Synthase Knockout Mice Research Articles

The expression and function of glutamate aspartate transporters in Bergmann glia are decreased in neuronal nitric oxide synthase‐knockout mice during postnatal development

Bergmann glia (BG) predominantly use glutamate/aspartate transporters (GLAST) for glutamate uptake in the cerebellum. Recently, nitric oxide (NO) treatment has been shown to upregulate GLAST function and increase glutamate uptake in vitro. We previously discovered that neuronal nitric oxide synthase knockout (nNOS−/−) mice displayed structural and functional neuronal abnormalities in the cerebellum during development, in addition to previously reported motor deficits. Although these developmental deficits have been identified in the nNOS−/− cerebellum, it is unknown whether BG morphology and GLAST expression are also affected in the absence of nNOS in vivo. This study is the first to characterize BG morphology and GLAST expression during development in nNOS−/− mice using immunohistochemistry and western blotting across postnatal development. Results showed that BG in nNOS−/− mice exhibited abnormal morphology and decreased GLAST expression compared with wildtype (WT) mice across postnatal development. Treating ex vivo WT cerebellar slices with the NOS inhibitor L‐NAME decreased GLAST expression while treating nNOS−/− slices with the slow‐release NO‐donor NOC‐18 increased GLAST expression when compared with their respective controls. In addition, treating primary BG isolated from WT mice with the selective nNOS inhibitor 7N decreased the membrane expression of GLAST and influx of Ca2+/Na+, while treating nNOS−/− BG with SNAP increased the membrane expression of GLAST and Ca2+/Na+ influx. Moreover, the effects of SNAP on GLAST expression and Ca2+/Na+ influx in nNOS−/− BG were significantly reduced by a PKG inhibitor. Together, these results reveal a novel role for nNOS/NO signaling in BG development, regulated by a PKG‐mediated mechanism.

Lipocalin-type Prostaglandin D2 Synthase appears to function as a Novel Adipokine Preventing Adipose Dysfunction in response to a High Fat Diet

Adipose dysfunction is the primary defect in obesity that contributes to the development of dyslipidemia, insulin resistance, cardiovascular diseases, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) and some cancers. Previously, we demonstrated the development of NAFLD in lipocalin-type prostaglandin D2 synthase (L-PGDS) knockout mice regardless of diet. In the present study, we examined the role of L-PGDS in adipose in response to a high fat diet. We observed decreased expression of L-PGDS in adipose tissue and concomitant lower plasma levels in a dietary model of obesity as well as in insulin resistant 3T3-L1 adipocytes. We show reduced adiponectin expression and phosphorylation of AMPK in white adipose tissue of L-PGDS KO mice after 14 weeks on a high fat diet as compared to control C57BL/6 mice. We also observe an increased fat content in L-PGDS KO mice as demonstrated by adipocyte hypertrophy and increased expression of lipogenenic genes. We confirmed our in vivo findings in in vitro 3T3-L1 adipocytes, using an enzymatic inhibitor of L-PGDS (AT56). Rosiglitazone treatment drastically increased L-PGDS expression in insulin resistant 3T3-L1 adipocytes and increased adiponectin expression and AMPK phosphorylation in AT56 treated 3T3-L1 adipocytes. We conclude that the absence of L-PGDS has a deleterious effect on adipose tissue functioning, which further reduces insulin sensitivity in adipose tissue. Consequently, we propose L-PGDS appears to function as a potential member of the adipokine secretome involved in the regulation of the obesity-associated metabolic syndrome.

St8sia1-deficiency in mice alters tumor environments of gliomas, leading to reduced disease severity.

ABSTRACTGanglioside GD3/GD2 are over-expressed in various neuroectoderm-derived tumors. Previous studies indicated that GD3 is involved in the enhancement of cancer properties such as rapid growth and increased invasiveness. However, little is known about the functions of GD3/GD2 in glioma cells and glioma microenvironments. To clarify the functions of GD3/GD2 in gliomas, we used a mouse glioma model based on the RCAS/Gtv-a system. At first, we compared the gliomas size between wild-type (WT) and GD3 synthase (GD3S) knockout (KO) mice, showing a less malignant histology and slower tumor growth in GD3S-KO mice than in WT mice. Immunohistochemistry of glioma sections from WT and GD3S-KO mice revealed that reactive microglia/macrophages showed different localization patterns between the two genetic types of mice. CD68+ cells were more frequently stained inside glioma tissues of GD3S-KO mice, while they were stained mainly around glioma tissues in WT mice. The number of CD68+ cells markedly increased in tumor tissues of GD3S-KO mice at 2 weeks after injection of transfectant DF-1 cells. Furthermore, CD68+ cells in GD3S(-/-) glioma tissues expressed higher levels of inducible nitric oxide synthase. We observed higher expression levels of pro-inflammatory cytokine genes in primary-cultured glioma cells of WT mice than in GD3S-KO mice. DNA microarray data also revealed differential expression levels of various cytokines and chemokines in glioma tissues between WT and GD3S-KO mice. These results suggest that expression of GD3S allows glioma cells to promote polarization of microglia/macrophages towards M2-like phenotypes by modulating the expression levels of chemokines and cytokines.

