Abstract MP141: Role of Cryptochrome in Angiogenesis

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Kazuhito Tsuzuki ◽  
Yuuki Shimizu ◽  
Zhongyue Pu ◽  
Jyunya Suzuki ◽  
Toyoaki Murohara
Keyword(s):  
Growth Factors ◽  
Knockout Mice ◽  
Clock Genes ◽  
Umbilical Vein ◽  
Blood Perfusion ◽  
Gene Expressions ◽  
Double Knockout ◽  
Tissue Ischemia ◽  
Peripheral Clock

Background: Although clock genes generate oscillations in about 10 % of all gene expressions as a 24-hour cycle in the cellular level cooperating with the central clock, little is known about the role of clock genes in angiogenesis. Objective: The aim of this study was to determine if a peripheral clock would modulate angiogenesis in a hind limb ischemia (HLI) model. Methods and Results: First, we surgically induced the HLI model in C57BL/6J (wild type; WT) mice (N=6 for each time point) and checked mRNA expressions of clock genes in a local ischemic tissue by PCR array in the setting of blood perfusion recovery. In vitro study, we next investigated those gene expressions in human umbilical vein endothelial cells (HUVECs) with or without stimulation by growth factors. Those data demonstrated that the peripheral clock genes were modulated in ECs and tissues in the presence of growth factors and tissue ischemia. Then, we examined whether inhibition of clock gene expressions had any effects on angiogenesis. For this study, we focused on Cryptochrome (Cry), which is well known as one of the core-loop forming clock genes producing circadian rhythm in mammals. Our loss-of-function study revealed that the abilities of proliferation, migration and tube formation were significantly inhibited by Cry1 and Cry2 double knockdown in HUVECs. Interestingly, although the knockdown of Cry1 and Cry2 changed the mRNA expression of Period2, it did not affect those of BMAL1 and Clock in HUVECs. Finally, we tested if Cry1 and Cry2 double hetero knockout mice of HLI models displayed worse blood perfusion recoveries with deterioration of angiogenesis. As results, compared with control WT mice, Cry1 and Cry2 double knockout mice showed the low capillary density detected by CD31-immunohistochemistry and low tissue blood perfusion demonstrated by laser Doppler perfusion imaging (LDPI) in a HLI. Conclusion: Our data indicated that Cryptochrome as a peripheral clock plays an important role in angiogenesis of the reparative tissue ischemia model.

Abstract 13133: Impact of Circadian Rhythm Disorders on Reparative Angiogenesis

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Kazuhito Tsuzuki ◽  
Yuuki Shimizu ◽  
Zhongyue Pu ◽  
Junya Suzuki ◽  
Shukuro Yamaguchi ◽  
...  
Keyword(s):  
Circadian Rhythm ◽  
Clock Genes ◽  
Blood Perfusion ◽  
Jet Lag ◽  
Double Knockout ◽  
Tissue Ischemia ◽  
Circadian Rhythm Disorder ◽  
Rhythm Disorder ◽  
Reparative Angiogenesis ◽  
Circadian Rhythm Disorders

