SWEET11 and 15 as key players in seed filling in rice

SummaryDespite the relevance of seed filling mechanisms for crop yield, we still have only a rudimentary understanding of the pathways and transport processes for supplying the caryopsis with sugars. We hypothesized that the recently identified SWEET sucrose transporters may play important roles in nutrient import pathways in the rice caryopsis.We used a combination of mRNA quantification, histochemical analyses, translational promoter-reporter fusions and analysis of knock out mutants created by genomic editing to evaluate the contribution of SWEET transporters to seed filling.In rice caryopses, SWEET11 and 15 had the highest mRNA levels and proteins localized to four key sites: the nucellus proper at early stages, the nucellar projection close to the dorsal vein, the nucellar epidermis that surrounds the endosperm, and the aleurone. ossweet11;15 double knock-out lines accumulated starch in the pericarp while caryopses did not contain a functional endosperm.Jointly, SWEET11 and 15 show all hallmarks of being responsible for seed filling with sucrose efflux function at the nucellar projection and transfer across the nucellar epidermis/aleurone interface, delineating two major steps for apoplasmic seed filling, observations that are discussed in relation to observations made in rice and barely on the relative prevalence of these two potential import routes.

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The Role of Serotonin in the Development of Psychiatric Disorders: Studies on Drug Dependence and Anxiety-like Behaviour Following the Genetic Reduction of the Serotonin Transporter

10.26686/wgtn.17013845 ◽

2021 ◽

Author(s):

◽

Alana Oakly

Keyword(s):

Psychiatric Disorders ◽

Early Development ◽

Drug Dependence ◽

Primary Objective ◽

Behavioural Response ◽

Mrna Levels ◽

Self Administration ◽

Knock Out ◽

Enhanced Sensitivity

<p>Rationale: Given the high prevalence and large burden of psychiatric disorders it is imperative to determine the underling etiology in order for better understanding and treatment. The neurotransmitter serotonin (5-HT) has been associated with mental disorders in humans both pharmacologically and genetically. Individuals with the short-allele of a prominent polymorphism within the 5-HT transporter (SERT) show increased incidence of mood disorders and drug dependence. However, whether or not dysregulation in the 5-HT system causes, or is just associated with, psychiatric disorders is impossible to determine from human studies alone. Consequently, it is imperative to employ an animal model of down-regulated SERT function. To better understand the role of 5-HT in drug dependence, the rat’s behavioural response to the psychostimulant (±) 3, 4-methylenedioxymethamphetamine (MDMA), a preferentially serotonergically mediated drug, was assessed. Finally, the ability to rescue the anxiety-like phenotype in the SERT-/- rat by altering extracellular 5-HT during early development was also evaluated. Objective: The primary objective of the current thesis was to determine whether dysregulation of 5-HT is directly linked to the occurrence of psychiatric disorders, particularly drug dependence and anxiety. Methods: A model of down-regulated SERT function, the SERT knock-out (SERT-/-) rat, was used for all experiments in order to determine a causal relationship between 5-HT dysregulation and psychiatric disorders. In Chapter 2, the response of the SERT-/- rats to various tasks usually disrupted by MDMA was assessed. In Chapter 3, the sensitivity of the SERT-/- rats to the reinforcing effects of MDMA was determined using the self-administration paradigm. Finally, in Chapter 4, whether the anxiety-like behaviour of the SERT-/- rat could be rescued through normalising excessive extracellular 5-HT neonatally was assessed. An attempt was also made to determine a mechanism by which 5-HT dysregulation could alter behaviour. To this end, gene expression previously found to be up- or down-regulated in the SERT-/- rat was assessed in the neonatally treated rats. Results: The results of Chapter 2 indicated the SERT is necessary for MDMA’s disruption of startle habituation but not its psychomotor effects. Moreover, for those rats that could discriminate low dose MDMA from saline, genetic removal of the SERT resulted in the inability to discriminate MDMA from amphetamine, implying that, in these rats, MDMA was now subjectively indistinguishable from amphetamine. Indeed, this alteration also resulted in enhanced sensitivity to the reinforcing properties of MDMA, giving MDMA the qualities of a traditional psychostimulant in SERT-/- rats (Chapter 3). Finally, lowering the excessive 5-HT during neonatal development in SERT-/- rats led to a rescue of mild, but not high, anxiety-like behaviour in males. However, mRNA levels of long 3’NTR BDNF and 5-HT1a, genes associated with neurodevelopment, remained unchanged across genotypes and treatment groups (Chapter 4). Conclusions: Genetic removal of the 5-HT transporter results in an altered behavioural response to MDMA, in particular an increased sensitivity to its reinforcing properties. However, while the genetic removal of the SERT results in enhanced extracellular 5-HT, the pathological phenotypes present in this rat are likely due to this increase occurring in early development, not its continued presence in adulthood. Overall, these findings contribute to the growing body of literature indicating that enhanced brain 5-HT during early development can lead to pathological behaviour in adulthood.</p>