GD3 synthase deletion alters retinal structure and impairs visual function in mice.

Gangliosides are glycosphingolipids abundantly expressed in the vertebrate nervous system, and are classified into a-, b-, or c-series according to the number of sialic acid residues. The enzyme GD3 synthase converts GM3 (an a-series ganglioside) into GD3, a b-series ganglioside highly expressed in the developing and adult retina. The present study evaluated the visual system of GD3 synthase knockout mice (GD3s-/- ), morphologically and functionally. The absence of b- series gangliosides in the retinas of knockout animals was confirmed by mass spectrometry imaging, which also indicated an accumulation of a-series gangliosides, such as GM3. Retinal ganglion cell (RGC) density was significantly reduced in GD3s-/- mice, with a similar reduction in the number of axons in the optic nerve. Knockout animals also showed a 15% reduction in the number of photoreceptor nuclei, but no difference in the bipolar cells. The area occupied by GFAP-positive glial cells was smaller in GD3s-/- retinas, but the number of microglial cells/macrophages did not change. In addition to the morphological alterations, a 30% reduction in light responsiveness was detected through quantification of pS6-expressing RGC, an indicator of neural activity. Furthermore, electroretinography (ERG) indicated a significant reduction in RGC and photoreceptor electrical activity in GD3s-/- mice, as indicated by scotopic ERG and pattern ERG (PERG) amplitudes. Finally, evaluation of the optomotor response demonstrated that GD3s-/- mice have reduced visual acuity and contrast sensitivity. These results suggest that b-series gangliosides play a critical role in regulating the structure and function of the mouse visual system.

Involvement of 3',5'-cyclic inosine monophosphate in cystathionine γ-lyase-dependent regulation of the vascular tone.

Background and Purposel‐cysteine or hydrogen sulfide (H2S) donors induce a biphasic effect on precontracted isolated vessels. The contractile effect occurs within a concentration range of 10 nM to 3 μM followed by vasodilatation at 30–100 μM. Here, we have investigated the signalling involved in the H2S‐induced contraction.Experimental ApproachVascular response to NaHS or l‐cysteine is evaluated on isolated precontracted with phenylephrine vessel rings harvested from wild type, cystathionine γ‐lyase (CSE−/−), soluble guanylyl cyclase (sGCα1 −/−) and endothelial nitric oxide synthase (eNOS−/−) knock‐out mice. The cAMP, cGMP and inosine 3′,5′‐cyclic monophosphate (cIMP) levels are simultaneously quantified using ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) analysis. The involvement of sGC, phosphodiesterase (PDE) 4A and PDE5 are also evaluated.Key ResultsCSE‐derived H2S‐induced contraction requires an intact eNOS/NO/sGC pathway and involves cIMP as a second messenger. H2S contractile effect involves a transient increase of cGMP and cAMP metabolism caused by PDE5 and PDE4A, thus unmasking cIMP contracting action. The stable cell‐permeable analogue of cIMP elicits concentration‐dependent contraction on a stable background tone induced by phenylephrine. The lack of cIMP, coupled to the hypocontractility displayed by vessels harvested from CSE−/− mice, confirms that H2S‐induced contraction involves cIMP.Conclusion and ImplicationsThe endothelium dynamically regulates vessel homeostasis by modulating contractile tone. This also involves CSE‐derived H2S that is mediated by cIMP.

No evidence of the unfolded protein response in the placenta of two rodent models of preeclampsia and intrauterine growth restriction.

In humans, intrauterine growth restriction (IUGR) and preeclampsia (PE) are associated with induction of the unfolded protein response (UPR) and increased placental endoplasmic reticulum (ER) stress. Especially in PE, oxidative stress occurs relative to the severity of maternal vascular underperfusion (MVU) of the placental bed. On the premise that understanding the mechanisms of placental dysfunction could lead to targeted therapeutic options for human IUGR and PE, we investigated the roles of the placental UPR and oxidative stress in two rodent models of these human gestational pathologies. We employed a rat IUGR model of gestational maternal protein restriction, as well as an endothelial nitric oxide synthase knockout mouse model (eNOS-/-) of PE/IUGR. Placental expression of UPR members was analyzed via qRT-PCR (Grp78, Calnexin, Perk, Chop, Atf6, and Ern1), immunohistochemistry, and Western blotting (Calnexin, ATF6, GRP78, CHOP, phospho-eIF2α, and phospho-IRE1). Oxidative stress was determined via Western blotting (3-nitrotyrosine and 4-hydroxy-2-nonenal). Both animal models showed a significant reduction of fetal and placental weight. These effects did not induce placental UPR. In contrast to human data, results from our rodent models suggest retention of placental plasticity in the setting of ER stress under an adverse gestational environment. Oxidative stress was significantly increased only in female IUGR rat placentas, suggesting a sexually dimorphic response to maternal malnutrition. Our study advances understanding of the involvement of the placental UPR in IUGR and PE. Moreover, it emphasizes the appropriate choice of animal models researching various aspects of these pregnancy complications.

