The Shear Stress-Induced Transcription Factor KLF2 Affects Dynamics and Angiopoietin-2 Content of Weibel-Palade Bodies

BACKGROUND: The shear-stress induced transcription factor KLF2 has been shown to induce an atheroprotective phenotype in endothelial cells (EC) that are exposed to prolonged laminar shear. In this study we characterized the effect of the shear stress-induced transcription factor KLF2 on regulation and composition of Weibel-Palade bodies (WPBs) using peripheral blood derived ECs. METHODOLOGY AND PRINCIPAL FINDINGS: Lentiviral expression of KLF2 resulted in a 4.5 fold increase in the number of WPBs per cell when compared to mock-transduced endothelial cells. Unexpectedly, the average length of WPBs was significantly reduced: in mock-transduced endothelial cells WPBs had an average length of 1.7 µm versus 1.3 µm in KLF2 expressing cells. Expression of KLF2 abolished the perinuclear clustering of WPBs observed following stimulation with cAMP-raising agonists such as epinephrine. Immunocytochemistry revealed that WPBs of KLF2 expressing ECs were positive for IL-6 and IL-8 (after their upregulation with IL-1β) but lacked angiopoietin-2 (Ang2), a regular component of WPBs. Stimulus-induced secretion of Ang2 in KLF2 expressing ECs was greatly reduced and IL-8 secretion was significantly lower. CONCLUSIONS AND SIGNIFICANCE: These data suggest that KLF2 expression leads to a change in size and composition of the regulated secretory compartment of endothelial cells and alters its response to physiological stimuli

Effect of laminar shear stress on the distribution of Weibel-Palade bodies in endothelial cells

Background: Vascular endothelial cells (ECs) provide a highly interactive barrier between blood and the underlying tissues. It is well established that ECs exposed to laminar flow align in the direction of flow and also arrange their actin stress fibers in a parallel manner in the direction of flow. Also the organization of the microtubule network is altered in response to flow with repositioning of the microtubule-organizing centre (MTOC) in the direction of flow. Weibel-Palade bodies (WPBs) are endothelial cell specific storage organelles that contain a number of important homeostatic and inflammatory components. Dynamics of WPBs are controlled by microtubules and the actin cytoskeleton. Objectives: Here, we monitored flow-induced changes in distribution of WPBs. Methods: ECs were exposed for five days to laminar shear stress of 10 dyne/cm(2). Subsequently we measured the distance of individual WPBs with respect to the centre of the nucleus using Image Pro Plus. Results: ECs aligned in the direction of flow under these conditions. After 5 days the MTOC was positioned downstream of the nucleus in the direction of the flow. The number of WPBs per cell was slightly reduced as a result of the application of flow. Unexpectedly, only minor differences in the distribution of WPBs in ECs cultured under laminar flow were observed when compared to that of cells grown under static conditions. Conclusions: Our findings suggest that laminar flow does not induce major changes in number and distribution of WPBs in ECs. (C) 2012 Elsevier Ltd. All rights reserve

Regulation of VDR expression in rat and human intestine and liver - Consequences for CYP3A expression

The vitamin D receptor (VDR) regulates the expression of drug metabolizing enzymes and transporters in intestine and liver, but the regulation of VDR expression in intestine and liver is incompletely understood. We studied the regulation of VDR mRNA expression by ligands for VDR, farnesoid X receptor (FXR), glucocorticoid receptor (GR) and protein kinase C alpha (PKC alpha) in rat and human ileum and liver using precision-cut slices. 1,25(OH)(2)D(3) induced VDR expression in rat ileum and liver, and human ileum but not in liver. Chenodeoxycholic acid (CDCA), but not lithocholic acid (LCA) and GW4064 induced VDR mRNA expression in rat ileum and liver. The PKCa activator, phorbol-12-myristate-13-acetate (PMA) induced the expression of VDR in the rat liver, and the induction of VDR by 1,25(OH)(2)D(3) and COCA was inhibited by the PKCa inhibitor, bisindolyl maleimide 1 (Bis 1). These results show that the expression of VDR is likely to be regulated by PKC but not by FXR or VDR activation at least in the rat liver. The VDR mediated induction of its target genes CYP3A1 and CYP3A2 by 1,25(OH)(2)D(3) or LCA in the rat ileum was strongly reduced in the presence of CDCA despite the higher VDR expression. Thus, CDCA might potentiate the toxicity of LCA by inhibiting its metabolism. (C) 2009 Elsevier Ltd. All rights reserved

IL-6 and IL-8 content of KLF2-transduced BOECs.

<p>(A) Immunofluorescence image showing co-localization of IL-6 (green) and VWF (red) in IL-1β-treated KLF2- and mock-transduced BOECs. Nuclei were visualized with DAPI (blue). Scale bars: 10 µm. (B) Western blot analysis for VWF, KLF2, IL-8 and IL-6 expression in lysates of mock- and KLF2-transduced BOECs; α-tubulin was shown as a loading control. (C) Immunofluorescence image showing co-localization of IL-8 (green) and VWF (red) in IL-1β-treated KLF2- and mock-transduced BOECs. Nuclei were visualized with DAPI (blue). Scale bars: 10 µm. (D) Release of VWF from PMA-stimulated KLF2 (black bars)- and mock (white bars)-transduced cells (IL-1β-treated), measured by determining the concentration of VWF in the conditioned medium by ELISA. **P<0.001; ***P<0.0001 by Students t-test (E-F) Release of IL-6 and IL-8 from PMA-stimulated KLF2 (black bars)- and mock (white bars)-transduced cells (IL-1β-treated), measured by determining the concentration of IL-6 and IL-8 in the conditioned medium by ELISA. The amount of IL-6 released without stimulation was slightly reduced in KLF2 expressing cells when compared to mock-transduced cells. NS: non-significant; *P<0.01; ***P<0.0001 by Students t-test.</p

Expression of KLF2 in BOECs.