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Effect of Oral Iron Treatment in Tmprss6 Knock-out Mouse Model

Blood ◽

10.1182/blood-2019-122416 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2235-2235

Author(s):

Elisa Brilli ◽

Michela Asperti ◽

Annalisa Castagna ◽

Claudio Cerchione ◽

Domenico Girelli ◽

...

Keyword(s):

Iron Deficiency ◽

Mouse Model ◽

Ferrous Sulfate ◽

Iron Status ◽

Iron Concentration ◽

Oral Iron ◽

Mrna Levels ◽

Advisory Committees ◽

Knock Out ◽

Parenteral Iron

Introduction: Iron Refractory Iron Deficiency Anemia (IRIDA) is an autosomal recessive iron metabolism disorder caused by mutations in Tmprss6 gene which encodes for Matriptase2 (MT2) that, by activating hemojuvelin (HJV), regulates the production of hepcidin, the master iron regulatory hormone. Altered MT2 cannot suppress hepatic BMP6/SMAD signaling in low iron condition, hence the resulting hepcidin excess blocks dietary iron absorption and cells release, leading to a form of iron deficiency that is typically refractory to oral iron supplementation. IRIDA is characterized by moderate/severe microcytic anemia (Hemoglobin 6-9 g/dL; MCV 45-65 fL); low transferrin saturation (<5%); impaired oral iron absorption and only a transient response to parenteral iron. Nonetheless, the current treatment is mainly based on parenteral iron therapy. A case study on a child with IRIDA showed for the first time the ability of Sucrosomial® Iron, to increase hemoglobin and MCV values over time (Capra et al., 2017). This oral iron formulation is an innovative preparation of ferric pyrophosphate, covered by a phospholipids plus sucrester matrix, with gastro-resistance properties, high bioavailability and tolerability due to alternative absorption pathways as endocytosis and M cells mediated route (Gomez-Ramirez et al., 2018). Moreover, Sucrosomial® Iron has been successfully used to treat iron deficiency in various clinical conditions, including inflammatory bowel diseases (Abbati et al., 2019). To confirm and characterize the ability of Sucrosomial® Iron to increase Hb in IRIDA disease we studied the response to Sucrosomial® Iron in a IRIDA mouse model (Mask) comparing the efficacy of Sucrosomial® Iron and Sulfate Iron at two different doses and in chronic treatment. Aim: to study Sucrosomial® Iron effect in IRIDA using the Tmprss6 knock-out mouse model Material and Methods: m/m homozygous mice (9-weeks old male mice, four mice per experimental group) were kept at iron balance diet and treated with 0.5 or 4 mg/Kg of Ferrous sulfate, Sucrosomial® Iron (patent n° PCT/IB2013/001659 owned by Alesco s.r.l, Italy), or vehicle by gavage for 35 days. Four 9-weeks old m/- male mice per experimental group were daily treated and Hb and Ht were monitored weekly. Mice were sacrificed at the end of treatments; blood, and different organs were collected for analysis. Total RNA was isolated from tissues using TRIzol Reagent (Ambion), cDNA was generated by Reverse transcription (Promega, Milan, Italy) and samples were analyzed for Hepcidin and Socs3 mRNA levels by qRT-PCR using PowerUp SYBR Green Master Mix (Life Technologies). Results: we analyzed the iron status of anemic homozygous Mask mice from 3 to 35 weeks of age by studying serological and tissue iron content. Interestingly only Sucrosomial® Iron (not Ferrous Sulfate), increased hemoglobin level from 11-12 to 13-14 g/dL in the first week with a tendency to increase until the fourth week, when it stabilized at 13 g/dL (Figure 1A-B). Serum iron concentration was higher in the Sucrosomial® Iron treated animals than in those treated with vehicle, while was lower in the Ferrous sulfate treated animals. Similar pattern was observed for spleen iron content that increased in mice treated with Sucrosomial® Iron but not in those receiving Ferrous sulfate. Liver iron concentration did not apparently varied after the treatments, but duodenal iron increased significantly only in the mice treated with the higher dose of Ferrous sulfate (Figure 1 C-F). Interestingly, we found that the mice treated with both doses of Ferrous sulfate, but not those treated with Sucrosomial® Iron, had a higher mRNA levels of hepcidin and of the inflammatory marker Socs3 (Figure 1 G-H). Conclusion: this study showed for the first time that Sucrosomial® Iron is able to increase hemoglobin level in a mouse model of IRIDA, probably due to its alternative absorption pathway. Sucrosomial® Iron could be used as effective iron supplement to improve iron status in IRIDA patients. Disclosures Girelli: La Jolla Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Vifor Pharma: Other: honoraria for lectures; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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Abstract 1064: Pellino-1: A Novel Candidate In Vegf/Flk-1/MKK2 Signaling In Ischemic Preconditioning Induced Cardioprotection: A Study Using Flk +/− and MKK2 −/− Knockout Mice.