Evaluation of Neutrophil Dynamics Change by Protective Effect of Tadalafil After Renal Ischemia/Reperfusion Using In Vivo Real-time Imaging.

Neutrophils play a major role in ischemia/reperfusion injury (IRI) in renal transplantation and acute kidney injury. However, it has been difficult to observe changes in neutrophil dynamics over time in living mice kidney. We investigate neutrophil dynamics in IRI in living mice using novel in vivo multiphoton microscope imaging techniques and characterize the renoprotective effects of a selective phosphodiesterase 5 inhibitor, tadalafil. Wild-type and endothelial nitric oxide synthase knockout mice, a model of endothelial dysfunction, were used to establish in vivo real-time imaging in living mouse kidneys. Neutrophils were labeled green with Ly-6G monoclonal antibody, and plasma flow was labeled red with BSA. Tadalafil was administered orally 1 h before surgery. Both kidney pedicles were reperfused after 37°C warm ischemia for 45 min. Our novel approach revealed that neutrophils were trapped in glomerulus within a few minutes after reperfusion. They gradually increased over time and infiltrated neutrophils were observed in the tubular lumen and peritubular capillary. The neutrophils were clearly visualized rolling on peritubular capillary plexus at 3 μm/min. The administration of tadalafil significantly reduced neutrophil influx into the glomerulus in both wild-type and endothelial nitric oxide synthase knockout mice. Reduced neutrophil infiltration in tadalafil groups, which was confirmed by flow cytometry, resulted in histopathologically decreased tubular injury. The expression of vascular cell adhesion molecule 1 and kidney injury molecule 1 was partially prevented by tadalafil. Use of a novel technique contributed to elucidation of neutrophil dynamics after reperfusion. Tadalafil has a potential for inhibiting neutrophil infiltration in renal IRI.

Molecular Mechanisms of Adenosine Stress T1 Mapping.

Adenosine stress T1 mapping is an emerging magnetic resonance imaging method to investigate coronary vascular function and myocardial ischemia without application of a contrast agent. Using gene-modified mice and 2 vasodilators, we elucidated and compared the mechanisms of adenosine myocardial perfusion imaging and adenosine T1 mapping. Wild-type (WT), A2AAR-/- (adenosine A2A receptor knockout), A2BAR-/- (adenosine A2B receptor knockout), A3AR-/- (adenosine A3 receptor knockout), and eNOS-/- (endothelial nitric oxide synthase knockout) mice underwent rest and stress perfusion magnetic resonance imaging (n=8) and T1 mapping (n=10) using either adenosine, regadenoson (a selective A2AAR agonist), or saline. Myocardial blood flow and T1 were computed from perfusion imaging and T1 mapping, respectively, at rest and stress to assess myocardial perfusion reserve and T1 reactivity (ΔT1). Changes in heart rate for each stress agent were also calculated. Two-way ANOVA was used to detect differences in each parameter between the different groups of mice. Myocardial perfusion reserve was significantly reduced only in A2AAR-/- compared to WT mice using adenosine (1.06±0.16 versus 2.03±0.52, P<0.05) and regadenoson (0.98±026 versus 2.13±0.75, P<0.05). In contrast, adenosine ΔT1 was reduced compared with WT mice (3.88±1.58) in both A2AAR-/- (1.63±1.32, P<0.05) and A2BAR-/- (1.55±1.35, P<0.05). Furthermore, adenosine ΔT1 was halved in eNOS-/- (1.76±1.46, P<0.05) versus WT mice. Regadenoson ΔT1 was approximately half of adenosine ΔT1 in WT mice (1.97±1.50, P<0.05), and additionally, it was significantly reduced in eNOS-/- mice (-0.22±1.46, P<0.05). Lastly, changes in heart rate was 2× greater using regadenoson versus adenosine in all groups except A2AAR-/-, where heart rate remained constant. The major findings are that (1) although adenosine myocardial perfusion reserve is mediated through the A2A receptor, adenosine ΔT1 is mediated through the A2A and A2B receptors, (2) adenosine myocardial perfusion reserve is endothelial independent while adenosine ΔT1 is partially endothelial dependent, and (3) ΔT1 mediated through the A2A receptor is endothelial dependent while ΔT1 mediated through the A2B receptor is endothelial independent.