Introduction: Circadian rhythm disorder seen in shift-worker or jet-lag is major social health concerns in advanced industrialized countries. The aim of this study was to examine if circadian rhythm disorders would influence on angiogenesis and blood perfusion recovery in a mouse model of hind limb ischemia (HLI). Methods and Results: First, we established a jet-lag model in C57BL/6J (wild type; WT) mice (8-10 weeks old, N=10 for each) using a light controlled isolation box. Mice were exposed to advanced 8-hr light phase once every 4 days in a jet-lag group as previously described. Conversely, control mice were kept a regular condition of LD 12:12 (12-hr light and 12-hr dark). Then, we surgically induced HLI in each group. The results showed that the condition jet-lag deteriorated capillary formation detected by CD31-immunohistochemistry at post-operative day (POD) 28 and tissue blood perfusion recovery demonstrated by laser Doppler perfusion imaging (LDPI) in HLI. The expression of clock genes (i.e. Clock, Bmal1, Per2, Cry1 and 2 ) in ischemic muscles were regulated by jet-lag condition at POD7.Next, we examined whether inhibition of clock gene had any effects on angiogenesis. For this study, we focused on Cryptochrome ( Cry ), which is well known as one of the core-loop forming clock genes producing circadian rhythm in mammals. Our loss-of-function study revealed that the abilities of proliferation, migration and tube formation were significantly inhibited by CRY1 and CRY2 double knockdown in HUVECs. Interestingly, although the knockdown of CRY1 and CRY2 changed the mRNA expression of PERIOD2 , it did not affect those of BMAL1 and CLOCK in HUVECs. Finally, we tested if Cry1 and Cry2 double knockout ( Cry1/2 -DKO) mice of HLI models displayed worse blood perfusion recoveries with deterioration of angiogenesis. Cry1/2 -DKO mice were reported to display circadian rhythm disorder in previous reports. As results, compared with control WT mice, Cry1/2 -DKO mice revealed suppressed capillary density and tissue blood perfusion recovery in HLI model. Conclusion: Our data suggest that a maintenance of circadian rhythm plays an important role in reparative angiogenesis of the tissue ischemia model.

scholarly journals Rhythmic changes in colonic motility are regulated by period genes

2010 ◽  
Vol 298 (2) ◽  
pp. G143-G150 ◽  
Author(s):  
Willemijntje A. Hoogerwerf ◽  
Vahakn B. Shahinian ◽  
Germaine Cornélissen ◽  
Franz Halberg ◽  
Jonathon Bostwick ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Clock Genes ◽  
Ex Vivo ◽  
Colonic Motility ◽  
Biological Clock ◽  
Circular Muscle ◽  
Organ Bath ◽  
Wild Type ◽  
Double Knockout ◽  
Muscle Contractility

Human bowel movements usually occur during the day and seldom during the night, suggesting a role for a biological clock in the regulation of colonic motility. Research has unveiled molecular and physiological mechanisms for biological clock function in the brain; less is known about peripheral rhythmicity. This study aimed to determine whether clock genes such as period 1 ( per1) and period2 ( per2) modulate rhythmic changes in colonic motility. Organ bath studies, intracolonic pressure measurements, and stool studies were used to examine measures of colonic motility in wild-type and per1per2 double-knockout mice. To further examine the mechanism underlying rhythmic changes in circular muscle contractility, additional studies were completed in neuronal nitric oxide synthase (nNOS) knockout mice. Intracolonic pressure changes and stool output in vivo, and colonic circular muscle contractility ex vivo, are rhythmic with greatest activity at the start of night in nocturnal wild-type mice. In contrast, rhythmicity in these measures was absent in per1per2 double-knockout mice. Rhythmicity was also abolished in colonic circular muscle contractility of wild-type mice in the presence of Nω-nitro-l-arginine methyl ester and in nNOS knockout mice. These findings suggest that rhythms in colonic motility are regulated by both clock genes and a nNOS-mediated inhibitory process and suggest a connection between these two mechanisms.

scholarly journals Polyamine homeostasis in arginase knockout mice

2007 ◽  
Vol 293 (4) ◽  
pp. C1296-C1301 ◽  
Author(s):  
Joshua L. Deignan ◽  
Justin C. Livesay ◽  
Lisa M. Shantz ◽  
Anthony E. Pegg ◽  
William E. O'Brien ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Polyamine Metabolism ◽  
Mrna Levels ◽  
Ornithine Aminotransferase ◽  
Double Knockout ◽  
Mammalian Tissues ◽  
Knockout Animals ◽  
Regulatory Functions ◽  
Arginase I