Circulation ◽

10.1161/circ.116.suppl_16.ii_213 ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Mahesh Thirunavukkarasu ◽

Samson Mathews Samuel ◽

Sankar Addya ◽

Lijun Zhan ◽

Chi-Kuang Huang ◽

...

Keyword(s):

Real Time ◽

Ischemic Preconditioning ◽

Knockout Mice ◽

Real Time Pcr ◽

Left Ventricular ◽

List Type ◽

Mrna Levels ◽

Clinical Issue ◽

Endothelial Cell Function ◽

Vegf Signaling

VEGF modulates the complex process of angiogenesis and other various aspects of endothelial cell function through either of its two tyrosine kinase receptors, VEGFR1/Flt-1 or VEGFR2/Flk1/ KDR via its target protein MKK2. In the present study we used Flk1 +/− and MKK2 −/− knockout mice in an attempt to address an important clinical issue by identifying potential downstream candidates of VEGF signaling through Flk1 receptor that trigger cardioprotective signal during ischemic preconditioning (IP). Mouse hearts were subjected to 30 min of global ischemia and 2 hours of reperfusion (IR) in the Isolated Working Heart model. It is known that IP (4min of ischemia + 6min reperfusion, 4 cycles, before 30 min of ischemia) induces cardioprotection through the activation of the VEGF signaling cascade. The mice were randomly divided into 6 groups for both the gene knockout (KO) studies: Wild Type-Baseline (WTBL), FlkBL/ MKK2BL (KOBL), WTIR , KOIR, WTIP and KOIP. Significant reduction in left ventricular functional recovery through out reperfusion (dp/dt = 605 vs 884), diminished coronary flow (1.9 vs 2.4) and aortic flow (0.16 vs 1.2) and increased infarct size (38.4% vs. 28.41%) after reperfusion were observed in FlkIR, compared to WTIR. As expected we observed disruption in IP induced cardioprotection in FlkIP compared to WTIP. Affymetrix gene chip analysis demonstrated significant downregulation of genes (Pellino-1, MKK2, NF-Î°B) which are thought to play important roles in cardioprotection after ischemic insults in the Flk +/− mice compared to WT. These results were further validated at the mRNA expression level with Real Time PCR. Pellino-1 (Pel-1) was found to be significantly downregulated in FlkBL (0.74 vs 1), FlkIR (1.29 vs 1.35) and FlkPC (1.35 vs 1.49) as compared to their respective controls. We further validated the mRNA levels of Pel-1using Real Time PCR and RT-PCR in the MKK2 −/− mice and found that it, remained unaffected in MKK2BL as compared to its WTBL, and increased as expected in MKK2PC as compared to both MKK2BL and FlkPC (2.48 vs 1.2 and 2.48 vs 1.35, respectively). Therefore this study validated for the first time that Pel-1 is a novel downstream mediator in VEGF/FLK1 signaling and it induces IP mediated cardioprotection via MKK2 signaling.