Insulin-like growth factor 1 signaling in the placenta requires endothelial nitric oxide synthase to support trophoblast function and normal fetal growth.

Currently, there is no effective treatment for placental dysfunction in utero. In a ligated mouse model of fetal growth restriction (FGR), nanoparticle-mediated human insulin-like 1 growth factor (hIGF1) gene delivery (NP-Plac1-hIGF1) increased hIGF1 expression and maintained fetal growth. However, whether it can restore fetal growth remains to be determined. Using the endothelial nitric oxide synthase knockout (eNOS-/-) mouse model, a genetic model of FGR, we found that despite inducing expression of hIGF1 in the placentas treated with NP-Plac1-hIGF1 (P = 0.0425), FGR did not resolve. This was associated with no change to the number of fetal capillaries in the placental labyrinth; an outcome which was increased with NP-Plac1-hIGF1 treatment in the ligated mouse model, despite increased expression of angiopoietin 1 (P = 0.05), and suggested IGF1 signaling in the placenta requires eNOS to modulate placenta angiogenesis. To further assess this hypothesis, BeWo choriocarcinoma cell line and human placental explant cultures were treated with NP-Plac1-hIGF1, oxidative stress was induced with hydrogen peroxide (H2O2), and NOS activity was inhibited using the inhibitor NG-monomethyl-l-arginine (l-NMMA). In both BeWo cells and explants, the protective effect of NP-Plac1-hIGF1 treatment against H2O2-induced cell death/lactate dehydrogenase release was prevented by eNOS inhibition (P = 0.003 and P < 0.0001, respectively). This was associated with an increase in mRNA expression of oxidative stress markers hypoxia inducing factor 1α (HIF1α; P < 0.0001) and ADAM10 (P = 0.0002) in the NP-Plac1-hIGF1 + H2O2 + l-NMMA-treated BeWo cells. These findings show for the first time the requirement of eNOS/NOS in IGF1 signaling in placenta cells that may have implications for placental angiogenesis and fetal growth.

Nitric oxide attenuates microglia proliferation by sequentially facilitating calcium influx through TRPV2 channels, activating NFATC2, and increasing p21 transcription

ABSTRACT Microglia proliferation is critical for proper development and function of the central nervous system (CNS), while dysregulation of proliferation contributes to pathology. We recently reported that male inducible nitric oxide synthase knockout (iNOS−/−) mice displayed significantly more proliferating microglia in their postnatal cortex than age-matched wildtype (WT) male mice. Moreover, nitric oxide (NO) signaling in mouse microglia greatly upregulates calcium entry through transient receptor potential vanilloid type 2 (TRPV2) channels. Considering that TRPV2 activity restricts astrocytic proliferation within glioma tissues, we investigated the roles of iNOS/NO signaling and TRPV2 expression in the regulation of microglial proliferation in vitro using assays of calcium imaging, immunocytochemistry, western blot, and polymerase chain reaction. Results showed that non-dividing microglia exhibited substantially higher expression of TRPV2 on the plasma membrane and significantly larger calcium influx through TRPV2 channels in comparison to dividing microglia. Additionally, non-dividing WT microglia exhibited significantly more NO production than dividing WT microglia. Furthermore, the NO-donor NOC18 increased the nuclear translocation of nuclear factor of activated T-cells cytoplasmic 2 (NFATC2) and the mRNA of the cyclin-dependent kinase inhibitor p21 and decreased the percentage of dividing WT and iNOS–/– microglia in culture. Importantly, the presence of the TRPV2 inhibitor tranilast abolished these effects of NOC18. Together, results from this study indicated that iNOS/NO signaling inhibits microglial proliferation through TRPV2-mediated calcium influx, nuclear translocation of the transcription factor NFATC2, and p21 expression. Proposed molecular mechanism by which NO-TRPV2 signaling regulates microglia proliferation in vitro.

High-salt intake reduces renal tissue levels of inflammatory cytokines in mice.

