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Resuscitation with 0.9% Normal Saline (NS), a non-buffered acidic solution, leads to increased morbidity and mortality in the critically ill. The goal of this study was to determine the molecular mechanisms of endothelial injury after exposure to NS. The hypothesis of this investigation is that exposure of endothelium to NS would lead to loss of cell membrane integrity, resulting in release of ATP, activation of the purinergic receptor (P2X7R), and subsequent activation of stress activated signaling pathways and inflammation. Human saphenous vein endothelial cells (HSVEC) incubated in NS, but not buffered electrolyte solution (Plasma-Lyte, PL), exhibited abnormal morphology and increased release of lactate dehydrogenase (LDH), adenosine triphosphate (ATP), and decreased transendothelial resistance (TEER), suggesting loss of membrane integrity. Incubation of intact rat aorta (RA) or human saphenous vein in NS but not PL led to impaired endothelial-dependent relaxation which was ameliorated by apyrase (hydrolyzes ATP) or SB203580 (p38 MAPK inhibitor). Exposure of HSVEC to NS but not PL led to activation of p38 MAPK and its downstream substrate, MAPKAP kinase 2 (MK2). Treatment of HSVEC with exogenous ATP led to interleukin 1β (IL-1β) release and increased vascular cell adhesion molecule (VCAM) expression. Treatment of RA with IL-1β led to impaired endothelial relaxation. IL-1β treatment of HSVEC led to increases in p38 MAPK and MK2 phosphorylation, and increased levels of arginase II. Incubation of porcine saphenous vein (PSV) in PL with pH adjusted to 6.0 or less also led to impaired endothelial function, suggesting that the acidic nature of NS is what contributes to endothelial dysfunction. Volume overload resuscitation in a porcine model after hemorrhage with NS, but not PL, led to acidosis and impaired endothelial function. These data suggest that endothelial dysfunction caused by exposure to acidic, non-buffered NS is associated with loss of membrane integrity, release of ATP, and is modulated by P2X7R-mediated inflammatory responses.
BACKGROUND - Vascular dysfunction is commonly seen during severe viral infections. Endothelial nitric oxide synthase (eNOS), has been postulated to play an important role in regulating vascular homeostasis as well as propagation of the inflammatory reaction. We hypothesized that the loss of eNOS would negatively impact toll-like receptor 3 (TLR3) signaling and worsen vascular function to viral challenge.
METHODS - Human microvascular endothelial cells (HMVECs) were exposed to either control or eNOS siRNA and then treated with Poly I:C, a TLR3 agonist and mimicker of dsRNA viruses. Cells were assessed for protein-protein associations, cytokine and chemokine analysis as well as transendothelial electrical resistance (TEER) as a surrogate of permeability.
RESULTS - HMVECs that had reduced eNOS expression had a significantly elevated increase in IL-6, IL-8 and IP-10 production after Poly I:C. In addition, the knockdown of eNOS enhanced the change in TEER after Poly I:C stimulation. Western blot analysis showed enhanced phosphorylation of p38 in sieNOS treated cells with Poly I:C compared to siControl cells. Proximity ligation assays further demonstrated direct eNOS-p38 protein-protein interactions. The addition of the p38 inhibitor, SB203580, in eNOS knockdown cells reduced both cytokine production after Poly I:C, and as well as mitigated the reduction in TEER, suggesting a direct link between eNOS and p38 in TLR3 signaling.
CONCLUSIONS - These results suggest that reduction of eNOS increases TLR3-mediated inflammation in human endothelial cells in a p38-dependent manner. This finding has important implications for understanding the pathogenesis of severe viral infections and the associated vascular dysfunction.
Diabetic gastroparesis (GP) is a clinical syndrome characterized by delayed gastric emptying (DGE). Loss of Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) led to reduced nNOSα mediated gastric motility and DGE. The molecular signaling of cinnamaldehyde (CNM) mediated Nrf2 activation and its mechanistic role on DGE were further investigated in obese/T2D female mice. Adult female homozygous Nfe2l2 (C57BL/6J) and their wild-type (WT) littermates (Nfe2l2) mice were fed with high fat diet (HFD; Obese/T2D model), or normal diet (ND) with or without CNM (50 mg/kg b.w; i.p). Supplementation of CNM attenuated (p < 0.05) DGE in WT female but not in Nrf2 KO Obese/T2D mice. CNM (1) normalized serum estradiol-17β levels, (2) induced gastric Nrf2 and phase II antioxidant enzymes through extracellular signal-regulated kinase, (ERK)/c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK), (3) reduced glucose synthase kinase 3 beta (GSK3β) and aryl hydrocarbon receptor (AhR) and this was associated with (4) increased estrogen receptor expression, BH (Cofactor of nNOS) biosynthesis enzyme GCH-1 and nNOSα dimerization in WT Obese/T2 diabetic female mice. In addition, CNM restored impaired nitrergic relaxation in hyperglycemic conditions. These findings emphasize the importance of Nrf2 in maintaining nNOSα mediated GE and may have a translational relevance to treat obese/diabetic gastroparesis in women.