The role of ornithine decarboxylase (ODC) in polyamine metabolism has long been established, but the exact source of ornithine has always been unclear. The arginase enzymes are capable of producing ornithine for the production of polyamines and may hold important regulatory functions in the maintenance of this pathway. Utilizing our unique set of arginase single and double knockout mice, we analyzed polyamine levels in the livers, brains, kidneys, and small intestines of the mice at 2 wk of age, the latest timepoint at which all of them are still alive, to determine whether tissue polyamine levels were altered in response to a disruption of arginase I (AI) and II (AII) enzymatic activity. Whereas putrescine was minimally increased in the liver and kidneys from the AII knockout mice, spermidine and spermine were maintained. ODC activity was not greatly altered in the knockout animals and did not correlate with the fluctuations in putrescine. mRNA levels of ornithine aminotransferase (OAT), antizyme 1 (AZ1), and spermidine/spermine- N1-acetyltransferase (SSAT) were also measured and only minor alterations were seen, most notably an increase in OAT expression seen in the liver of AI knockout and double knockout mice. It appears that putrescine catabolism may be affected in the liver when AI is disrupted and ornithine levels are highly reduced. These results suggest that endogenous arginase-derived ornithine may not directly contribute to polyamine homeostasis in mice. Alternate sources such as diet may provide sufficient polyamines for maintenance in mammalian tissues.

scholarly journals Dendritic spine morphology and memory formation depend on postsynaptic Caskin proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Norbert Bencsik ◽  
Szilvia Pusztai ◽  
Sándor Borbély ◽  
Anna Fekete ◽  
Metta Dülk ◽  
...  
Keyword(s):  
Dendritic Spine ◽  
Knockout Mice ◽  
Hippocampal Neurons ◽  
Pain Perception ◽  
Hippocampal Slices ◽  
Scaffold Proteins ◽  
Double Knockout ◽  
Learning Abilities ◽  
Spine Morphology

AbstractCASK-interactive proteins, Caskin1 and Caskin2, are multidomain neuronal scaffold proteins. Recent data from Caskin1 knockout animals indicated only a mild role of Caskin1 in anxiety and pain perception. In this work, we show that deletion of both Caskins leads to severe deficits in novelty recognition and spatial memory. Ultrastructural analyses revealed a reduction in synaptic profiles and dendritic spine areas of CA1 hippocampal pyramidal neurons of double knockout mice. Loss of Caskin proteins impaired LTP induction in hippocampal slices, while miniature EPSCs in dissociated hippocampal cultures appeared to be unaffected. In cultured Caskin knockout hippocampal neurons, overexpressed Caskin1 was enriched in dendritic spine heads and increased the amount of mushroom-shaped dendritic spines. Chemically induced LTP (cLTP) mediated enlargement of spine heads was augmented in the knockout mice and was not influenced by Caskin1. Immunocytochemistry and immunoprecipitation confirmed that Shank2, a master scaffold of the postsynaptic density, and Caskin1 co-localized within the same complex. Phosphorylation of AMPA receptors was specifically altered by Caskin deficiency and was not elevated by cLTP treatment further. Taken together, our results prove a previously unnoticed postsynaptic role of Caskin scaffold proteins and indicate that Caskins influence learning abilities via regulating spine morphology and AMPA receptor localisation.

scholarly journals Protective Role of Interleukin-1 in Mycobacterial Infection in IL-1 α/β Double-Knockout Mice

2000 ◽  
Vol 80 (5) ◽  
pp. 759-767 ◽  
Author(s):  
Hiroyuki Yamada ◽  
Satoru Mizumo ◽  
Reiko Horai ◽  
Yoichiro Iwakura ◽  
Isamu Sugawara
Keyword(s):  
Knockout Mice ◽  
Interleukin 1 ◽  
Mycobacterial Infection ◽  
Protective Role ◽  
Double Knockout

scholarly journals Individual Somatic H1 Subtypes Are Dispensable for Mouse Development Even in Mice Lacking the H10Replacement Subtype

2001 ◽  
Vol 21 (23) ◽  
pp. 7933-7943 ◽  
Author(s):  
Yuhong Fan ◽  
Allen Sirotkin ◽  
Robert G. Russell ◽  
Julianna Ayala ◽  
Arthur I. Skoultchi
Keyword(s):  
Knockout Mice ◽  
Knockout Mouse ◽  
Embryonic Stem ◽  
Mouse Development ◽  
Double Knockout ◽  
Linker Histones ◽  
Deficient Mice ◽  
Mouse Lines