High salt (HS) intake is usually considered as an aggravating factor to induce inflammatory renal injury. However, the changes in the renal levels of inflammatory cytokines during HS intake is not yet clearly defined. We hypothesize that HS increases renal levels of tumor necrosis factor‐alpha (TNF‐α) and interleukin‐6 (IL‐6) but decreases interleukin‐10 (IL‐10; anti‐inflammatory cytokine) and these responses exacerbate in NO deficient conditions. Both wild‐type (WT) and endothelial NO synthase knockout (eNOSKO) mice (~8 weeks old, n = 6 in each group) were given normal‐salt (NS; 0.3% NaCl) and HS (4% NaCl) containing diets for 2 weeks. Systolic blood pressure (SBP) was determined by tail‐cuff plethysmography and urine collections were made using metabolic cages. Basal SBP was higher in eNOSKO than WT mice (131 ± 7 vs 117 ± 3 mmHg; p < .05). HS intake for 2 weeks increased SBP in eNOSKO (161 ± 5 mmHg) but not in WT mice. In NS groups, the cytokine levels in renal tissues (measured using ELISA kits and expressed in pg/mg protein) were significantly higher in eNOSKO than WT mice (TNF‐α, 624 ± 67 vs. 325 ± 73; IL‐6, 619 ± 106 vs. 166 ± 61; IL‐10, 6,087 ± 567 vs. 3,929 ± 378). Interestingly, these cytokine levels in HS groups were significantly less both in WT (TNF‐α, 114 ± 17; IL‐6, 81 ± 14; IL‐10, 865 ± 130) and eNOSKO (TNF‐α, 115 ± 18; IL‐6, 56 ± 7; IL‐10, 882 ± 141) mice. These findings indicate that HS induces downregulation of cytokines in the kidney. Such HS‐induced reduction in cytokines, particularly TNF‐α (a natriuretic agent), would facilitate more salt‐retention, and thus, leading to salt‐sensitive hypertension in NO deficient conditions.

Inflammation Precedes Fat Deposition in an Experimental Model of Lymphedema.

Background: Chronic lymphedema is a common complication of lymphatic obstruction, particularly after cancer treatment, characterized by an increased volume of the affected extremity, partly caused by the accumulation of excessive adipose tissue. The relationship between lymph vessels' obstruction and fat deposit is, however, poorly understood. Objective: Our central hypothesis was that the inflammatory process caused by lymph stasis precedes the adipocyte differentiation and fat deposition. Methods and Results: We used a modified mouse tail model to produce secondary lymphedema. Animals were treated with dexamethasone, or the procedure was performed in nitric oxide synthase 2 (NOS2)-deficient mice to evaluate the role of inflammation in lymphedema formation. Adipose tissue (Lipin) and inflammatory markers (IL-6, MCP-1, and F4-80) were analyzed in histological samples and by quantitative polymerase chain reaction. We observed an increased deposition of fat into the affected area that starts 3 weeks after lymph vessel ligation; it further increased after 6 weeks. Genes involved in the inflammatory process were upregulated before adipocyte maturation. Treatment with dexamethasone or the use of inducible nitric oxide synthase knockout mice blocked the inflammatory reaction and inhibited the accumulation of fat distal to the lymphatic obstruction. Conclusion: In the modified mouse tail lymphedema, inflammation precedes adipogenesis. Our data suggest that MCP-1 and nitric oxide may be potential targets for lymphedema management.

Grape Seed Extract Polyphenols Improve Resistance Artery Function in Pregnant eNOS-/- Mice.

Hypertension during pregnancy is a leading cause of maternal and fetal morbidity and mortality worldwide, increasing the risk of complications including preeclampsia, intracerebral hemorrhage and fetal growth restriction. Increased oxidative stress is known to contribute to poor vascular function; however, trials of antioxidant supplementation have raised concerns about fetal outcomes, including risk of low birthweight. Grape seed extract polyphenols (GSEP) have been suggested to promote cardiovascular protection, at least in part through antioxidant actions. We tested the hypothesis that administration of GSEP during pregnancy would reduce oxidative stress and improve resistance artery function with no detrimental effects on fetal growth, in an established model of maternal hypertension associated with vascular dysfunction, the endothelial NO synthase knockout (eNOS–/–) mouse. Pregnant C57BL/6J (WT) and eNOS–/– mice received either GSEP (200 mg/kg/day) or drinking water, between gestational (GD) day 10.5 and GD18.5. At GD17.5, maternal systolic blood pressure (SBP) was measured; at GD18.5, maternal malondialdehyde (MDA) concentrations, vascular function of aortic, mesenteric, uterine and posterior cerebral arteries was assessed, and fetal outcome evaluated. GSEP reduced maternal SBP (P < 0.01) and plasma MDA concentrations (P < 0.01) in eNOS–/– mice. Whilst there was no effect of GSEP on vascular reactivity of aortas, GSEP improved endothelial-dependent relaxation in mesenteric and uterine arteries of eNOS–/– mice (P < 0.05 and P < 0.001, respectively) and normalized lumen diameters of pressurized posterior cerebral arteries in eNOS–/– mice (P < 0.001). Supplementation with GSEP had no effect in WT mice and did not affect fetal outcomes in either genotype. Our data suggest that GSEP improve resistance artery function, potentially through antioxidant actions, and provide a basis to further investigate these beneficial effects including in the prevention of intracerebral hemorrhage. Maternal supplementation with GSEP may be a safe intervention to improve outcomes in pregnancies associated with hypertension and vascular dysfunction.

Clinically relevant high levels of human C-reactive protein induces endothelial dysfunction and hypertension by inhibiting the AMPK-eNOS axis.