Copyright © 2019. Published by Elsevier Inc.
A viable vascular endothelial layer prevents vasomotor dysfunction, thrombosis, inflammation, and intimal hyperplasia. Injury to the endothelium occurs during harvest and "back table" preparation of human saphenous vein prior to implantation as an arterial bypass conduit. A subfailure overstretch model of rat aorta was used to show that subfailure stretch injury of vascular tissue leads to impaired endothelial-dependent relaxation. Stretch-induced impaired relaxation was mitigated by treatment with purinergic P2X7 receptor (P2X7R) inhibitors, brilliant blue FCF (FCF) and A740003, or apyrase, an enzyme that catalyzes the hydrolysis of ATP. Alternatively, treatment of rat aorta with exogenous ATP or 2'(3')-O-(4-Benzoyl benzoyl)-ATP (BzATP) also impaired endothelial-dependent relaxation. Treatment of human saphenous vein endothelial cells (HSVEC) with exogenous ATP led to reduced nitric oxide production which was associated with increased phosphorylation of the stress activated protein kinase, p38 MAPK. ATP- stimulated p38 MAPK phosphorylation of HSVEC was inhibited by FCF and SB203580. Moreover, ATP inhibition of nitric oxide production in HSVEC was prevented by FCF, SB203580, L-arginine supplementation and arginase inhibition. Finally, L-arginine supplementation and arginase inhibition restored endothelial dependent relaxation after stretch injury of rat aorta. These results suggest that vascular stretch injury leads to ATP release, activation of P2X7R and p38 MAPK resulting in endothelial dysfunction due to arginase activation. Endothelial function can be restored in both ATP treated HSVEC and intact stretch injured rat aorta by P2X7 receptor inhibition with FCF or L-arginine supplementation, implicating straightforward therapeutic options for treatment of surgical vascular injury.
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.
BACKGROUND - Human saphenous veins used for arterial bypass undergo stretch injury at the time of harvest and preimplant preparation. Vascular injury promotes intimal hyperplasia, the leading cause of graft failure, but the molecular events leading to this response are largely unknown. This study investigated adenosine triphosphate (ATP) as a potential molecular mediator in the vascular response to stretch injury, and the downstream effects of the purinergic receptor, P2X7R, and p38 MAPK activation.
MATERIALS AND METHODS - A subfailure stretch rat aorta model was used to determine the effect of stretch injury on release of ATP and vasomotor responses. Stretch-injured tissues were treated with apyrase, the P2X7R antagonist, A438079, or the p38 MAPK inhibitor, SB203580, and subsequent contractile forces were measured using a muscle bath. An exogenous ATP (eATP) injury model was developed and the experiment repeated. Change in p38 MAPK phosphorylation after stretch and eATP tissue injury was determined using Western blotting. Noninjured tissue was incubated in the p38 MAPK activator, anisomycin, and subsequent contractile function and p38 MAPK phosphorylation were analyzed.
RESULTS - Stretch injury was associated with release of ATP. Contractile function was decreased in tissue subjected to subfailure stretch, eATP, and anisomycin. Contractile function was restored by apyrase, P2X7R antagonism, and p38-MAPK inhibition. Stretch, eATP, and anisomycin-injured tissue demonstrated increased phosphorylation of p38 MAPK.
CONCLUSIONS - Taken together, these data suggest that the vascular response to stretch injury is associated with release of ATP and activation of the P2X7R/P38 MAPK pathway, resulting in contractile dysfunction. Modulation of this pathway in vein grafts after harvest and before implantation may reduce the vascular response to injury.
Copyright © 2017 Elsevier Inc. All rights reserved.
In familial pulmonary arterial hypertension (FPAH), the autosomal dominant disease-causing BMPR2 mutation is only 20% penetrant, suggesting that genetic variation provides modifiers that alleviate the disease. Here, we used comparison of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from three families with unaffected mutation carriers (UMCs), FPAH patients, and gender-matched controls to investigate this variation. Our analysis identified features of UMC iPSC-ECs related to modifiers of BMPR2 signaling or to differentially expressed genes. FPAH-iPSC-ECs showed reduced adhesion, survival, migration, and angiogenesis compared to UMC-iPSC-ECs and control cells. The "rescued" phenotype of UMC cells was related to an increase in specific BMPR2 activators and/or a reduction in inhibitors, and the improved cell adhesion could be attributed to preservation of related signaling. The improved survival was related to increased BIRC3 and was independent of BMPR2. Our findings therefore highlight protective modifiers for FPAH that could help inform development of future treatment strategies.
Copyright © 2017 Elsevier Inc. All rights reserved.
