelial dysfunction is induced by both endosomal and cell surface events, including NF-κB and mitogen-activated protein kinases (MAPKs) activation, and endocytosis of the TNFR modifies signaling. Mesenteric artery segments from C57BL/6 mice were treated with TNF-α (10 ng/ml) for 22 h in tissue culture, with or without signaling inhibitors (dynasore for endocytosis, SP600125 for JNK, SB203580 for p38, U0126 for ERK), and vascular function was assessed. Endothelium-dependent relaxation to acetylcholine (ACh) was impaired by TNF-α, and dynasore exacerbated this, whereas JNK or p38 inhibition prevented these effects. In cultured endothelial cells from murine mesenteric arteries, dynasore potentiated JNK and p38 but not ERK phosphorylation and promoted cell death. NF-κB activation by TNF-α was decreased by dynasore. JNK inhibition dramatically increased both the magnitude and duration of TNF-α-induced NF-κB activation and potentiated intercellular adhesion molecule-1 (ICAM-1) activation. Dynasore still inhibited NF-κB activation in the presence of SP600125. Thus TNF-α-induced endothelial dysfunction is both JNK and p38 dependent. Endocytosis modulates the balance of NF-κB and MAPK signaling, and inhibition of NF-κB activation by JNK limits this pro-proliferative signal, which may contribute to endothelial cell death in response to TNF-α.

O-GlcNAcylation is an unusual form of protein glycosylation, where a single-sugar [GlcNAc (N-acetylglucosamine)] is added (via β-attachment) to the hydroxyl moiety of serine and threonine residues of nuclear and cytoplasmic proteins. A complex and extensive interplay exists between O-GlcNAcylation and phosphorylation. Many phosphorylation sites are also known glycosylation sites, and this reciprocal occupancy may produce different activities or alter the stability in a target protein. The interplay between these two post-translational modifications is not always reciprocal, as some proteins can be concomitantly phosphorylated and O-GlcNAcylated, and the adjacent phosphorylation or O-GlcNAcylation can regulate the addition of either moiety. Increased cardiovascular production of ROS (reactive oxygen species), termed oxidative stress, has been consistently reported in various chronic diseases and in conditions where O-GlcNAcylation has been implicated as a contributing mechanism for the associated organ injury/protection (for example, diabetes, Alzheimer's disease, arterial hypertension, aging and ischaemia). In the present review, we will briefly comment on general aspects of O-GlcNAcylation and provide an overview of what has been reported for this post-translational modification in the cardiovascular system. We will then specifically address whether signalling molecules involved in redox signalling can be modified by O-GlcNAc (O-linked GlcNAc) and will discuss the critical interplay between O-GlcNAcylation and ROS generation. Experimental evidence indicates that the interactions between O-GlcNAcylation and oxidation of proteins are important not only for cell regulation in physiological conditions, but also under pathological states where the interplay may become dysfunctional and thereby exacerbate cellular injury.

Vascular dysfunction, including reduced endothelium-dependent dilation, is a major characteristic of hypertension. We previously investigated that thioredoxin reductase (TrxR) inhibition impairs vasodilation via soluble guanylyl cyclase S-nitrosylation, but S-nitrosylation and TrxR function are not known in hypertension. We hypothesized that S-nitrosylation is associated with reduced vasodilation in hypertensive mice.Aortic rings from normotensive (sham) and angiotensin II (AngII)-induced hypertensive C57BL/6 mice were treated with a TrxR inhibitor, 1-chloro-2,4-dinitrobenzene (DNCB) for 30 min, and relaxation to acetylcholine (ACh) was measured in the rings following contraction with phenylephrine.DCNB reduced relaxation to ACh compared with vehicle in sham aorta but not in AngII (sham-vehicle E(max) = 77 ± 2, sham-DNCB E(max) = 59 ± 4, P 0.05). DNCB shifted the concentration-response relaxation to sodium nitroprusside (SNP) to the right in both sham and AngII aortic rings (sham-vehicle pD(2) = 8.8±0.1, sham-DNCB pD(2) = 8.4±0.1, *P 0.05; AngII-vehicle pD(2) = 8.5±0.1, AngII-DNCB pD(2) = 8.3 ± 0.1, P 0.05). As downstream signaling of nitric oxide, cyclic GMP level was reduced by DNCB during activation with SNP. The effect of DNCB to increase S-nitrosylation was confirmed by the biotin-switch method and western blot analysis, and total protein S-nitrosylation was increased in AngII aorta (1.5-fold) compared with sham. TrxR activity was inhibited in AngII aorta compared with sham.We conclude that increased S-nitrosylation contributes to impaired relaxation in aorta from AngII-induced hypertensive mice. AngII treatment resulted in inactivation of TrxR and increased S-nitrosylation, indicating that TrxR and S-nitrosylation may provide a critical mechanism in hypertension associated with abnormal vascular reactivity.