Successful treatment of resistant hypertension accompanied by elevated human C-reactive protein (hCRP) remains a key challenge in reducing the burden of cardiovascular diseases. It is still unclear whether clinically relevant high-level hCRP is merely a marker or a key driver of hypertension. Here, we investigated the role and mechanism of clinically relevant high level of hCRP in hypertension. Elevated blood pressure was observed in all three hCRP overexpression models, including adeno-associated virus 9 (AAV9)-transfected mice, AAV9-transfected rats and hCRP transgenic (hCRPtg) rats. hCRPtg rats expressing clinically relevant high-level hCRP developed spontaneous hypertension, cardiac hypertrophy, myocardial fibrosis and impaired endothelium-dependent relaxation. Mechanistically, studies in endothelial nitric oxide (NO) synthase (eNOS) knockout mice transfected with AAV9-hCRP and phosphoproteomics analysis of hCRP-treated endothelial cells revealed that hCRP inhibited AMP-activated protein kinase (AMPK)-eNOS phosphorylation pathway. Further, activation of AMPK by metformin normalized endothelial-dependent vasodilation and decreased the blood pressure of hCRPtg rats. Our results show that clinically relevant high-level hCRP induces hypertension and endothelial dysfunction by inhibiting AMPK-eNOS signaling, and highlight hCRP is not only an inflammatory biomarker but also a driver of hypertension. Treatment with metformin or a synthetic AMPK activator may be a potential strategy for vaso-dysfunction and hypertension in patients with high hCRP levels.

Beetroot juice lowers blood pressure and improves endothelial function in pregnant eNOS-/- mice: importance of nitrate-independent effects.

Maternal hypertension is associated with increased rates of pregnancy pathologies, including fetal growth restriction, due at least in part to reductions in nitric oxide (NO) bioavailability and associated vascular dysfunction. Dietary nitrate supplementation, from beetroot juice (BRJ), has been shown to increase NO bioavailability and improve cardiovascular function in both preclinical and clinical studies. This study is the first to investigate effects of dietary nitrate supplementation in a pregnant animal model. Importantly, the effects of nitrate-containing BRJ were compared with both 'placebo' (nitrate-depleted) BRJ as well as water to control for potential nitrate-independent effects. Our data show novel, nitrate-independent effects of BRJ to lower blood pressure and improve vascular function in endothelial nitric oxide synthase knockout (eNOS-/- ) mice. These findings suggest potential beneficial effects of BRJ supplementation in pregnancy, and emphasize the importance of accounting for nitrate-independent effects of BRJ in study design and interpretation. Maternal hypertension is associated with adverse pregnancy outcomes, including fetal growth restriction (FGR), due in part to reductions in nitric oxide (NO) bioavailability. We hypothesized that maternal dietary nitrate administration would increase NO bioavailability to reduce systolic blood pressure (SBP), improve vascular function and increase fetal growth in pregnant endothelial NO synthase knockout (eNOS-/- ) mice, which exhibit hypertension, endothelial dysfunction and FGR. Pregnant wildtype (WT) and eNOS-/- mice were supplemented with nitrate-containing beetroot juice (BRJ+) from gestational day (GD) 12.5. Control mice received an equivalent dose of nitrate-depleted BRJ (BRJ-) or normal drinking water. At GD17.5, maternal SBP was measured; at GD18.5, maternal nitrate/nitrite concentrations, uterine artery (UtA) blood flow and endothelial function were assessed, and pregnancy outcomes were determined. Plasma nitrate concentrations were increased in both WT and eNOS-/- mice supplemented with BRJ+ (P < 0.001), whereas nitrite concentrations were increased only in eNOS-/- mice (P < 0.001). BRJ- did not alter nitrate/nitrite concentrations. SBP was lowered and UtA endothelial function was enhanced in eNOS-/- mice supplemented with either BRJ+ or BRJ-, indicating nitrate-independent effects of BRJ. Improvements in endothelial function in eNOS-/- mice were abrogated in the presence of 25 mm KCl, implicating enhanced EDH signalling in BRJ- treated animals. At GD18.5, eNOS-/- fetuses were significantly smaller than WT animals (P < 0.001), but BRJ supplementation did not affect fetal weight. BRJ may be a beneficial intervention in pregnancies associated with hypertension, endothelial dysfunction and reduced NO bioavailability. Our data showing biological effects of non-nitrate components of BRJ have implications for both interpretation of previous findings and in the design of future clinical trials.

