Promoted Nitric Oxide Production Research Articles

Hyperoside ameliorates depression-like behavior in ovariectomized mice

The physiological changes caused by the decline in estrogen levels due to menopause are linked to an increased risk of depression. This study investigated the antidepressant effects of hyperoside (HYP), a natural flavonol glycoside, and its associated molecular mechanisms in primary hippocampal neurons and ovariectomized (OVX) mice. HYP treatment increased nitric oxide (NO) production and neuronal nitric oxide synthase (nNOS) expression in primary hippocampal neurons; additionally, it upregulated the expression of brain-derived neurotrophic factor (BDNF) and phosphorylated tropomyosin receptor kinase B (TrkB). In OVX mice, HYP treatment significantly improved depression-like behaviors in an open field test to a level comparable to estrogen treatment. Furthermore, HYP treatment upregulated OVX-induced decreased nNOS expression and BDNF-TrkB signaling in the hippocampus. Therefore, this study suggests that HYP exhibits antidepressant potential by addressing estrogen deficiency-induced alterations, specifically by restoring nNOS expression, promoting NO production, and concurrently enhancing BDNF-TrkB signaling in OVX mice.

Minimalist Nanocomplex with Dual Regulation of Endothelial Function and Inflammation for Targeted Therapy of Inflammatory Vascular Diseases.

Vascular disorders, characterized by vascular endothelial dysfunction combined with inflammation, are correlated with numerous fatal diseases, such as coronavirus disease-19 and atherosclerosis. Achieving vascular normalization is an urgent problem that must be solved when treating inflammatory vascular diseases. Inspired by the vascular regulatory versatility of nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) catalyzing l-arginine (l-Arg), the eNOS-activating effects of l-Arg, and the powerful anti-inflammatory and eNOS-replenishing effects of budesonide (BUD), we constructed a bi-prodrug minimalist nanoplatform co-loaded with BUD and l-Arg via polysialic acid (PSA) to form BUD-l-Arg@PSA. This promoted vascular normalization by simultaneously regulating vascular endothelial dysfunction and inflammation. Mediated by the special affinity between PSA and E-selectin, which is highly expressed on the surface of activated endothelial cells (ECs), BUD-l-Arg@PSA selectively accumulated in activated ECs, targeted eNOS expression and activation, and promoted NO production. Consequently, the binary synergistic regulation of the NO/eNOS signaling pathway occurred and improved vascular endothelial function. NO-induced nuclear factor-kappa B alpha inhibitor (IκBα) stabilization and BUD-induced nuclear factor-kappa B (NF-κB) response gene site occupancy achieved dual-site blockade of the NF-κB signaling pathway, thereby reducing the inflammatory response and inhibiting the infiltration of inflammation-related immune cells. In a renal ischemia-reperfusion injury mouse model, BUD-l-Arg@PSA reduced acute injury. In an atherosclerosis mouse model, BUD-l-Arg@PSA decreased atherosclerotic plaque burden and improved vasodilation. This represents a revolutionary therapeutic strategy for inflammatory vascular diseases.

Hesperidin Preferentially Stimulates Transient Receptor Potential Vanilloid 1, Leading to NO Production and Mas Receptor Expression in Human Umbilical Vein Endothelial Cells.

Here, the mechanism of vasorelaxant Mas receptor (MasR) expression elevated by hesperidin in spontaneously hypertensive rats was investigated in human umbilical vein endothelial cells (HUVECs). HUVECs were cultured with 1 μM hesperidin for 2 h, following the measurements of nitric oxide (NO) production and vasomotor-related receptors' expression. Hesperidin significantly promoted NO production (224.1 ± 18.3%, P < 0.01 vs control) in the HUVECs. Only the MasR expression was upregulated (141.2 ± 12.5%, P < 0.05 vs control), whereas a MasR antagonist did not alter the hesperidin-induced NO production. When a transient receptor potential vanilloid 1 (TRPV1) was knocked down by silencing RNA or Ca2+/calmodulin-dependent kinase II (CaMKII) and p38 mitogen-activated protein kinase (p38 MAPK) were inhibited, the increased MasR expression by hesperidin was abrogated. The inhibitions of CaMKII and endothelial NO synthase (eNOS) abolished the hesperidin-induced NO production. The structure-activity relationship analysis of flavonoids demonstrated that the B ring of the twisted flavonoid skeleton with a hydroxy group at the 3' position was a crucial factor for TRPV1 stimulation. Taken together, it was demonstrated that hesperidin may stimulate TRPV1-mediated cascades, leading to the activation of two signaling axes, CaMKII/p38 MAPK/MasR expression and CaMKII/eNOS/NO production in HUVECs.

