Vascular dysfunction in S1P2 sphingosine 1-phosphate receptor knockout mice

2007 ◽  
Vol 292 (1) ◽  
pp. R440-R446 ◽  
Author(s):  
John N. Lorenz ◽  
Lois J. Arend ◽  
Rachel Robitz ◽  
Richard J. Paul ◽  
A. John MacLennan
Keyword(s):  
Knockout Mice ◽  
Vascular Function ◽  
Vascular Dysfunction ◽  
Vascular Morphology ◽  
Relative Contribution ◽  
Sphingosine 1 Phosphate ◽  
In Vivo Analysis ◽  
And Function ◽  
Isometric Forces

There is growing evidence that sphingosine 1-phosphate (S1P) plays an important role in regulating the development, morphology, and function of the cardiovascular system. There is little data, however, regarding the relative contribution of endogenous S1P and its cognate receptors (referred to as S1P1–5) to cardiovascular homeostasis. We used S1P2 receptor knockout mice (S1P2−/−) to evaluate the role of S1P2 in heart and vascular function. There were no significant differences in blood pressure between wild-type and S1P2−/− mice, measured in awake mice. Cardiac function, evaluated in situ by using a Millar catheter, was also not different in S1P2−/− mice under baseline or stimulated conditions. In vivo analysis of vascular function by flowmetry revealed decreases in mesenteric and renal resistance in S1P2−/− mice, especially during vasoconstriction with phenylephrine. In intact aortic rings, the concentration-force relations for both KCl and phenylephrine were right shifted in S1P2−/− mice, whereas the maximal isometric forces were not different. By contrast, in deendothelialized rings the concentration-force relations were not different but the maximal force was significantly greater in S1P2−/− aorta. Histologically, there were no apparent differences in vascular morphology. These data suggest that the S1P2 receptor plays an important role in the function of the vasculature and is an important mediator of normal hemodynamics. This is mediated, at least in part, through an effect on the endothelium, but direct effects on vascular smooth muscle cannot be ruled out and require further investigation.

scholarly journals WASP confers selective advantage for specific hematopoietic cell populations and serves a unique role in marginal zone B-cell homeostasis and function

Blood ◽  
2008 ◽  
Vol 112 (10) ◽  
pp. 4139-4147 ◽  
Author(s):  
Lisa S. Westerberg ◽  
Miguel A. de la Fuente ◽  
Fredrik Wermeling ◽  
Hans D. Ochs ◽  
Mikael C. I. Karlsson ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Marginal Zone ◽  
Hematopoietic Cells ◽  
Selective Advantage ◽  
Relative Contribution ◽  
B Cell Homeostasis ◽  
And Function

Abstract Development of hematopoietic cells depends on a dynamic actin cytoskeleton. Here we demonstrate that expression of the cytoskeletal regulator WASP, mutated in the Wiskott-Aldrich syndrome, provides selective advantage for the development of naturally occurring regulatory T cells, natural killer T cells, CD4+ and CD8+ T lymphocytes, marginal zone (MZ) B cells, MZ macrophages, and platelets. To define the relative contribution of MZ B cells and MZ macrophages for MZ development, we generated wild-type and WASP-deficient bone marrow chimeric mice, with full restoration of the MZ. However, even in the presence of MZ macrophages, only 10% of MZ B cells were of WASP-deficient origin. We show that WASP-deficient MZ B cells hyperproliferate in vivo and fail to respond to sphingosine-1-phosphate, a crucial chemoattractant for MZ B-cell positioning. Abnormalities of the MZ compartment in WASP−/− mice lead to aberrant uptake of Staphylococcus aureus and to a reduced immune response to TNP-Ficoll. Moreover, WASP-deficient mice have increased levels of “natural” IgM antibodies. Our findings reveal that WASP regulates both development and function of hematopoietic cells. We demonstrate that WASP deficiency leads to an aberrant MZ that may affect responses to blood-borne pathogens and peripheral B-cell tolerance.