Pleiotropic Activation of Endothelial Function by Angiotensin II Receptor Blockers is Crucial to Their Protective Anti‐vascular Remodeling Effects

RationaleEndothelial function is strongly associated with vascular health and homeostasis. However, therapeutically relevant approaches capable of stimulating the protective properties of the endothelium remain elusive.ObjectiveTo determine whether modulation of the Angiotensin II (AngII) system with Angiotensin receptor blockers (ARBs) can improve endothelial function and promote downstream vascular homeostasis.Methods and ResultsWe show that all 4 ARBs tested (losartan, telmisartan, olmesartan, and valsartan) can promote robust chronic activation of endothelial nitric oxide (NO) in mice. With telmisartan, a well‐known pleiotropic ARB, in vivo treatment lead to a 58% inhibition of vascular contractility after two weeks of treatment and up to 77% at 6 months, in a fully L‐NAME sensitive fashion. Endothelial NO synthase (eNOS) knockout (KO) mice showed complete resistance against the endothelial function effects of telmisartan, whereas the Angiotensin converting enzyme inhibitor (ACEi) captopril showed little to no endothelial function activity at a similar blood pressure (BP) lowering dose. In a myograph chamber, direct stimulation of aortic vessels with telmisartan in the absence of AngII stimulated endothelial function, confirming pleiotropism. Moreover, in vitro stimulation of cultured endothelial cells caused rapid intracellular Calcium ([Ca2+]i) mobilization, which is a known upstream event of eNOS activation. In vivo, telmisartan completely abolished aortic wall remodeling in aging animals as well as a well‐established mouse model of Marfan syndrome (MFS), which causes accelerated and pre‐mature vascular aging and remodeling of the aortic root. In addition, in vivo inhibition of NOS activity with L‐NAME rendered telmisartan inactive in a BP‐independent manner.ConclusionARBs have unique and robust endothelial function activation properties independent of their inhibitory effects on the AngII system. Direct stimulation of the endothelial NO system with ARBs results in vascular protection and homeostasis independent of BP lowering effects, which supports a broader prophylactic use of this class of medication.Support or Funding InformationSupported by CIHR, HSFC, BCKDF, Marfan Foundation, SPH Foundation and Rare Disease Foundation.

Kruppel‐like Factor 4 is Critical to the Maintenance of Glomerular Endothelial Integrity

BackgroundThrombotic microangiopathy (TMA) represents the most severe manifestation of glomerular endothelial injury consisting of endothelial cell (EC) swelling, subendothelial expansion, inflammation and microthrombi. TMA occurs subsequent to various etiologies and is associated with dysregulation of thrombotic and inflammatory transcripts and complement activation. Expression of membrane bound (CD55, CD59, CD46) complement regulators, is altered in many subtypes of TMA and it is unclear if this is a driver or consequence of EC injury. To investigate transcriptional mediators involved in TMA, we reviewed expression arrays of both global and single cell RNA‐seq from human kidney biopsies with complement mediated renal microvascular injury and observed that Krüppel‐Like Factor 4 (KLF4), a zinc finger transcription factor, is both differentially expressed in ECs and predicted to regulate other differentially expressed genes. Previous studies demonstrate that KLF4 is a critical mediator of anti‐thrombotic and anti‐inflammatory phenotype in systemic vascular beds. We aimed to investigate its role in modulating renal microvascular injury and complement activation in TMA.MethodsEndothelial‐specific Klf4 knockout mice (Klf4ΔEC) were generated by crossing Klf4fl/fl mice withCdh5‐Cre mice. As two separate models of endothelial dysfunction, Klf4ΔEC mice were both aged to 1 year and crossed with Nos3−/− mice to generate double knockout mice (DKO). Periodic acid‐Schiff, electron microscopy, immunofluorescence, ELISA and RT‐PCR were performed to investigate the effects of endothelial Klf4 on the renal microvasculature. Renal histology and ultrastructure were evaluated, blinded, by renal pathology (MPR).ResultsAt 12 weeks of age, while no overt histologic changes were observed, Klf4ΔEC mice exhibited increased glomerular Vcam‐1 and Pai‐1 expression and complement activation (increased C3 and C5b‐9 deposition) as compared to Klf4fl/fl mice. To investigate the basis for this increased complement activation, we measured the expression of the membrane‐bound complement regulators and noted reduced glomerular Cd55 and Cd59 expression in Klf4ΔEC as compared with Klf4fl/fl mice. Interestingly, 1‐year‐old Klf4ΔEC mice exhibited pathology consistent with subacute TMA (glomerular capillary congestion, EC swelling, subendothelial expansion and mesangiolysis as well as increased intraglomerular von Willebrand Factor and albuminuria) as compared to age‐matched Klf4fl/fl mice. In addition, Klf4ΔEC mice crossed with nitric oxide synthase knockout (Nos3−/−) mice also exhibited increased glomerular complement activation and histological and ultrastructural features consistent with subacute TMA (loss of fenestrations, glomerular capillary congestion, tuft collapse with basement membrane wrinkling) as compared to all other groups (12 weeks). Finally, knockdown of KLF4 in cultured human microvascular endothelial cells reduced the expression of CD55, whereas induction of KLF4 increased CD55 expression in HMEC‐1s.ConclusionsLoss of endothelial Klf4 in both aged mice and in Nos3−/− mice recapitulates many pathologic features of renal‐TMA, including complement activation and is potentially mediated through its regulation of Cd55.Support or Funding InformationNIDDK