New progress in understanding roles of nitric oxide during hepatic ischemia-reperfusion injury.

Hepatic ischemia-reperfusion injury (HIRI) is a major clinical cause of morbidity and mortality in liver surgery and transplantation. Many studies have found that nitric oxide (NO) plays an important role in the HIRI and its increase or decrease can affect the progression and outcome of HIRI. However, the role of NO in HIRI is controversial and complicated. NO derived by endothelial NO synthase (eNOS) shows a protective role in HIRI, while excessive NO derived by inducible NO synthase (iNOS) accelerates inflammation and increases oxidative stress, further aggravating HIRI. Nevertheless, the overexpression of eNOS may exacerbate HIRI and iNOS-derived NO in some cases reduces HIRI. Here we review the new progress in the understanding of the roles of NO during HIRI: (1) NO possesses different roles in HIRI by increasing NO bioavailability, down-regulating leukotriene C4 synthase, inhibiting the activation of the nuclear factorκB (NFκB) pathway, enhancing cell autophagy, and reducing inflammatory cytokines and reactive oxygen species (ROS). And NO has both protective and deleterious effects by regulating apoptotic factors; (2) eNOS promotes NO production and suppresses its own overexpression, exerting a hepatoprotective effect reversely. Its activation is regulated by the PI3K/Akt and KLF2/AMPK pathways; and (3) iNOS derived NO mainly has deteriorating effects on HIRI, while it may have a protective function under some conditions. Their expression should reach a balance to reduce the adverse side and make NO protective in the treatment of HIRI. Thus, it can be inferred that NO modulating drugs may be a new direction in the treatment of HIRI or may be used as an adjunct to mitigate HIRI for the purpose of protecting the liver.

Acetate Improves the Killing of Streptococcus pneumoniae by Alveolar Macrophages via NLRP3 Inflammasome and Glycolysis-HIF-1α Axis.

Short-chain fatty acids (SCFAs) are metabolites produced mainly by the gut microbiota with a known role in immune regulation. Acetate, the major SCFA, is described to disseminate to distal organs such as lungs where it can arm sentinel cells, including alveolar macrophages, to fight against bacterial intruders. In the current study, we explored mechanisms through which acetate boosts macrophages to enhance their bactericidal activity. RNA sequencing analyses show that acetate triggers a transcriptomic program in macrophages evoking changes in metabolic process and immune effector outputs, including nitric oxide (NO) production. In addition, acetate enhances the killing activity of macrophages towards Streptococcus pneumoniae in an NO-dependent manner. Mechanistically, acetate improves IL-1β production by bacteria-conditioned macrophages and the latter acts in an autocrine manner to promote NO production. Strikingly, acetate-triggered IL-1β production was neither dependent of its cell surface receptor free-fatty acid receptor 2, nor of the enzymes responsible for its metabolism, namely acetyl-CoA synthetases 1 and 2. We found that IL-1β production by acetate relies on NLRP3 inflammasome and activation of HIF-1α, the latter being triggered by enhanced glycolysis. In conclusion, we unravel a new mechanism through which acetate reinforces the bactericidal activity of alveolar macrophages.

Conjugated Metabolites of Hydroxytyrosol and Tyrosol Contribute to the Maintenance of Nitric Oxide Balance in Human Aortic Endothelial Cells at Physiologically Relevant Concentrations.

Nitric oxide (NO) is an important signaling molecule involved in many pathophysiological processes. NO mediates vasodilation and blood flow in the arteries, and its action contributes to maintaining vascular homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium. Dietary antioxidants and their metabolites have been found to be directly and/or indirectly involved in the modulation of the intracellular signals that lead to the production of NO. The purpose of this study was to investigate the contribution of conjugated metabolites of hydroxytyrosol (HT) and tyrosol (TYR) to the release of NO at the vascular level, and the related mechanism of action, in comparison to their parental forms. Experiments were performed in human aortic endothelial cells (HAEC) to evaluate the superoxide production, the release of NO and production of cyclic guanosine monophosphate (cGMP), the activation of serine/threonine-protein kinase 1 (Akt1), and the activation state of endothelial nitric oxide synthase (eNOS). It was observed that the tested phenolic compounds enhanced NO and cGMP concentration, inhibiting its depletion caused by superoxide overproduction. Moreover, some of them enhanced the activation of Akt (TYR, HT metabolites) and eNOS (HT, HVA, TYR-S, HT-3S). Overall, the obtained data showed that these compounds promote NO production and availability, suggesting that HT and TYR conjugated metabolites may contribute to the effects of parental extra virgin olive oil (EVOO) phenolics in the prevention of cardiovascular diseases.