Abstract 12555: The Dual PPAR-α/γ Agonist Aleglitazar Enhances Arteriogenesis, Angiogenesis and Endothelial Function

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Christian Werner ◽  
Stephan H Schirmer ◽  
Valerie Pavlickova ◽  
Michael Böhm ◽  
Ulrich Laufs
Keyword(s):  
Endothelial Function ◽  
Vascular Function ◽  
Daily Injection ◽  
Peroxisome Proliferator ◽  
Fluorescent Microspheres ◽  
Artery Ligation ◽  
Peroxisome Proliferator Activated Receptor ◽  
Beneficial Effects ◽  
And Function

Objective: Peroxisome proliferator-activated receptor (PPAR)-α and -γ agonists modify lipid and glucose metabolism. The aim of the study was to characterize the effects of the dual PPAR-α/γ agonist aleglitazar on endothelial function, neoangiogenesis and arteriogenesis in mice and on human endothelial progenitor cells (EPC). Methods and Results: Male C57Bl/6 wild-type (WT, normal chow) and apolipoprotein E-deficient (apoE-/-) mice on Western-type diet (WTD) were treated with aleglitazar (10 mg/kg i.p.) or vehicle by daily injection. Hindlimb ischemia was induced by right femoral artery ligation (FAL). ApoE-/- mice on WTD treated with aleglitazar before FAL were characterized by an improvement of endothelial-dependent laser Doppler perfusion (right/left foot ratio 0.40±0.03) 1 week after FAL compared to controls (R/L foot ratio 0.24±0.01; p<0.001). Collateral-dependent perfusion measured under conditions of maximal vasodilatation 1 week after FAL using fluorescent microspheres was impaired in apoE-/- on WTD compared to WT mice (R/L leg ratio in WT 78±13 vs. apoE-/- 56±6; p<0.001) and was normalized by aleglitazar treatment. Neoangiogenesis was measured in-vivo by subcutaneously implanting discs covered with cell-impermeable filters. The vascularized area of the discs was quantified after 14 days by perfusion of the animals with space-filling fluorescent microspheres. Aleglitazar increased neoangiogenesis in WT mice by 178±18% compared to vehicle (p<0.05). Endothelium-dependent relaxation of aortic rings was impaired in apoE-/- mice on WTD for 6 weeks (relaxation to 52±5% of max. contraction) compared to WT animals (relaxation to 18±5% of max. contraction) (p<0.001). Aleglitazar treatment improved endothelial function (relaxation to 39±5% of max. contraction; p<0.05). In parallel, number and function of EPC were improved in mice. Studies in human EPC showed that 1) aleglitazar’s effects were mediated by both PPAR-α and -γ signalling and Akt and 2) migration and colony forming units were up-regulated by aleglitazar in cultivated EPC from CAD patients. Conclusion: The study provides evidence for beneficial effects of the dual PPAR-α/γ agonist aleglitazar on vascular function in addition to or mediated by its metabolic actions.

scholarly journals In Vivo Analysis of the Major Exocytosis-sensitive Phosphoprotein in Tetrahymena

1997 ◽  
Vol 139 (5) ◽  
pp. 1197-1207 ◽  
Author(s):  
N. Doane Chilcoat ◽  
Aaron P. Turkewitz
Keyword(s):  
Membrane Fusion ◽  
Protein Function ◽  
Potential Role ◽  
Secretory Granules ◽  
Rabbit Muscle ◽  
Paramecium Tetraurelia ◽  
Post Translational Modifications ◽  
In Vivo Analysis ◽  
And Function

Phosphoglucomutase (PGM) is a ubiquitous highly conserved enzyme involved in carbohydrate metabolism. A number of recently discovered PGM-like proteins in a variety of organisms have been proposed to function in processes other than metabolism. In addition, sequence analysis suggests that several of these may lack PGM enzymatic activity. The best studied PGM-like protein is parafusin, a major phosphoprotein in the ciliate Paramecium tetraurelia that undergoes rapid and massive dephosphorylation when cells undergo synchronous exocytosis of their dense-core secretory granules. Indirect genetic and biochemical evidence also supports a role in regulated exocytotic membrane fusion. To examine this matter directly, we have identified and cloned the parafusin homologue in Tetrahymena thermophila, a ciliate in which protein function can be studied in vivo. The unique T. thermophila gene, called PGM1, encodes a protein that is closely related to parafusin by sequence and by characteristic post-translational modifications. Comparison of deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like proteins have PGM activity. We evaluated the activity and function of PGM1 through gene disruption. Surprisingly, ΔPGM1 cells displayed no detectable defect in exocytosis, but showed a dramatic decrease in PGM activity. Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like proteins in regulated exocytosis is unlikely to precede membrane fusion.