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The integrated stress response promotes B7H6 expression

Journal of Molecular Medicine ◽

10.1007/s00109-019-01859-w ◽

2019 ◽

Vol 98 (1) ◽

pp. 135-148 ◽

Cited By ~ 3

Author(s):

Akram Obiedat ◽

Yoav Charpak-Amikam ◽

Julie Tai-Schmiedel ◽

Einat Seidel ◽

Mohamed Mahameed ◽

...

Keyword(s):

Stress Response ◽

Er Stress ◽

Protease Inhibitors ◽

Hiv Protease ◽

Hiv Protease Inhibitors ◽

List Type ◽

Mrna Levels ◽

Integrated Stress Response ◽

Eif2α Phosphorylation ◽

Car T

Abstract The B7 family member, B7H6, is a ligand for the natural killer cell receptor NKp30. B7H6 is hardly expressed on normal tissues, but undergoes upregulation on different types of tumors, implicating it as an attractive target for cancer immunotherapy. The molecular mechanisms that control B7H6 expression are poorly understood. We report that in contrast to other NK cell ligands, endoplasmic reticulum (ER) stress upregulates B7H6 mRNA levels and surface expression. B7H6 induction by ER stress requires protein kinase R-like ER kinase (PERK), one of the three canonical sensors of the unfolded protein response. PERK phosphorylates eIF2α, which regulates protein synthesis and gene expression. Because eIF2α is phosphorylated by several kinases following different stress conditions, the program downstream to eIF2α phosphorylation is called the integrated stress response (ISR). Several drugs were reported to promote the ISR. Nelfinavir and lopinavir, two clinically approved HIV protease inhibitors, promote eIF2α phosphorylation by different mechanisms. We show that nelfinavir and lopinavir sustainably instigate B7H6 expression at their pharmacologically relevant concentrations. As such, ER stress and ISR conditions sensitize melanoma targets to CAR-T cells directed against B7H6. Our study highlights a novel mechanism to induce B7H6 expression and suggests a pharmacological approach to improve B7H6-directed immunotherapy. Key messages B7H6 is induced by ER stress in a PERK-dependent mechanism. Induction of B7H6 is obtained pharmacologically by HIV protease inhibitors. Exposure of tumor cells to the HIV protease inhibitor nelfinavir improves the recognition by B7H6-directed CAR-T.

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AHSP Is a Quantitative Trait Gene That Modifies the Phenotype of β Thalassemia.

Blood ◽

10.1182/blood.v106.11.3638.3638 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3638-3638

Author(s):

Mei I. Lai ◽

Jie Jiang ◽

Nicholas Silver ◽

Steve Best ◽

Stephan Menzel ◽

...

Keyword(s):