Bai-Hu-Tang regulates endothelin-1 and its signalling pathway in vascular endothelial cells

Ethnopharmacological relevanceBai-Hu-Tang (BHT) is traditionally used to treat human and animal fever syndrome with four symptoms: large and vigorous pulse, large thirst, high sweat, and high heat. Aim of the studyTo investigate the mechanism of vasodilation regulation of Bai-Hu-Tang in primary vascular endothelial cells stimulated by lipopolysaccharide (LPS). Materials and methodsA hydrophilic concentrate of BHT was prepared, and the main components of mangiferin and timosaponin BⅡ were determined by HLPC analysis. The rabbit fever model was constructed by intravenous injection of LPS (15 μg/kg body weight), and BHT was gavaged to treat febrile rabbits. After treatment for 6 h, animal peripheral blood was collected, and serum was isolated for endothelin-1 (ET-1) and nitric oxide (NO) assays. Rabbit vascular endothelial cells (RVECs) were isolated and stimulated with 1 μg/mL LPS, and then inflammatory cells were treated with 125 or 250 μg/mL BHT for 24 h. The supernatant cytokines TNF-ɑ, IL-1β, IL-6, and ET-1 were detected by ELISA kits. Gene expression levels of endothelin receptor type B (ETB receptor) were analysed by real-time polymerase chain reaction (RT-PCR), and protein expression levels of PI3K and Akt were detected by Western blot. A nitrite assay was used to measure intracellular nitric oxide (NO) production, and nitric oxide synthase (NOS) was measured by the T-NOS colorimetric method. ResultsAnimal experiments demonstrated that BHT significantly restored ET-1 and NO in animal peripheral blood, which were disordered in LPS-induced fever rabbits. Moreover, a cytotoxicity assay demonstrated that BHT ≤700 μg/mL is innoxious to RVECs. BHT significantly repressed cellular TNF-α, IL-1β, and ET-1, which were originally elevated by LPS in RVECs. Meanwhile, BHT elevated the gene expression level of the ETB receptor and promoted NOS and NO production in RVECs induced by LPS. ConclusionBHT can inhibit excessive ET-1 secretion induced by LPS in vascular endothelial cells and activate the classic ET-1 signalling pathway to promote NO production, which may facilitate vasodilation of smooth muscle cells.