<p>(A) Immunofluorescent analysis of mock- and KLF2-transduced BOECs. Cells were immunostained for CD31 (red) and KLF2 (green); nuclei were stained using DAPI (blue). Scale bars: 20 µm; (B) Western blot analysis of KLF2 expression in lysates of KLF2-transduced and mock-transduced BOECs; α-tubulin is shown as a loading control.</p

Angiopoietin-2 content of WPBs in mock- and KLF2-transduced BOECs.

<p>(A) Immunofluorescence image showing the co-localization of Ang2 (green) and P-selectin (red) in WPBs of mock-transduced BOECs. Nuclei were visualized with DAPI (blue). Scale bars: 10 µm. (B) Western blot analysis of VWF, KLF2 and Ang2 expression in lysates of mock- and KLF2-transduced BOECs; α-tubulin is shown as a loading control. (C) Mock- and KLF2-transduced BOECs stained for VWF (red) and Ang2 (green). Nuclei were stained using DAPI (blue). Scale bars: 10 µm. (D-E) Release of Ang2 and VWF from PMA-stimulated KLF2 (black bars)- and mock (white bars)-transduced BOECs measured by determining the concentration of Ang2 in the medium by ELISA. **P<0.001; ***P<0.0001 by Students t-test. (F) Time course of regulated VWF and Ang2 secretion after PMA stimulation of mock- and KLF2-transduced BOECs.</p

Reduced average length of WPBs in KLF2-transduced BOECs.

<p>(A) Confocal microscopy analysis of WPBs in mock- and KLF2-transduced BOECs stained for VWF (red) and DAPI (blue). Scale bars: 10 µm. (B) Average amount of WPBs per cell in unstimulated and stimulated mock-transduced BOECs and KLF2-transduced BOECs. ***P<0.0001 using Student’s t- test (C) The average length of the WPBs in individual mock- or KLF2-transduced BOECs. WPBs from 20 randomly selected cells were analyzed. ***P<0.0005 by Student’s t-test (D) Release of VWF from forskolin/IBMX- and epinephrine/IBMX-stimulated KLF2 (black bars)- and mock (white bars)-transduced BOECs. ***P<0.0001 by Students t-test. Error bars represent SEM.</p

OPG content of mock- or KLF2-transduced BOECs.

<p>(A) Immunofluorescence image showing co-localization of OPG (green) and VWF (red) in both mock- and KLF2-tranduced BOECs. Nuclei were stained using DAPI (blue). Scale bars: 10 µm.(B) Western blot analysis for VWF, KLF2, IL-8 and IL-6 expression in lysates of mock- and KLF2-transduced BOECs; α-tubulin was shown as a loading control.</p

Proteomic Screen Identifies IGFBP7 as a Novel Component of Endothelial Cell-Specific Weibel-Palade Bodies

Vascular endothelial cells contain unique storage organelles, designated Weibel-Palade bodies (WPBs), that deliver inflammatory and hemostatic mediators to the vascular lumen in response to agonists like thrombin and vasopressin. The main component of WPBs is von Willebrand factor (VWF), a multimeric glycoprotein crucial for platelet plug formation. In addition to VWF, several other components are known to be stored in WPBs, like osteoprotegerin, monocyte chemoattractant protein-1 and angiopoetin-2 (Ang-2). Here, we used an unbiased proteomics approach to identify additional residents of WPBs. Mass spectrometry analysis of purified WPBs revealed the presence of several known components such as VWF, Ang-2, and P-selectin. Thirty-five novel candidate WPB residents were identified that included insulin-like growth factor binding protein-7 (IGFBP7), which has been proposed to regulate angiogenesis. Immunocytochemistry revealed that IGFBP7 is a bona fide WPB component. Cotransfection studies showed that IGFBP7 trafficked to pseudo-WPB in HEK293 cells. Using a series of deletion variants of VWF, we showed that targeting of IGFBP7 to pseudo-WPBs was dependent on the carboxy-terminal D4-C1-C2-C3-CK domains of VWF. IGFBP7 remained attached to ultralarge VWF strings released upon exocytosis of WPBs under flow. The presence of IGFBP7 in WPBs highlights the role of this subcellular compartment in regulation of angiogenesis

A physiological role for glucuronidated thyroid hormones: Preferential uptake by H9c2(2-1) myotubes

Contains fulltext : 35115.pdf (publisher's version ) (Closed access)Conjugation reactions are important pathways in the peripheral metabolism of thyroid hormones. Rat cardiac fibroblasts produce and secrete glucuronidated thyroxine (T4G) and 3,3',5-triiodothyronine (T3G). We here show that, compared to fibroblasts from other anatomical locations, the capacity of cardiofibroblasts to secrete T4G and T3G is highest. H9c2(2-1) myotubes, a model system for cardiomyocytes, take up T4G and T3G at a rate that is 10-15 times higher than that for the unconjugated thyroid hormones. T3 and T4, and their glucuronides, stimulate H9c2(2-1) myoblast-to-myotube differentiation. A substantial beta-glucuronidase activity was measured in H9c2(2-1) myotubes, and this confers a deconjugating capacity to these cells, via which native thyroid hormones can be regenerated from glucuronidated precursors. This indicates that the stimulatory effects on myoblast differentiation are exerted by the native hormones. We suggest that glucuronidation represents a mechanism to uncouple local thyroid hormone action in the heart from that in other peripheral tissues and in the systemic circulation. This could represent a mechanism for the local fine-tuning of cardiac thyroid hormone action