Strong Association ◽

Luciferase Reporter ◽

Healthy Population ◽

Mrna Levels ◽

Globin Genes ◽

Thalassemia Intermedia ◽

Altered Expression ◽

Knock Out ◽

Minor Alleles

Abstract Alpha hemoglobin stabilizing protein (AHSP) was identified in a screen for genes that are activated by the erythroid transcription factor, GATA-1. Studies have shown that: AHSP binds specifically to the α chain of hemoglobin (Hb) but not to β Hb or Hb A; AHSP knock-out mice demonstrated pathological features similar to β thalassemia; and loss of AHSP exacerbates severity of disease in β thalassemia mice. The evidence suggests that AHSP acts as a chaperone for free α Hb and that altered AHSP expression levels could modify the severity of β thalassemia in humans. To assess variation of AHSP expression, mRNA levels were measured in peripheral blood reticulocytes of 103 healthy individuals by quantitative real-time RT-PCR. The sample was approximately 90% (91/103) female with an average age of 52 years (SD=0.14, min=18, max=76). AHSP expression relative to GAPDH, varied up to 3-fold, and did not correlate with age or sex. A systematic survey of the genomic region encompassing the AHSP locus revealed 8 sequence variants of which six were common - five single nucleotide polymorphisms (SNPs), and one homopolymer (Tn) at position 160 bp upstream of exon 1. Four variants (c.-69–237A, c.-69–160 T18, c.-4–27G, and c. 337T) showed strong association with AHSP expression and had nearly equal frequencies. The four variants are in near complete linkage disequilibrium with the minor alleles in coupling. The haplotype consisting of the four minor alleles termed clade B, was associated with relatively lower expression of AHSP. Relative expression of AHSP and AHSP/α globin ratio were both significantly higher (p<0.001) in homozygotes for clade A haplotypes compared to heterozygotes. A potential functional role of one of the variable sites, a T-homopolymer (the T18 variant being part of clade A) in the promoter was investigated in-vitro using luciferase reporter assays in K562 cells. Luciferase activity was 1.30±0.08 times higher in the T18 promoter compared to T14, consistent with genetic studies. We investigated if a shorter homopolymer could have an adverse effect on β thalassemia. Nine patients with thalassemia intermedia and a genotypic combination of heterozygous β thalassemia and 5 α globin genes (aaa/aa) were studied. Based on the allele frequencies in the healthy population, 6–7 of the 9 patients are expected to be homozygous for T18 but only two were observed. In contrast, while 2–3 patients are expected to be heterozygous for the shorter homopolymer, 3 patients were homozygous and 4 heterozygous. In conclusion, AHSP gene expression is variable, with cis control accounting for some of its variance. The subtle altered expression might precipitate a phenotype of thalassemia intermedia in β thalassemia heterozygotes with α globin overload.

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Efficient long fragment editing technique enables rapid construction of genetically stable bacterial strains

10.21203/rs.2.22299/v1 ◽

2020 ◽

Author(s):

Chaoyong Huang ◽

Liwei Guo ◽

Jingge Wang ◽

Ning Wang ◽

Yi-Xin Huo

Keyword(s):

Open Reading Frames ◽

Deletion Mutants ◽

Dna Fragments ◽

Bacterial Strains ◽

Chromosome Engineering ◽

Knock Out ◽

Rapid Construction ◽

Stable Plasmid ◽

Chromosomal Engineering ◽

Genomic Editing

Abstract Background Bacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable molecules. Long fragment editing techniques are of great importance for accelerating bacterial chromosome engineering to obtain desirable and genetically stable strains. However, the existing genomic editing methods cannot meet the needs of researchers. Results We herein report an efficient long fragment editing technique for complex chromosomal engineering in Escherichia coli. The technique enabled us to integrate DNA fragments up to 12 kb into the chromosome, and to knock out DNA fragments up to 187 kb from the chromosome, with over 95% positive rates. We applied this technique for E. coli chromosomal simplification, resulting in twelve individual deletion mutants and four cumulative deletion mutants. The simplest chromosome lost a 370.6 kb DNA sequence containing 364 open reading frames. In addition, we applied the technique to metabolic engineering and constructed a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask micro-aerobic fermentation. Conclusions These results suggested that the technique is a powerful chromosomal engineering tool, highlighting its potential to be applied in different fields of synthetic biology.

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ZYG11A Is Expressed in Epithelial Ovarian Cancer and Correlates With Low Grade Disease

Frontiers in Endocrinology ◽

10.3389/fendo.2021.688104 ◽

2021 ◽

Vol 12 ◽

Author(s):

Laris Achlaug ◽

Lina Somri-Gannam ◽

Shilhav Meisel-Sharon ◽

Rive Sarfstein ◽

Manisha Dixit ◽

...