In This Issue

HomeCirculation ResearchVol. 129, No. 10In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published28 Oct 2021https://doi.org/10.1161/RES.0000000000000516Circulation Research. 2021;129:891is related toMacrophage MST1/2 Disruption Impairs Post-Infarction Cardiac Repair via LTB4Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NGChaperone-Mediated Autophagy of eNOS in Myocardial Ischemia-Reperfusion InjuryEfficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG (p 895)Ma et al perform base editing in mouse embryos to prevent hypertrophic cardiomyopathy.Download figureDownload PowerPointInherited hypertrophic cardiomyopathy (HCM), characterized by thickening and dysfunction of the heart muscle, is the most common genetic heart disorder. Mutations causing HCM often affect a single gene, such as the myosin heavy chain (MHC) gene, and as such have the potential to be corrected by gene editing style approaches. Not all editing techniques are the same, however, with some potentially introducing unwanted insertions or deletions of nucleotides (indels) at the target site. Ma and colleagues wondered whether base editing—in which a single nucleotide basepair is corrected and which typically doesn’t lead to indels—might be a safer approach. The team created a base-editor system to convert an HCM-causing A-T basepair into the wildtype G-C at a single point in the mouse Mhc6 gene. Transfection of the base-editing system into single-cell zygotes resulted in a correction rate of approximately 60-70 percent and, after transfer of the corrected embryos into surrogate females, prevented HCM in the postnatal animals. The team also performed base editing on mouse embryos in utero, achieving a 25 percent gene correction rate. The approach did not cause detectable indels or off-target editing, the team showed, suggesting it might have potential for development as a clinical tool.Macrophage MST1/2 Disruption Impairs Post-Infarction Cardiac Repair via LTB4 (p 909)Heart-repair via MST1 inhibition may be hindered by inflammatory effects, say Liu et al, who suggest a pharmacological work-around.Download figureDownload PowerPointAfter a myocardial infarction, the injured heart muscle is largely unable to regenerate and instead forms a dysfunctional scar that can ultimately lead to heart failure. Cardiomyocyte-specific inhibition of the kinase MST1—shown to boost repair and regeneration in other organs—can prevent infarction-induced death of these cells and preserve heart function, suggesting it may have clinical utility. However, MST1 also has anti-inflammatory properties in macrophages. Its inhibition in those cells may thus delay inflammation resolution after infarction and impair proper healing. Liu and colleagues have now examined mice lacking MST1 in macrophages and found that, sure enough, after myocardial infarction the inflammatory mediator leukotriene B4 was upregulated in macrophages and the animals’ heart function was reduced compared to that of wildtype controls. Importantly, blocking the action of leukotriene B4 in mice reduced infarction injuries in the hearts of MST1-lacking animals and enhanced repair in the injured hearts of wildtype animals given an MST1 inhibitor. The results suggest that, if MST1 inhibition is used as a future post-infarction regenerative therapy, then leukotriene B4 blockade may prevent its inflammatory side-effects.Chaperone-Mediated Autophagy of eNOS in Myocardial Ischemia-Reperfusion Injury (p 930)Maintaining nitric oxide synthase function after reperfusion injury protects the heart from damage, say Subramani et al.Download figureDownload PowerPointRestoring blood flow to ischemic heart muscle after a myocardial infarction is critical for salvaging muscle function, but can itself cause damage—so-called reperfusion injury. The generation of reactive oxygen species (ROS) and loss of nitric oxide (NO) both contribute to such injury, and the situation is exacerbated by the NO-producing enzyme, endothelial NO synthase (eNOS), producing damaging superoxide anions instead of NO. This switch in eNOS function is caused by glutathionylation of the enzyme (SG-eNOS), but how long the malfunction lasts and how it is rectified is unclear. Subramani and colleagues show that in human endothelial cells exposed to several hours of hypoxia followed by reoxygenation, the level of SG-eNOS steadily increases until, at 16 hours, it decreases sharply. By blocking several different cellular degradation pathways, the team discovered that this decrease in SG-eNOS was due to chaperone-mediated autophagy, with chaperone protein HSC70 being responsible for SG-eNOS destruction. Importantly, the team went on to show that pharmacological deglutathionylation of eNOS in mice promoted NO production and reduced reperfusion injury, suggesting this approach may be of clinical benefit after a myocardial infarction. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesMacrophage MST1/2 Disruption Impairs Post-Infarction Cardiac Repair via LTB4Mingming Liu, et al. Circulation Research. 2021;129:909-926Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NGShuhong Ma, et al. Circulation Research. 2021;129:895-908Chaperone-Mediated Autophagy of eNOS in Myocardial Ischemia-Reperfusion InjuryJaganathan Subramani, et al. Circulation Research. 2021;129:930-945 October 29, 2021Vol 129, Issue 10Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000516 Originally publishedOctober 28, 2021 PDF download Advertisement

Chaperone-Mediated Autophagy of eNOS in Myocardial Ischemia-Reperfusion Injury.

[Figure: see text].

Estrogen receptor (ESR1 and ESR2)-mediated activation of eNOS–NO–cGMP pathway facilitates high altitude acclimatization

Higher levels of circulatory nitric oxide (NO) and NO metabolites reportedly facilitate high altitude acclimatization. But the underlying factors and molecular pathways promoting NO production at high altitude has been poorly characterized. Studying healthy lowlanders at sea level (C, lowlander) and high altitude (3500 m, after day 1, 4 and 7 of ascent), we report higher protein levels of eNOS and eNOSSer1177, higher plasma levels of BH4, NOx (nitrate and nitrites), cGMP and lower levels of endogenous eNOS inhibitor ADMA during healthy high altitude acclimatization. Our qRT-PCR-based gene expression studies identified higher levels of eNOS/NOS3 mRNA along with several other eNOS pathway genes like CALM1, SLC7A1 and DNM2. In addition, we observed higher mRNA levels of estrogen (E2) receptors ERα/ESR1 and ERβ/ESR2 at high altitude that transcriptionally activates NOS3. We also observed higher mRNA level of membrane receptor ERBB2 that phosphorylates eNOS at Ser1177 and thus augments NO availability. Evaluating E2 biosynthesis at high altitude, we report higher plasma levels of CYP11A1, CYP19A1, E2, lower levels of testosterone (T) and T/E2 ratio as compared to sea level. Correlation studies revealed moderate positive correlation between E2 and NOx (R = 0.68, p = 0.02) after day 4 and cGMP (R = 0.69, p = 0.02) after day 7 at high altitude. These findings suggest a causative role of E2 and its receptors ESR1 and ESR2 in augmenting eNOS activity and NO availability during healthy high altitude ascent. These results will aid in better understanding of NO production during hypobaric hypoxia and help in designing better high altitude acclimatization protocols.