Abstract 14820: Sirt7 Deficiency in Blood Vessel Components Impairs Vascular Function by Inhibiting Cell Cycle- and Inflammatory-Related Protein Expression

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Yuichi Kimura ◽  
Yasuhiro Izumiya ◽  
Satoshi Araki ◽  
Satoru Yamamura ◽  
Yoshiro Onoue ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Blood Flow ◽  
Vascular Function ◽  
Vascular Dysfunction ◽  
Vascular Diseases ◽  
Tube Formation ◽  
Hindlimb Ischemia ◽  
Proliferation Assay

Introduction: Aging is a well-established cardiovascular risk factor and associated with vascular dysfunction. Sirt7, one of the members of mammalian sirtuin family, is thought to be involved in age-related diseases. However, little is known about the relative contribution of Sirt7 in vascular dysfunction. Hypothesis: Sirt7 maintains vascular cell functions and its deficiency plays a critical role in vascular diseases. Methods: Sirt7 loss- and gain-of-function experiments were performed with human aortic smooth muscle cells (HAoSMCs) and human umbilical vein endothelial cells (HUVECs). In vivo, blood flow recovery was evaluated by hindlimb ischemia model in homozygous Sirt7 deficient (Sirt7-/-) and wild-type (WT) mice. Irradiated WT mice were intravenously received bone marrow (BM) cells from WT or Sirt7 -/- mouse to achieve BM transfer. Results: An RNAi-medicated Sirt7 knockdown resulted in a significant inhibition of HAoSMCs proliferation following serum or Platelet-derived growth factor BB (PDGF-BB) stimulation as determined by cell count, BrdU cell proliferation assay and MTS proliferation assay. Knockdown of endogenous Sirt7 also reduced cell migration as revealed by Boyden chamber migration assay. The Cyclin D1 and Cyclin dependent kinase 2 (CDK2) protein levels were significantly decreased in Sirt7 siRNA-treated HAoSMCs in response to serum or PDGF-BB stimulation. In endothelial cells, knockdown of Sirt7 attenuated tube formation, proliferation and migration. These changes were accompanied by reduced ERK activation and VCAM-1 mRNA and protein expression in Sirt7 siRNA-treated HUVECs. Conversely, overexpression of Sirt7 by adenovirus enhanced tube formation and cell proliferation. In vivo, blood flow recovery in response to hindlimb ischemia was significantly attenuated in Sirt7-/- mice compared with WT mice. There was no difference in blood flow recovery between WT mice transplanted with WT or Sirt7-/- BM cells suggesting that Sirt7 deficiency in vascular cells have a predominant effect on attenuated blood flow recovery in response to hindlimb ischemia. Conclusions: Sirt7 in blood vessel components have an important role in maintenance of vascular function. Sirt7 could be a promising therapeutic target for vascular diseases.

Endothelin-mediated in vivo pressor responses following TRPV1 activation

2011 ◽  
Vol 301 (3) ◽  
pp. H1135-H1142 ◽  
Author(s):  
Vahagn A. Ohanyan ◽  
Giacinta Guarini ◽  
Charles K. Thodeti ◽  
Phani K. Talasila ◽  
Priya Raman ◽  
...  
Keyword(s):  
Vascular Function ◽  
Vascular Reactivity ◽  
Transient Receptor Potential ◽  
Vascular Dysfunction ◽  
Vascular Complications ◽  
Receptor Potential ◽  
Immunoblot Analysis ◽  
Trpv1 Channels ◽  
Transient Receptor