Keyword(s):

Ovarian Cancer ◽

Epithelial Ovarian Cancer ◽

Growth Hormone Receptor ◽

Histological Grade ◽

Inverse Correlation ◽

Low Grade ◽

Mrna Levels ◽

Knock Out ◽

Coordinated Expression ◽

Candidate Target

The insulin-like growth factors (IGF) are important players in the development of gynecological malignancies, including epithelial ovarian cancer (EOC). The identification of biomarkers that can help in the diagnosis and scoring of EOC patients is of fundamental importance in clinical oncology. We have recently identified the ZYG11A gene as a new candidate target of IGF1 action. The aim of the present study was to evaluate the expression of ZYG11A in EOC patients and to correlate its pattern of expression with histological grade and pathological stage. Furthermore, and in view of previous analyses showing an interplay between ZYG11A, p53 and the IGF1 receptor (IGF1R), we assessed a potential coordinated expression of these proteins in EOC. In addition, zyg11a expression was assessed in ovaries and uteri of growth hormone receptor (GHR) knock-out mice. Tissue microarray analysis was conducted on 36 patients with EOC and expression of ZYG11A, IGF1R and p53 was assessed by immunohistochemistry. Expression levels were correlated with clinical parameters. qPCR was employed to assess zyg11a mRNA levels in mice tissues. Our analyses provide evidence of reduced ZYG11A expression in high grade tumors, consistent with a putative tumor suppressor role. In addition, an inverse correlation between ZYG11A and p53 levels in individual tumors was noticed. Taken together, our data justify further exploration of the role of ZYG11A as a novel biomarker in EOC.

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The E3 ubiquitin ligase RNF216/TRIAD3 is a central regulator of the hypothalamic-pituitary-gonadal axis

10.1101/2021.03.21.436306 ◽

2021 ◽

Author(s):

Arlene J. George ◽

Yarely C. Hoffiz ◽

Christopher Ware ◽

Bin Dong ◽

Ning Fang ◽

...

Keyword(s):

Hypogonadotropic Hypogonadism ◽

Cell Activity ◽

Calcium Transient ◽

Therapeutic Interventions ◽

List Type ◽

Dependent Manner ◽

Hpg Axis ◽

Knock Out ◽

Soma Size

SummaryRNF216/TRIAD3 is an E3 ligase that ubiquitinates substrates in the nervous system. Recessive mutations in RNF216/TRIAD3 cause Gordon Holmes syndrome (GHS), where hypogonadotropic hypogonadism is a core phenotype. However, the functions of RNF216/TRIAD3 within the neuroendocrine system are not well-understood. Here, we used the CRISPR-Cas9 system to knock out Rnf216/Triad3 in GT1-7 cells, a GnRH immortalized cell line derived from mouse hypothalamus. Rnf216/Triad3 knockout cells had decreased steady state Gnrh and reduced calcium transient frequency. To address functions of RNF216/TRIAD3 in vivo, we generated a Rnf216/Triad3 constitutive knockout (KO) mouse. KO mice of both sexes showed reductions in GnRH and soma size. Furthermore, KO mice exhibited sex-specific phenotypes with males showing gonadal impairment and derangements in gonadotropin release compared to KO females, which only had irregular estrous cyclicity. Our work shows that dysfunction of RNF216/TRIAD3 affects the HPG axis in a sex-dependent manner, implicating sex-specific therapeutic interventions for GHS.HighlightsRnf216/Triad3 controls Gnrh and intrinsic hypothalamic cell activityRnf216/Triad3 knockout male mice have greater reproductive impairments than femalesRnf216/Triad3 controls the HPG axis at multiple levels

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Tissue-Specific Function of Thyroid Hormone Transporters: New Insights from Mouse Models

Experimental and Clinical Endocrinology & Diabetes ◽

10.1055/a-1032-8328 ◽

2019 ◽

Vol 128 (06/07) ◽

pp. 423-427 ◽

Cited By ~ 2

Author(s):

Eva Salveridou ◽

Steffen Mayerl ◽

Sivaraj Mohana Sundaram ◽

Boyka Markova ◽

Heike Heuer

Keyword(s):