Identification of the MAPK Cascade and its Relationship with Nitrogen Metabolism in the Green Alga Chlamydomonas reinhardtii

The mitogen activated protein kinases (MAPKs) form part of a signaling cascade through phosphorylation reactions conserved in all eukaryotic organisms. The MAPK cascades are mainly composed by three proteins, MAPKKKs, MAPKKs and MAPKs. Some signals induce MAPKKK-mediated phosphorylation and activation of MAPKK that phosphorylate and activate MAPK. Afterward, MAPKs can act either in the cytoplasm or be imported into the nucleus to activate other proteins or transcription factors. In the green microalga Chlamydomonas reinhardtii the pathway for nitrogen (N) assimilation is well characterized, yet its regulation still has many unknown features. Nitric oxide (NO) is a fundamental signal molecule for N regulation, where nitrate reductase (NR) plays a central role in its synthesis. The MAPK cascades could be regulating N assimilation, since it has been described that the phosphorylation of NR by MAPK6 promotes NO production in Arabidopsis thaliana. We have identified the proteins involved in the MAPK cascades in Chlamydomonas reinhardtii, finding 17 MAPKs, 2 MAPKKs and 108 MAPKKKs (11 MEKK-, 94 RAF- and 3 ZIK-type) that have been structurally and phylogenetically characterized. The genetic expressions of MAPKs and the MAPKK were slightly regulated by N. However, the genetic expressions of MAPKKKs RAF14 and RAF79 showed a very strong repression by ammonium, which suggests that they may have a key role in the regulation of N assimilation, encouraging to further analyze in detail the role of MAPK cascades in the regulation of N metabolism.

Potential immune-modulatory effects of wheat phytase on the performance of a mouse macrophage cell line, Raw 264.7, exposed to long-chain inorganic polyphosphate.

ObjectiveThis experiment was conducted to find out the immunological effects of wheat phytase when long-chain inorganic polyphosphate (polyP) treated with wheat phytase was added to a macrophage cell line, Raw 264.7, when compared to intact long-chain polyP.MethodsNitric oxide (NO) production of Raw 264.7 cells exposed to P700, a long-chain polyP with an average of 1,150 phosphate residues, treated with or without wheat phytase, was measured by Griess method. Phagocytosis assay of P700 treated with or without phytase in Raw 264.7 cells was investigated using neutral red uptake. The secretion of tumor necrosis factor α (TNF-α) by Raw 264.7 cells with wheat phytase-treated P700 compared to intact P700 was observed by using Mouse TNF-α enzyme-linked immunosorbent assay kit.ResultsP700 treated with wheat phytase effectively increased NO production of Raw 264.7 cells by 172% when compared with intact P700 at 12 h exposure. At 5 mM of P700 concentration, wheat phytase promoted NO production of macrophages most strongly. P700, treated with wheat phytase, stimulated phagocytosis in macrophages at 12 h exposure by about 1.7-fold compared to intact P700. In addition, P700 treated with wheat phytase effectively increased in vitro phagocytic activity of Raw 264.7 cells at a concentration above 5 mM when compared to intact P700. P700 dephosphorylated by wheat phytase increased the release of TNF-α from Raw 264.7 cells by 143% over that from intact P700 after 6 h exposure. At the concentration of 50 μM P700, wheat phytase increased the secretion of cytokine, TNF-α, by 124% over that from intact P700.ConclusionIn animal husbandry, wheat phytase can mitigate the long-chain polyP causing damage by improving the immune capabilities of macrophages in the host. Thus, wheat phytase has potential as an immunological modulator and future feed additive for regulating immune responses caused by inflammation induced by long-chain polyP from bacterial infection.

