scholarly journals Spatiotemporal restriction of endothelial cell calcium signaling is required during leukocyte transmigration

2020 ◽  
Vol 218 (1) ◽  
Author(s):  
Prarthana J. Dalal ◽  
David P. Sullivan ◽  
Evan W. Weber ◽  
David B. Sacks ◽  
Matthias Gunzer ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Calcium Signaling ◽  
Dominant Negative ◽  
Calcium Flux ◽  
Inhibitory Peptide ◽  
Leukocyte Transmigration ◽  
Cell Calcium ◽  
Leukocyte Transendothelial Migration ◽  
Calmodulin Kinase

Endothelial cell calcium flux is critical for leukocyte transendothelial migration (TEM), which in turn is essential for the inflammatory response. Intravital microscopy of endothelial cell calcium dynamics reveals that calcium increases locally and transiently around the transmigration pore during TEM. Endothelial calmodulin (CaM), a key calcium signaling protein, interacts with the IQ domain of IQGAP1, which is localized to endothelial junctions and is required for TEM. In the presence of calcium, CaM binds endothelial calcium/calmodulin kinase IIδ (CaMKIIδ). Disrupting the function of CaM or CaMKII with small-molecule inhibitors, expression of a CaMKII inhibitory peptide, or expression of dominant negative CaMKIIδ significantly reduces TEM by interfering with the delivery of the lateral border recycling compartment (LBRC) to the site of TEM. Endothelial CaMKII is also required for TEM in vivo as shown in two independent mouse models. These findings highlight novel roles for endothelial CaM and CaMKIIδ in transducing the spatiotemporally restricted calcium signaling required for TEM.

scholarly journals Visualizing calcium responses to acetylcholine convection along endothelium of arteriolar networks in Cx40BAC-GCaMP2 transgenic mice

2011 ◽  
Vol 301 (3) ◽  
pp. H794-H802 ◽  
Author(s):  
Pooneh Bagher ◽  
Michael J. Davis ◽  
Steven S. Segal
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Transgenic Mice ◽  
Calcium Signaling ◽  
Muscarinic Receptors ◽  
Side Branch ◽  
Widespread Application ◽  
Cell Calcium ◽  
Flow Stream

Acetylcholine evokes endothelium-dependent vasodilation subsequent to a rise in intracellular calcium. Despite widespread application in human and animal studies, calcium responses to intravascular ACh have not been visualized in vivo. Microiontophoresis of ACh in tissue adjacent to an arteriole activates abluminal muscarinic receptors on endothelial cells within a “local” region of diffusion, but it is unknown whether ACh released in such fashion gains access to the flow stream resulting in further actions downstream. To test this hypothesis and provide new insight into calcium signaling in vivo, we studied the cremaster muscle microcirculation of anesthetized male Cx40BAC-GCaMP2 transgenic mice ( n = 22; 5–9 mo; 33 ± 1 g) expressing the fluorescent calcium sensor GCaMP2 selectively in arteriolar endothelial cells. Submaximal ACh stimuli were delivered using microiontophoresis (1-μm pipette tip, 500 nA). With stimulus duration <500 ms or with the micropipette positioned within one vessel diameter (∼30 μm) away from an arteriole, endothelial cell calcium fluorescence was restricted to the region of ACh diffusion (<200 μm). In contrast, with the micropipette tip positioned immediately adjacent to an arteriole or within its lumen, calcium fluorescence encompassed entire networks downstream. The velocity of downstream calcium signaling in response to ACh increased with centerline velocity of fluorescent tracer microbeads ( r2 > 0.99; range: <1 mm/s to >10 mm/s). Diverting arteriolar blood flow into a side branch redirected downstream fluorescence responses to ACh; occluding flow abolished responses. Blocking luminal muscarinic receptors (intravascular glycopyrrolate; 6 μg/kg) inhibited downstream responses reversibly. Through visualizing the actions of a “local” ACh stimulus on endothelial cell calcium fluorescence in vivo, the present findings illustrate that transmural diffusion and convection of an agonist can activate entire networks of arteriolar endothelial cells concomitant with its delivery in the flow stream.

scholarly journals Glomerular Endothelial Cell Calcium Dynamics Visualized in vivo

2018 ◽  
Vol 32 (S1) ◽  
Author(s):  
Ina Maria Schiessl ◽  
Georgina Gyarmati ◽  
Janos Peti‐Peterdi
Keyword(s):  
Endothelial Cell ◽  
Calcium Dynamics ◽  
Glomerular Endothelial Cell ◽  
Cell Calcium

scholarly journals Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis

2008 ◽  
Vol 295 (5) ◽  
pp. C1292-C1301 ◽  
Author(s):  
Anke C. Webler ◽  
U. Ruth Michaelis ◽  
Rüdiger Popp ◽  
Eduardo Barbosa-Sicard ◽  
Andiappan Murugan ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Protein Expression ◽  
Second Messengers ◽  
Tube Formation ◽  
Dominant Negative ◽  
Endothelial Cell Proliferation ◽  
Angiogenic Response

Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5( Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.