Thyroid Hormone ◽

Elevated Serum ◽

Transport Processes ◽

Tissue Specific ◽

Knock Out ◽

Serum T3 ◽

A Cell ◽

Knock Out Mice ◽

Mct8 Deficiency ◽

Shed Light

AbstractThyroid hormone (TH) transporters are required for cellular transmembrane passage of TH and are thus mandatory for proper TH metabolism and action. Consequently, inactivating mutations in TH transporters such as MCT8 or OATP1C1 can cause tissue- specific changes in TH homeostasis. As the most prominent example, patients with MCT8 mutations exhibit elevated serum T3 levels, whereas their CNS appear to be in a TH deficient state. Here, we will briefly summarize recent studies of mice lacking Mct8 alone or in combination with the TH transporters Mct10 or Oatp1c1 that shed light on many aspects and pathogenic events underlying global MCT8 deficiency and also underscore the contribution of Mct10 and Oatp1c1 in tissue-specific TH transport processes. Moreover, development of conditional knock-out mice that allow a cell-specific inactivation of TH transporters in distinct tissues, disclosed cell-specific changes in TH signaling, thereby highlighting the pathophysiological significance of local control of TH action.

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Abstract 347: eNOS Deficiency Ameliorates Vascular Remodeling Induced by Periadventitial Injury in SOD1 Knock-Out Mice

Circulation ◽

10.1161/circ.116.suppl_16.ii_52-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Keiichiro Kataoka ◽

Masaya Fukuda ◽

Eiichiro Yamamoto ◽

Taishi Nakamura ◽

Hisao Ogawa ◽

...

Keyword(s):

Nitric Oxide ◽

Vascular Remodeling ◽

List Type ◽

Neointimal Formation ◽

Knock Out ◽

Copper Zinc ◽

Knock Out Mice ◽

Oxide Synthase ◽

Endothelial Nitric Oxide ◽

Deficient Mice

Background: Copper/zinc SOD (SOD1) is a major enzyme which deactivates superoxide radicals (O2−), and endothelial nitric oxide synthase (eNOS) synthesizes nitric oxide (NO) in endothelial cells. Reduction of each enzyme can increase oxidative stress, leading to cardiovascular dysfunction. Herein, we established SOD1 and eNOS double deficient mice, and examined their physiological and pathological cardiovascular phenotypes to clarify the function of eNOS in cardiovascular system in the case of SOD1 deficiency. Methods and Results: SOD1 deficient mice (SOD-KO) were crossbred with eNOS deficient mice (eNOS-KO), and SOD1 and eNOS double-deficient mice (Do-KO) were established. Do-KO had significantly higher blood pressure (BP) than SOD-KO (129.6 ± 4.7 vs. 102.1 ± 1.2 mmHg, p<0.0001). Do-KO had significantly higher heart weights than SOD-KO (3.12 ± 0.09 vs. 2.89 ± 0.03 mg/g, p<0.01). Relaxation of carotid arteries due to acetylcholine was mildly impaired in SOD-KO when compared with wild type mice (WT), while relaxation to acetylcholine was completely ablated in Do-KO. These data indicated that targeted ablation of eNOS in SOD-KO impaired their vascular relaxation, and caused hypertension. Next, we examined vascular remodeling induced by periadventitial cuff-injuries. Four weeks after cuff replacement, marked neointimal formation was induced in SOD-KO; however, eNOS deficiency in SOD-KO ameliorated the vascular remodeling of SOD-KO, and significantly decreased the ratio of intimal to medial areas (1.23 ± 0.23 vs. 2.23 ± 0.38, p<0.05). This data shows that eNOS enhances the vascular remodeling of SOD-KO caused by cuff injury. NO and O2− react to form the strong oxidant peroxynitrite, which is involved in vascular injury. The levels of 3-nitrotyrosine, a marker of peroxynitrite generation, were significantly elevated in the injured arteries of SOD-KO, while their elevation were attenuated in Do-KO. This indicates that NO derived from eNOS enhances the peroxynitrite formation in injured arteries of SOD-KO, so that vascular remodeling may be markedly enhanced. Conclusion: Our results demonstrate that peroxynitrite, generated from O2− and eNOS derived-NO, plays a key role in vascular remodeling induced by periadventitial injury.

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