Panax notoginseng saponins promotes cutaneous wound healing and suppresses scar formation in mice.

Panax notoginseng saponins are believed to promote wound healing due to its anti-proliferative effect on fibroblasts. The present work was therefore aimed to examine the beneficial effect of PNS on wound healing in vitro and in a murine model of cutaneous wound. The in vitro effects of Panax notoginseng saponins on the proliferation of and nitric oxide (NO) synthesis in human fibroblast 3T3 cells were studied. The in vivo effects of Panax notoginseng saponins were examined in C57 mice with dorsal cutaneous wound. The healing rate and scar formation were followed after treatment with Panax notoginseng saponins. The histology and fibroblast accumulation in the wounds were studied using hematoxylin and eosin (H&E) staining. Expression of α-smooth muscle actin (α-SMA) was examined by immunohistochemistry. Panax notoginseng saponins inhibited the proliferation of human fibroblast 3T3 with an EC50 of 1.825mM Panax notoginseng saponins (0.1mM) significantly promoted NO production (P<0.01) and NO synthase activity (P<0.01) of 3T3. In C57 mice with dorsal cutaneous wounds, 0.1mM Panax notoginseng saponins significant expedited wound healing by reducing the size of lesions and suppressing the formation of scar. H&E staining revealed that treatment with Panax notoginseng saponins suppressed fibroblast accumulation in wound areas, while immunohistochemistry showed a significant reduction in α-SMA expression by 0.1mM Panax notoginseng saponins. Panax notoginseng saponins are a promising drug candidate that can accelerate wound healing and reduce scar formation.

Fumarase Overexpression Abolishes Hypertension Attributable to endothelial NO synthase Haploinsufficiency in Dahl Salt-Sensitive Rats.

Human blood pressure salt sensitivity is associated with changes in urinary metabolites related to fumarase (Fh) and nitric oxide (NO) metabolism, and fumarase promotes NO production through an arginine regeneration pathway. We examined the role of the fumarase-NO pathway in the development of hypertension using genetically engineered rat models. Dahl salt-sensitive (SS) rats with heterozygous mutation of eNOS (endothelial NO synthase or Nos3; SS-Nos3+/-) were bred with SS rats with a hemizygous Fh transgene. SS-Nos3+/- rats without the Fh transgene (SS-Nos3+/-/Fh0/0) developed substantial hypertension with a mean arterial pressure of 134.2±3.7 mm Hg on a 0.4% NaCl diet and 178.0±3.5 mm Hg after 14 days on a 4% NaCl diet. Mean arterial pressure decreased remarkably to 123.1±1.4 mm Hg on 0.4% NaCl, and 143.3±1.5 mm Hg on 4% NaCl in SS-Nos3+/- rats with a Fh transgene (SS-Nos3+/-/Fh0/1), and proteinuria, renal fibrosis, and tubular casts were attenuated in SS-Nos3+/-/Fh0/1 rats compared with SS-Nos3+/-/Fh0/0 rats. eNOS protein abundance decreased in rats with the Nos3 heterozygous mutation, which was not influenced by Fh overexpression in rats on the 0.4% NaCl diet. However, the decrease in NO metabolite in the renal outer medulla of SS-Nos3+/-/Fh0/0 rats on the 0.4% NaCl diet was reversed in SS-Nos3+/-/Fh0/1 rats, and levels of L-arginine, but not the other 12 amino acids analyzed, were significantly higher in SS-Nos3+/-/Fh0/1 rats than in SS-Nos3+/+/Fh0/0 rats. In conclusion, fumarase has potent effects in restoring NO production and blunting the development of hypertension attributable to eNOS haploinsufficiency.

Recombinant LCAT (Lecithin:Cholesterol Acyltransferase) Rescues Defective HDL (High-Density Lipoprotein)-Mediated Endothelial Protection in Acute Coronary Syndrome.