Transient receptor potential vanilliod 1 (TRPV1) channels have recently been postulated to play a role in the vascular complications/consequences associated with diabetes despite the fact that the mechanisms through which TRPV1 regulates vascular function are not fully known. Accordingly, our goal was to define the mechanisms by which TRPV1 channels modulate vascular function and contribute to vascular dysfunction in diabetes. We subjected mice lacking TRPV1 [TRPV1(−/−)], db/ db, and control C57BLKS/J mice to in vivo infusion of the TRPV1 agonist capsaicin or the α-adrenergic agonist phenylephrine (PE) to examine the integrated circulatory actions of TRPV1. Capsaicin (1, 10, 20, and 100 μg/kg) dose dependently increased MAP in control mice (5.7 ± 1.6, 11.7 ± 2.1, 25.4 ± 3.4, and 51.6 ± 3.9%), which was attenuated in db/db mice (3.4 ± 2.1, 3.9 ± 2.1, 7.0 ± 3.3, and 17.9 ± 6.2%). TRPV1(−/−) mice exhibited no changes in MAP in response to capsaicin, suggesting the actions of this agonist are specific to TRPV1 activation. Immunoblot analysis revealed decreased aortic TRPV1 protein expression in db/db compared with control mice. Capsaicin-induced responses were recorded following inhibition of endothelin A and B receptors (ETA /ETB). Inhibition of ETA receptors abolished the capsaicin-mediated increases in MAP. Combined antagonism of ETA and ETB receptors did not further inhibit the capsaicin response. Cultured endothelial cell exposure to capsaicin increased endothelin production as shown by an endothelin ELISA assay, which was attenuated by inhibition of TRPV1 or endothelin-converting enzyme. TRPV1 channels contribute to the regulation of vascular reactivity and MAP via production of endothelin and subsequent activation of vascular ETA receptors. Impairment of TRPV1 channel function may contribute to vascular dysfunction in diabetes.

MAP1 structural organization in Drosophila: in vivo analysis of FUTSCH reveals heavy- and light-chain subunits generated by proteolytic processing at a conserved cleavage site

2008 ◽  
Vol 414 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Beiyan Zou ◽  
Huaru Yan ◽  
Fumiko Kawasaki ◽  
Richard W. Ordway
Keyword(s):  
Light Chain ◽  
Neural Development ◽  
Cleavage Site ◽  
Structural Organization ◽  
Single Gene ◽  
Proteolytic Cleavage ◽  
Proteolytic Processing ◽  
In Vivo Analysis ◽  
And Function

The MAP1 (microtubule-associated protein 1) family is a class of microtubule-binding proteins represented by mammalian MAP1A, MAP1B and the recently identified MAP1S. MAP1A and MAP1B are expressed in the nervous system and thought to mediate interactions of the microtubule-based cytoskeleton in neural development and function. The characteristic structural organization of mammalian MAP1s, which are composed of heavy- and light-chain subunits, requires proteolytic cleavage of a precursor polypeptide encoded by the corresponding map1 gene. MAP1 function in Drosophila appears to be fulfilled by a single gene, futsch. Although the futsch gene product is known to share several important functional properties with mammalian MAP1s, whether it adopts the same basic structural organization has not been addressed. Here, we report the identification of a Drosophila MAP1 light chain, LCf, produced by proteolytic cleavage of a futsch-encoded precursor polypeptide, and confirm co-localization and co-assembly of the heavy chain and LCf cleavage products. Furthermore, the in vivo properties of MAP1 proteins were further defined through precise MS identification of a conserved proteolytic cleavage site within the futsch-encoded MAP1 precursor and demonstration of light-chain diversity represented by multiple LCf variants. Taken together, these findings establish conservation of proteolytic processing and structural organization among mammalian and Drosophila MAP1 proteins and are expected to enhance genetic analysis of conserved MAP1 functions within the neuronal cytoskeleton.

scholarly journals Cystic fibrosis transmembrane conductance regulator dysfunction in VIP knockout mice

2014 ◽  
Vol 307 (2) ◽  
pp. C195-C207 ◽  
Author(s):  
Nicole G. Alcolado ◽  
Dustin J. Conrad ◽  
Diogo Poroca ◽  
Mansong Li ◽  
Walaa Alshafie ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Knockout Mice ◽  
Cellular Localization ◽  
Intraperitoneal Administration ◽  
Smooth Muscles ◽  
Primary Role ◽  
Innate Defense ◽  
And Function ◽  
The Impact