Lipocalin-type prostaglandin D2 synthase deletion induces dyslipidemia and non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is an emerging risk factor for type 2 diabetes mellitus, cardiovascular disease, and all-cause mortality. Previously, we demonstrated that lipocalin-type prostaglandin D2 synthase (L-PGDS) knockout mice show increased glucose intolerance and accelerated atherosclerosis. In the present study, we investigated the role of L-PGDS in mediating NAFLD utilizing L-PGDS knockout (KO) and control C57BL/6 mice fed either low fat (LFD) or high fat diet (HFD) for 14 weeks. Our present study demonstrates that L-PGDS KO mice remain slightly lighter in weight compared to control mice, yet develop NAFLD faster and eventually progress to the more severe non-alcoholic steatohepatitis (NASH). We found increased lipid accumulation in the liver of KO mice over time on both diets, as compared to control mice. The L-PGDS KO mice showed elevated fasting glucose and insulin levels and developed insulin resistance on both LFD and HFD. Lipogenesis marker proteins such as SREBP-1c and LXRα were increased in L-PGDS KO mice after 14 weeks on both diets, when compared to control mice. We replicated our in vivo findings in vitro using HepG2 cells treated with a combination of free fatty acids (oleic and palmitic acid) and exposure to a L-PGDS inhibitor and prostaglandin D2 receptor (DP1) antagonists. We conclude that the absence or inhibition of L-PGDS results in dyslipidemia, altered expression of lipogenesis genes and the acceleration of NAFLD to NASH, independent of diet and obesity. We propose L-PGDS KO mice as a useful model to explore the pathogenesis of NAFLD and NASH, and L-PGDS as a potential therapeutic target for treatment.

Enhanced Susceptibility to Chemoconvulsant-Induced Seizures in Ganglioside GM3 Synthase Knockout Mice.

Ganglioside GM3 synthase (α-2,3-sialyltransferase, ST3GAL5, GM3S) is a key enzyme involved in the biosynthesis of gangliosides. ST3GAL5 deficiency causes an absence of GM3 and all downstream biosynthetic derivatives. The affected individuals manifest deafness, severe irritability, intractable seizures, and profound intellectual disability. To investigate whether deficiency of GM3 is involved in seizure susceptibility, we induced seizures with different chemoconvulsants in ST3GAL5 knockout mice. We report here that ST3GAL5 knockout mice are hyperactive and more susceptible to seizures induced by chemoconvulsants, including kainate and pilocarpine, compared with normal controls. In the hippocampal dentate gyrus, loss of GM3 aggravates seizure-induced aberrant neurogenesis. These data indicate that GM3 and gangliosides derived from GM3 may serve as important regulators of epilepsy and may play an important role in aberrant neurogenesis associated with seizures.

Nitric oxide signaling inhibits microglia proliferation by activation of protein kinase-G

Microglia population is primarily determined by a finely-regulated proliferation process during early development of the central nervous system (CNS). Nitric oxide (NO) is known to inhibit proliferation in numerous cell types. However, how NO signaling regulates microglia proliferation remains elusive. Using wildtype (WT) and inducible nitric oxide synthase knockout (iNOS-/-) mice, this study investigated the role and underlying mechanisms of iNOS/NO signaling in microglia proliferation. Here we reported that iNOS-/- mice displayed significantly more BrdU-labeled proliferating microglia in the cortex than that in WT mice at postnatal day 10. Compared to microglia isolated from WT mouse cortex, significantly more iNOS-/- microglia displayed the specific cell-cycle markers Ki67 and phospho-histone H3 (pH3) in their nuclei. In addition, treating WT microglia with the NOS inhibitor LNAME drastically increased the percentage of cells expressing Ki67 and pH3, whereas treating iNOS-/- microglia with NOC18, a slow-release NO-donor, significantly decreased the percentage of microglia expressing the two cell-cycle markers. Moreover, inhibition of protein kinase-G (PKG) in WT microglia increased the proportion of microglia expressing Ki67 and pH3, whereas activation of PKG signaling using 8Br-cGMP in iNOS-/- microglia significantly decreased the fraction of microglia displaying Ki67 and pH3. Interestingly, in the presence of a PKG inhibitor, NOC18 increased the quantity of iNOS-/- microglia expressing Ki67 and pH3. Together, these results indicate that basal activity of iNOS/NO signaling impedes microglial cell-cycle progression and attenuates proliferation through activation of the cGMP-PKG pathway. However, NO increases microglia cell-cycle progression in the absence of cGMP-PKG signaling.