Objective- Aim of this study was to evaluate changes in LCAT (lecithin:cholesterol acyltransferase) concentration and activity in patients with an acute coronary syndrome, to investigate if these changes are related to the compromised capacity of HDL (high-density lipoprotein) to promote endothelial nitric oxide (NO) production, and to assess if rhLCAT (recombinant human LCAT) can rescue the defective vasoprotective HDL function. Approach and Results- Thirty ST-segment-elevation myocardial infarction (STEMI) patients were enrolled, and plasma was collected at hospital admission, 48 and 72 hours thereafter, at hospital discharge, and at 30-day follow-up. Plasma LCAT concentration and activity were measured and related to the capacity of HDL to promote NO production in cultured endothelial cells. In vitro studies were performed in which STEMI patients' plasma was added with rhLCAT and HDL vasoprotective activity assessed by measuring NO production in endothelial cells. The plasma concentration of the LCAT enzyme significantly decreases during STEMI with a parallel significant reduction in LCAT activity. HDL isolated from STEMI patients progressively lose the capacity to promote NO production by endothelial cells, and the reduction is related to decreased LCAT concentration. In vitro incubation of STEMI patients' plasma with rhLCAT restores HDL ability to promote endothelial NO production, possibly related to significant modification in HDL phospholipid classes. Conclusions- Impairment of cholesterol esterification may be a major factor in the HDL dysfunction observed during acute coronary syndrome. rhLCAT is able to restore HDL-mediated NO production in vitro, suggesting LCAT as potential therapeutic target for restoring HDL functionality in acute coronary syndrome.

Fumarase Overexpression Abolishes Hypertension Attributable to eNOS Haploinsufficiency in Dahl Salt‐Sensitive Rats

BackgroundHuman blood pressure salt sensitivity is associated with changes in urinary metabolites related to fumarase (Fh) and nitric oxide (NO) metabolism, and fumarase promotes NO production through a malate‐aspartate‐arginine pathway.ObjectiveWe examined the role of the fumarase‐NO pathway in the development of hypertension in the genetic background of the Dahl salt‐sensitive (SS) rat, a model of human salt‐sensitive hypertension and renal injury used widely.Methods and ResultsSS rats with heterozygous mutation of endothelial NO synthase (eNOS or Nos3) (SS‐Nos3+/−) were bred with SS rats with a hemizygous Fh transgene. SS‐Nos3+/− rats without the Fh transgene (SS‐Nos3+/−/Fh0/0) developed substantial hypertension with a mean arterial pressure (MAP) of 138.5 ± 3.8 mmHg on a 0.4% NaCl diet, and 183.0 ± 3.7 mmHg after 14 days on a 4% NaCl diet. MAP decreased remarkably to 123.1 ± 1.4 mmHg on 0.4% NaCl, and 143.3 ± 1.5 mmHg on 4% NaCl in SS‐Nos3+/− rats with a Fh transgene (SS‐Nos3+/−/Fh0/1), and proteinuria, renal fibrosis, and tubular casts were attenuated in SS‐Nos3+/−/Fh0/1 rats compared to SS‐Nos3+/−/Fh0/0 rats. eNOS protein abundance decreased in rats with the Nos3 heterozygous mutation, which was not influenced by Fh overexpression. However, the decrease in NO metabolite in the renal outer medulla of SS‐Nos3+/−/Fh0/0 rats was reversed in SS‐Nos3+/−/Fh0/1 rats. Levels of L‐arginine, but not the other 12 amino acids analyzed, were significantly higher in SS‐Nos3+/−/Fh0/1 rats than in SS‐Nos3+/+/Fh0/0 rats.ConclusionsFumarase has potent effects in restoring NO production in renal outer medulla and blunting the development of hypertension attributable to eNOS haploinsufficiency.Support or Funding InformationThis work was supported by US National Institutes of Health grant HL116264‐5244 (M.L.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hydroxypropyltrimethyl ammonium chloride chitosan activates RAW 264.7 macrophages through the MAPK and JAK-STAT signaling pathways

Hydroxypropyltrimethyl ammonium chloride chitosan (HACC) is a water-soluble derivative of chitosan. To investigate the immunostimulatory effects of HACC, quaternized chitosans with different molecular weights were prepared and their effects on RAW 264.7 macrophages were compared. The results showed that HACC promoted nitric oxide (NO) production in a molecular weight- and dose-dependent manner. Lower molecular weight HACC was more active in promoting NO production. Furthermore, flow cytometry analysis showed that HACC significantly promoted the production of interleukin-6 and tumor necrosis factor-α. These results were further demonstrated by quantitive real-time reverse transcription polymerase chain reaction and western blot analysis. Moreover, western blotting revealed that HACC induced the phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38, and signal transducer and activator of transcription (STAT) proteins. In conclusion, HACC activated RAW 264.7 cells through the mitogen-activated protein kinases and Janus kinase/STAT pathways.