Oxidative stress is well known to lead to vascular dysfunction. Thioredoxin reductase (TrxR) catalyzes the reduction of oxidized thioredoxin. Reduced thioredoxin plays a role in cellular antioxidative defense and in decreasing S-nitrosylation. It is not known whether TrxR affects vascular reactivity. We hypothesized that TrxR inhibition decreases vascular relaxation via increased oxidative stress and S-nitrosylation. Aortic rings from C57BL/6 mice were treated with the TrxR inhibitor, 1-chloro-2,4-dinitrobenzene (DNCB), or auranofin for 30 minutes. Vascular relaxation to acetylcholine was measured in the rings contracted with phenylephrine. DNCB and auranofin reduced relaxation compared with vehicle (vehicle Emax = 71 ± 3%, DNCB Emax = 53 ± 3%; P < 0.05). The antioxidants, apocynin (nicotinamide adenine dinucleotide phosphate oxidase inhibitor), and tempol (superoxide dismutase mimetic) normalized impaired relaxation by DNCB in aorta (DNCB Emax = 53 ± 3%, DNCB + tempol Emax = 66 ± 3%; P < 0.05). In addition, DNCB reduced sodium nitroprusside-induced relaxation. DNCB increased soluble guanylyl cyclase (sGC) S-nitrosylation and decreased sGC activity. These data suggest that TrxR regulates vascular relaxation via antioxidant defense and sGC S-nitrosylation. TrxR may be an enzyme to approach for treatment of vascular dysfunction and arterial hypertension.

S-Nitrosylation is a ubiquitous protein modification in redox-based signaling and forms S-nitrosothiol (SNO) from nitric oxide (NO) on cysteine residues. Dysregulation of (S)NO signaling (nitrosative stress) leads to impairment of cellular function. Protein kinase C (PKC) is an important signaling protein that plays a role in the regulation of vascular function and it is not known whether (S)NO affects PKC’s role in vascular reactivity. We hypothesized that S-nitrosylation of PKC in vascular smooth muscle would inhibit its contractile activity. Aortic rings from male C57BL6 mice were treated with auranofin or 1-chloro-2,4-dinitrobenzene (DNCB) as pharmacological tools, which lead to stabilize S-nitrosylation, and propylamine propylamine NONOate (PANOate) or S-nitrosocysteine (CysNO) as NO donors. Contractile responses of aorta to phorbol-12,13-dibutyrate (PDBu), a PKC activator, were attenuated by auranofin, DNCB, PANOate, and CysNO. S-Nitrosylation of PKCα was increased by auranofin or DNCB and CysNO as compared to control protein. Augmented S-nitrosylation inhibited PKCα activity and subsequently downstream signal transduction. These data suggest that PKC is inactivated by S-nitrosylation and this modification inhibits PKC-dependent contractile responses. Since S-nitrosylation of PKC inhibits phosphorylation and activation of target proteins related to contraction, this post-translational modification may be a key player in conditions of decreased vascular reactivity.

Hyperglycemia, which increases O-linked β- N -acetylglucosamine (O-GlcNAc) proteins, leads to changes in vascular reactivity. Because vascular dysfunction is a key feature of arterial hypertension, we hypothesized that vessels from deoxycorticosterone acetate and salt (DOCA-salt) rats exhibit increased O-GlcNAc proteins, which is associated with increased reactivity to constrictor stimuli. Aortas from DOCA rats exhibited increased contraction to phenylephrine (E max [mN]=17.6±4 versus 10.7±2 control; n=6) and decreased relaxation to acetylcholine (47.6±6% versus 73.2±10% control; n=8) versus arteries from uninephrectomized rats. O-GlcNAc protein content was increased in aortas from DOCA rats (arbitrary units=3.8±0.3 versus 2.3±0.3 control; n=5). PugNAc (O-GlcNAcase inhibitor; 100 μmol/L; 24 hours) increased vascular O-GlcNAc proteins, augmented phenylephrine vascular reactivity (18.2±2 versus 10.7±3 vehicle; n=6), and decreased acetylcholine dilation in uninephrectomized (41.4±6 versus 73.2±3 vehicle; n=6) but not in DOCA rats (phenylephrine, 16.5±3 versus 18.6±3 vehicle, n=6; acetylcholine, 44.7±8 versus 47.6±7 vehicle, n=6). PugNAc did not change total vascular endothelial nitric oxide synthase levels, but reduced endothelial nitric oxide synthase Ser-1177 and Akt Ser-473 phosphorylation ( P Ser-1177 and Akt Ser-473 ( P P