JAM-A mediates neutrophil transmigration in a stimulus-specific manner in vivo: evidence for sequential roles for JAM-A and PECAM-1 in neutrophil transmigration

Blood ◽  
2007 ◽  
Vol 110 (6) ◽  
pp. 1848-1856 ◽  
Author(s):  
Abigail Woodfin ◽  
Christoph Andreas Reichel ◽  
Andrej Khandoga ◽  
Monica Corada ◽  
Mathieu-Benoit Voisin ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Transmembrane Protein ◽  
Ischemia Reperfusion ◽  
Platelet Activating Factor ◽  
Inhibitory Effect ◽  
Leukotriene B4 ◽  
Leukocyte Transmigration ◽  
Neutralizing Monoclonal Antibody ◽  
Transfer Method

Abstract Junctional adhesion molecule-A (JAM-A) is a transmembrane protein expressed at tight junctions of endothelial and epithelial cells and on the surface of platelets and leukocytes. The role of JAM-A in leukocyte transmigration in vivo was directly investigated by intravital microscopy using both a JAM-A–neutralizing monoclonal antibody (mAb) (BV-11) and JAM-A–deficient (knockout [KO]) mice. Leukocyte transmigration (but not adhesion) through mouse cremasteric venules as stimulated by interleukin 1β (IL-1β) or ischemia/reperfusion (I/R) injury was significantly reduced in wild-type mice treated with BV-11 and in JAM-A KO animals. In contrast, JAM-A blockade/genetic deletion had no effect on responses elicited by leukotriene B4 (LTB4) or platelet-activating factor (PAF). Furthermore, using a leukocyte transfer method and mice deficient in endothelial-cell JAM-A, evidence was obtained for the involvement of endothelial-cell JAM-A in leukocyte transmigration mediated by IL-1β. Investigation of the functional relationship between JAM-A and PECAM-1 (CD31) determined that dual blockade/deletion of these proteins does not lead to an inhibitory effect greater than that seen with blockade/deletion of either molecule alone. The latter appeared to be due to the fact that JAM-A and PECAM-1 can act sequentially to mediate leukocyte migration through venular walls in vivo.

Calmodulin kinase II inhibition protects against myocardial cell apoptosis in vivo

2006 ◽  
Vol 291 (6) ◽  
pp. H3065-H3075 ◽  
Author(s):  
Yingbo Yang ◽  
Wei-Zhong Zhu ◽  
Mei-ling Joiner ◽  
Rong Zhang ◽  
Carmine V. Oddis ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Myocardial Cell ◽  
Inhibitory Peptide ◽  
Transgenic Expression ◽  
Novel Approach ◽  
Calmodulin Kinase ◽  
Inactive Form ◽  
Dependent Protein ◽  
Induced Apoptosis

Inhibition of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) or depletion of sarcoplasmic reticulum (SR) Ca2+ stores protects against apoptosis from excessive isoproterenol (Iso) stimulation in cultured ventricular myocytes, suggesting that CaMKII inhibition could be a novel approach to reducing cell death in conditions of increased adrenergic tone, such as myocardial infarction (MI), in vivo. We used mice with genetic myocardial CaMKII inhibition due to transgenic expression of a highly specific CaMKII inhibitory peptide (AC3-I) to test whether CaMKII was important for apoptosis in vivo. A second line of mice expressed a scrambled, inactive form of AC3-I (AC3-C). AC3-C and wild-type (WT) littermates were used as controls. AC3-I mice have reduced SR Ca2+ content and are resistant to Iso- and MI-induced apoptosis compared with AC3-C and WT mice. Phospholamban (PLN) is a target for modulation of SR Ca2+ content by CaMKII. PLN−/− mice have increased susceptibility to Iso-induced apoptosis. Verapamil pretreatment prevented Iso-induced apoptosis in PLN−/− mice, indicating the involvement of a Ca2+-dependent pathway. AC3-I and AC3-C mice were bred into a PLN−/− background. Loss of PLN increased and equalized SR Ca2+ content in AC3-I, AC3-C, and WT mice and abolished the resistance to apoptosis in AC3-I mice after MI. There was a trend ( P = 0.07) for increased Iso-induced apoptosis in AC3-I mice lacking PLN compared with AC3-I mice with PLN. These findings indicate CaMKII is proapoptotic in vivo and suggest that regulation of SR Ca2+ content by PLN contributes to the antiapoptotic mechanism of CaMKII inhibition.

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