Kinases downstream of B-cell antigen receptor (BCR) represent attractive targets for therapy in non-Hodgkin lymphoma (NHL). As clinical responses vary, improved knowledge regarding activation and regulation of BCR signaling in individual patients is needed. Here, using phosphospecific flow cytometry to obtain malignant B-cell signaling profiles from 95 patients representing 4 types of NHL revealed a striking contrast between chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) tumors. Lymphoma cells from diffuse large B-cell lymphoma patients had high basal phosphorylation levels of most measured signaling nodes, whereas follicular lymphoma cells represented the opposite pattern with no or very low basal levels. MCL showed large interpatient variability in basal levels, and elevated levels for the phosphorylated forms of AKT, extracellular signal-regulated kinase, p38, STAT1, and STAT5 were associated with poor outcome. CLL tumors had elevated basal levels for the phosphorylated forms of BCR-signaling nodes (Src family tyrosine kinase, spleen tyrosine kinase [SYK], phospholipase Cγ), but had low α-BCR-induced signaling. This contrasted MCL tumors, where α-BCR-induced signaling was variable, but significantly potentiated as compared with the other types. Overexpression of CD79B, combined with a gating strategy whereby signaling output was directly quantified per cell as a function of CD79B levels, confirmed a direct relationship between surface CD79B, immunoglobulin M (IgM), and IgM-induced signaling levels. Furthermore, α-BCR-induced signaling strength was variable across patient samples and correlated with BCR subunit CD79B expression, but was inversely correlated with susceptibility to Bruton tyrosine kinase (BTK) and SYK inhibitors in MCL. These individual differences in BCR levels and signaling might relate to differences in therapy responses to BCR-pathway inhibitors.
© 2017 by The American Society of Hematology.
BACKGROUND - Impaired remyelination of demyelinated axons is a major cause of neurological disability. In inflammatory demyelinating disease of the central nervous system (CNS), although remyelination does happen, it is often incomplete, resulting in poor clinical recovery. Poly-IC a known TLR3 agonist and IL-33, a cytokine which is induced by poly-IC are known to influence recovery and promote repair in experimental models of CNS demyelination.
METHODOLOGY AND PRINCIPAL FINDINGS - We examined the effect of addition of poly-IC and IL-33 on the differentiation and maturation of oligodendrocyte precursor cells (OPC) cultured in vitro. Both Poly-IC and IL-33 induced transcription of myelin genes and the differentiation of OPC to mature myelin forming cells. Poly-IC induced IL-33 in OPC and addition of IL-33 to in vitro cultures, amplified further, IL-33 expression suggesting an autocrine regulation of IL-33. Poly-IC and IL-33 also induced phosphorylation of p38MAPK, a signaling molecule involved in myelination. Following the induction of gliotoxic injury with lysolecithin to the corpus callosum (CC), treatment of animals with poly-IC resulted in greater recruitment of OPC and increased staining for myelin in areas of demyelination. Also, poly-IC treated animals showed greater expression of IL-33 and higher expression of M2 phenotype macrophages in the CC.
CONCLUSION/SIGNIFICANCE - Our studies suggest that poly-IC and IL-33 play a role in myelin repair by enhancing expression of myelin genes and are therefore attractive therapeutic agents for use as remyelinating agents in human demyelinating disease.
Alterations in central serotonin (5-hydroxytryptamine, 5-HT) neurotransmission and peripheral immune activation have been linked to multiple neuropsychiatric disorders, including depression, schizophrenia and autism. The antidepressant-sensitive 5-HT transporter (SERT, SLC6A4), a critical determinant of synaptic 5-HT inactivation, can be regulated by pro-inflammatory cytokine signaling. Systemic innate immune system activation via intraperitoneal lipopolysaccharide (LPS) injection rapidly elevates brain SERT activity and 5-HT clearance. Moreover, the pro-inflammatory cytokine interleukin (IL)-1β rapidly stimulates SERT activity in raphe nerve terminal preparations ex vivo, effects that are attenuated by pharmacological p38 MAPK inhibition. To establish a role of serotonergic p38α MAPK signaling in LPS/IL-1β-induced SERT regulation and attendant behavioral responses, we pursued studies in mice that afford conditional elimination of p38α MAPK in 5-HT neurons (p38α(5HT-)). We found p38α(5HT-) and control (p38α(5HT+)) littermates to be indistinguishable in viability and growth and to express equivalent levels of SERT protein and synaptosomal 5-HT transport activity. Consistent with pharmacological studies, however, IL-1β fails to increase SERT activity in midbrain synaptosomes prepared from p38α(5HT-) animals. Moreover, although LPS elevated plasma corticosterone and central/peripheral pro-inflammatory cytokines in p38α(5HT-) animals, elevations in midbrain SERT activity were absent nor were changes in depressive and anxiety-like behaviors observed. Our studies support an obligate role of p38α MAPK signaling in 5-HT neurons for the translation of immune activation to SERT regulation and 5-HT-modulated behaviors.