ROS and NO Dynamics in Endothelial Cells Exposed to Exercise-Induced Wall Shear Stress.

Intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels are associated with vascular homeostasis and diseases. Exercise can modulate ROS and NO production through increasing frequency and magnitude of wall shear stress (WSS). However, the details of ROS and NO production in endothelial cells and their interplay under WSS induced by exercise at different intensities remain unclear. In this study, we developed an in vitro multicomponent nonrectangular flow chamber system to simulate pulsatile WSS waveforms induced by moderate and high intensity exercise. Furthermore, the dynamic responses of ROS and NO in endothelial cells and the relationship between ROS and NO were investigated under the WSS induced by different intensity exercise. After exposing to WSS induced by moderate intensity exercise, endothelial cells produced more NO than those under high intensity exercise-induced WSS. In this process, ROS was found to play a dual role in the generation of intracellular NO. Under WSS induced by moderate intensity exercise, modest elevated ROS promoted NO production, whereas excessive ROS in endothelial cells exposed to WSS induced by high intensity exercise attenuated NO bioavailability. Interestingly, antioxidant N-acetylcysteine (NAC) could increase NO production under WSS induced by high intensity exercise. Our results provide some cues for selecting appropriate exercise intensities and elevating benefits of exercise on endothelial function. Additionally, owing to the consistency of our results and some in vivo phenomena, this flow chamber system may serve as an in vitro exercise model of arterial vessel for future studies.

Abstract 035: Recombinant LCAT Restores Defective HDL mediated Endothelial Protection in Acute Coronary Syndrome

Recent evidence suggests that the vasoprotective effects of HDL are impaired in patients during an acute coronary syndrome (ACS). In particular, HDL from ACS patients are defective in promoting nitric oxide (NO) release from cultured endothelial cells (ECs). Lecithin:cholesterol acyltransferase (LCAT) plays a key role in HDL maturation and remodeling, and it is the enzyme responsible for cholesterol esterification in plasma. Very few studies have suggested that LCAT activity is decreased during an acute myocardial infarction, but the possible link between LCAT mass/activity and the HDL dysfunction observed in ACS has never been tested. To test this hypothesis, plasma from 30 STEMI patients was collected at admission, 48 and 72 hours and 30 days after event. LCAT concentration and activity significantly decreased 48 hours after event (-0.49±0.11 μg/ml, P&lt;0.001, and -3.15±1.05 nmol/ml/h, P=0.006) and remained reduced at 72 hours. HDL isolated from STEMI patients lose the capacity to promote NO production in ECs (-0.13 ± 0.06 fold, P=0.04) and the reduction is significantly correlated to decreased LCAT activity (R= 0.52, P=0.003). In vitro studies were performed in which STEMI patients’ plasma was added with rhLCAT and HDL vasoprotective activity assessed by measuring NO production in ECs. In vitro incubation of STEMI patients’ plasma with rhLCAT remodels phospholipids and protein content in HDL and restores HDL ability to promote endothelial NO production (+25%, P=0.03). Impairment of cholesterol esterification may be a major factor in the HDL dysfunction observed during ACS. rhLCAT is able to restore HDL-mediated NO production in vitro, suggesting LCAT as potential therapeutic target for restoring HDL functionality in ACS.

Effect of asiaticoside on endothelial cells in hypoxia‑induced pulmonary hypertension.

Pulmonary hypertension (PH) is a chronic progressive disease with limited treatment options. The exact etiology and pathogenesis of PH remain to be elucidated, however there is novel evidence that implicates abnormal endothelial cells (ECs) apoptosis and dysfunction of ECs to be involved in the initiation of PH. Asiaticoside (AS) is a saponin monomer extracted from a medicinal plant called Centella asiatica, which had a preventing effect of hypoxia-induced pulmonary hypertension (hypoxic PH) by blocking transforming growth factor-β1/SMAD family member 2/3 signaling in our previous study. The present study demonstrated that AS can prevent the development of hypoxic PH and reverse the established hypoxic PH. AS may activate the nitric oxide (NO)-mediated signals by enhancing the phosphorylation of serine/threonine-specific protein kinase/eNOS, thus promoting NO production, and prevent ECs from hypoxia-induced apoptosis. All these findings imply that AS may be a potential therapeutic option for hypoxic PH patients due to its effect on the vitality and function of endothelial cells.