Vasoactive intestinal peptide (VIP), a neuropeptide, controls multiple functions in exocrine tissues, including inflammation, and relaxation of airway and vascular smooth muscles, and regulates CFTR-dependent secretion, which contributes to mucus hydration and local innate defense of the lung. We had previously reported that VIP stimulates the VPAC1 receptor, PKCϵ signaling cascade, and increases CFTR stability and function at the apical membrane of airway epithelial cells by reducing its internalization rate. Moreover, prolonged VIP stimulation corrects the molecular defects associated with F508del, the most common CFTR mutation responsible for the genetic disease cystic fibrosis. In the present study, we have examined the impact of the absence of VIP on CFTR maturation, cellular localization, and function in vivo using VIP knockout mice. We have conducted pathological assessments and detected signs of lung and intestinal disease. Immunodetection methods have shown that the absence of VIP results in CFTR intracellular retention despite normal expression and maturation levels. A subsequent loss of CFTR-dependent chloride current was measured in functional assays with Ussing chamber analysis of the small intestine ex vivo, creating a cystic fibrosis-like condition. Interestingly, intraperitoneal administration of VIP corrected tissue abnormalities, close to the wild-type phenotype, as well as associated defects in the vital CFTR protein. The results show in vivo a primary role for VIP chronic exposure in CFTR membrane stability and function and confirm in vitro data.

scholarly journals In Vitro and In Vivo Analysis of the Immune System of Vitamin D Receptor Knockout Mice

2001 ◽  
Vol 16 (11) ◽  
pp. 2057-2065 ◽  
Author(s):  
Chantal Mathieu ◽  
Evelyne Van Etten ◽  
Conny Gysemans ◽  
Brigitte Decallonne ◽  
Shigeaki Kato ◽  
...  
Keyword(s):  
Vitamin D ◽  
Immune System ◽  
Vitamin D Receptor ◽  
Knockout Mice ◽  
In Vivo Analysis

Abstract 14: Caskin2 is a Novel Regulator of Endothelial Cell Quiescence

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Sarah B Mueller ◽  
Shubhangi Arora ◽  
Natalie Mattocks ◽  
Susan B Gurley ◽  
Christopher D Kontos
Keyword(s):  
Knockout Mice ◽  
Vascular Function ◽  
Fasting Blood Glucose ◽  
Scaffolding Protein ◽  
In Vivo Model ◽  
Serum Starvation ◽  
Chow Diet ◽  
Binding Motif

Maintenance of vascular homeostasis is an active process that is dependent on continuous signaling by the quiescent endothelial cells (ECs) that line mature vessels. The signaling pathways that are active in quiescent ECs are distinct from those that regulate angiogenesis but are comparatively poorly understood. Here we demonstrate that the previously uncharacterized scaffolding protein Caskin2 is a novel regulator of EC quiescence. Caskin2 is highly expressed in ECs from various vascular beds both in vitro and in vivo. Morpholino knockdown of Caskin2 in zebrafish results in abnormal vascular development characterized by overly branched intersegmental vessels and failure to form the dorsal longitudinal vessels. Interestingly, Caskin2 knockout mice are viable and fertile. However, compared to wild-type mice, adult Caskin2 knockout mice have significantly more abdominal adipose and higher fasting blood glucose levels even when fed a standard chow diet. In vitro, Caskin2 overexpression inhibits serum-induced EC proliferation and DNA synthesis but promotes EC survival during serum starvation. Caskin2 localizes to the nucleus as well as the cytoplasm and promotes the downregulation of genes associated with EC activation (e.g., IL-8, VEGFR2, ANG2) and the upregulation of genes associated with EC quiescence (e.g., Notch1, KLF2/4, ANG1). More broadly, pathway analysis of microarray data demonstrates that Caskin2 primarily regulates cell cycle and metabolic genes. Additional data suggest that these effects result from the interaction of Caskin2 with the Ser/Thr phosphatase PP1alpha, which is mediated by a consensus PP1 binding motif at the Caskin2 N-terminus. Taken together, our data demonstrate that Caskin2 is sufficient to suppress EC proliferation in vitro and necessary to prevent dysregulated angiogenesis in at least one in vivo model. These findings indicate that Caskin2 is a novel regulator of EC quiescence and suggest a role for Caskin2 in the prevention of EC dysfunction in vivo. Understanding Caskin2’s function on the molecular level may lead to the development of novel pharmacological approaches to prevent inappropriate angiogenesis, normalize dysfunctional vessels, and improve vascular function in a variety of cardiovascular diseases.

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