ABSTRACT H1 linker histones are involved in facilitating the folding of chromatin into a 30-nm fiber. Mice contain eight H1 subtypes that differ in amino acid sequence and expression during development. Previous work showed that mice lacking H10, the most divergent subtype, develop normally. Examination of chromatin in H10−/− mice showed that other H1s, especially H1c, H1d, and H1e, compensate for the loss of H10 to maintain a normal H1-to-nucleosome stoichiometry, even in tissues that normally contain abundant amounts of H10 (A. M. Sirotkin et al., Proc. Natl. Acad. Sci. USA 92:6434–6438, 1995). To further investigate the in vivo role of individual mammalian H1s in development, we generated mice lacking H1c, H1d, or H1e by homologous recombination in mouse embryonic stem cells. Mice lacking any one of these H1 subtypes grew and reproduced normally and did not exhibit any obvious phenotype. To determine whether one of these H1s, in particular, was responsible for the compensation present in H10−/− mice, each of the three H1 knockout mouse lines was bred with H10 knockout mice to generate H1c/H10, H1d/H10, or H1e/H10double-knockout mice. Each of these doubly H1-deficient mice also was fertile and exhibited no anatomic or histological abnormalities. Chromatin from the three double-knockout strains showed no significant change in the ratio of total H1 to nucleosomes. These results suggest that any individual H1 subtype is dispensable for mouse development and that loss of even two subtypes is tolerated if a normal H1-to-nucleosome stoichiometry is maintained. Multiple compound H1 knockouts will probably be needed to disrupt the compensation within this multigene family.

scholarly journals Activation of liver X receptors inhibits experimental fibrosis by interfering with interleukin-6 release from macrophages

2014 ◽  
Vol 74 (6) ◽  
pp. 1317-1324 ◽  
Author(s):  
Christian Beyer ◽  
Jingang Huang ◽  
Jürgen Beer ◽  
Yun Zhang ◽  
Katrin Palumbo-Zerr ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Interleukin 6 ◽  
Skin Fibrosis ◽  
Target Cells ◽  
Similar Response ◽  
Liver X Receptors ◽  
Double Knockout ◽  
X Receptors ◽  
And Control

ObjectivesTo investigate the role of liver X receptors (LXRs) in experimental skin fibrosis and evaluate their potential as novel antifibrotic targets.MethodsWe studied the role of LXRs in bleomycin-induced skin fibrosis, in the model of sclerodermatous graft-versus-host disease (sclGvHD) and in tight skin-1 (Tsk-1) mice, reflecting different subtypes of fibrotic disease. We examined both LXR isoforms using LXRα-, LXRβ- and LXR-α/β-double-knockout mice. Finally, we investigated the effects of LXRs on fibroblasts and macrophages to establish the antifibrotic mode of action of LXRs.ResultsLXR activation by the agonist T0901317 had antifibrotic effects in bleomycin-induced skin fibrosis, in the sclGvHD model and in Tsk-1 mice. The antifibrotic activity of LXRs was particularly prominent in the inflammation-driven bleomycin and sclGvHD models. LXRα-, LXRβ- and LXRα/β-double-knockout mice showed a similar response to bleomycin as wildtype animals. Low levels of the LXR target gene ABCA-1 in the skin of bleomycin-challenged and control mice suggested a low baseline activation of the antifibrotic LXR signalling, which, however, could be specifically activated by T0901317. Fibroblasts were not the direct target cells of LXRs agonists, but LXR activation inhibited fibrosis by interfering with infiltration of macrophages and their release of the pro-fibrotic interleukin-6.ConclusionsWe identified LXRs as novel targets for antifibrotic therapies, a yet unknown aspect of these nuclear receptors. Our data suggest that LXR activation might be particularly effective in patients with inflammatory disease subtypes. Activation of LXRs interfered with the release of interleukin-6 from macrophages and, thus, inhibited fibroblast activation and collagen release.

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