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Methods for Analysis of Matrix Metalloproteinase Regulation of Neutrophil-Endothelial Cell Adhesion.

Fernandez-Patron C, Zouki C, Whittal RM, Chan JS, Davidge ST - Biol Proced Online (2002)

Bottom Line: The neutrophil responses to ET-1[1-32] were mediated via activation of ET(A)receptors through activation of the Ras/Raf-1/MEK/ERK signaling pathway.These results suggest a novel role for gelatinase A and B in the regulation of neutrophil functions and their interactions with endothelial cells.Here we describe the methods in detail as they relate to our previously published work.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Alberta. Medical Sciences Building 3-08, Edmonton Alberta, Canada T6G 2S2. Canada.Research Center, Maisonneuve-Rosemont Hospital and Department of Medicine, University of Montréal. Montréal Québec, Canada H1T 2M4. Canada.Mass Spectrometry Facility, Department of Chemistry, University of Alberta. Edmonton Alberta, Canada T6G 2S2. Canada.Perinatal Research Center, University of Alberta. Edmonton Alberta, Canada T6G 2S2. Canada. janos.g.filep@umontreal.ca

ABSTRACT
Recent evidence indicates novel role for matrix metalloproteinases (MMPs), in particular gelatinase A (MMP-2), in the regulation of vascular biology that are unrelated to their well-known proteolytic breakdown of matrix proteins. We have previously reported that MMP-2 can modulate vascular reactivity by cleavage of the Gly32-Leu33 bound in big endothelin-1 (ET-1) yielding a novel vasoactive peptide ET-1[1-32]. These studies were conducted to investigate whether gelatinolytic MMPs could affect neutrophil-endothelial cell attachment. ET-1[1-32] produced by MMP-2 up-regulated CD11b/CD18 expression on human neutrophils, thereby promoted their adhesion to cultured endothelial cells. ET-1[1-32] evoked release of gelatinase B (MMP-9), which in turn cleaved big ET-1 to yield ET-1[1-32], thus revealing a self-amplifying loop for ET-1[1-32] generation. ET-1[1-32] was rather resistant to cleavage by neutrophil proteases and further metabolism of ET-1[1-32] was not a prerequisite for its biological actions on neutrophils. The neutrophil responses to ET-1[1-32] were mediated via activation of ET(A)receptors through activation of the Ras/Raf-1/MEK/ERK signaling pathway. These results suggest a novel role for gelatinase A and B in the regulation of neutrophil functions and their interactions with endothelial cells. Here we describe the methods in detail as they relate to our previously published work.

No MeSH data available.


Related in: MedlinePlus

ET-1[1-32] enhances adhesion of neutrophils (PMN) to monolayers of human coronary artery endothelial cells (HCAEC). HCAEC were incubated with LPS (1 μg/ml) for 4 h, washed and PMN without or together with ET-1[1-32] (30 nM) were then added for 30 min at 37°C. Values are means ± SEM (n=5).
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Figure 5: ET-1[1-32] enhances adhesion of neutrophils (PMN) to monolayers of human coronary artery endothelial cells (HCAEC). HCAEC were incubated with LPS (1 μg/ml) for 4 h, washed and PMN without or together with ET-1[1-32] (30 nM) were then added for 30 min at 37°C. Values are means ± SEM (n=5).

Mentions: To assess the biological significance of ET-1[1-32]-induced changes in expression of adhesion molecules on the neutrophil surface, we performed a neutrophil-endothelial cell adhesion assay. To mimic blood flow, the assay was performed under non-static conditions (26). While ET-1[1-32] did not stimulate neutrophil adhesion to monolayers of unstimulated HCAEC, about 1.5-fold more neutrophils adhered to LPS-stimulated HCAEC when neutrophils were added to HCAEC together with ET-1[1-32] than in the absence of ET-1[1-32] (Fig. 5). Since neutrophils were incubated in the adhesion assays for 30 min with activated HCAEC, stimulation of neutrophil adhesion by ET-1[1-32] can be attributed primarily to the effects of this peptide on neutrophils. Indeed, much longer periods of culture of HCAEC with ET-1[1-32] were required to detect slight increases in expression of E-selectin and ICAM-1 on HCAEC (18).


Methods for Analysis of Matrix Metalloproteinase Regulation of Neutrophil-Endothelial Cell Adhesion.

Fernandez-Patron C, Zouki C, Whittal RM, Chan JS, Davidge ST - Biol Proced Online (2002)

ET-1[1-32] enhances adhesion of neutrophils (PMN) to monolayers of human coronary artery endothelial cells (HCAEC). HCAEC were incubated with LPS (1 μg/ml) for 4 h, washed and PMN without or together with ET-1[1-32] (30 nM) were then added for 30 min at 37°C. Values are means ± SEM (n=5).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC145555&req=5

Figure 5: ET-1[1-32] enhances adhesion of neutrophils (PMN) to monolayers of human coronary artery endothelial cells (HCAEC). HCAEC were incubated with LPS (1 μg/ml) for 4 h, washed and PMN without or together with ET-1[1-32] (30 nM) were then added for 30 min at 37°C. Values are means ± SEM (n=5).
Mentions: To assess the biological significance of ET-1[1-32]-induced changes in expression of adhesion molecules on the neutrophil surface, we performed a neutrophil-endothelial cell adhesion assay. To mimic blood flow, the assay was performed under non-static conditions (26). While ET-1[1-32] did not stimulate neutrophil adhesion to monolayers of unstimulated HCAEC, about 1.5-fold more neutrophils adhered to LPS-stimulated HCAEC when neutrophils were added to HCAEC together with ET-1[1-32] than in the absence of ET-1[1-32] (Fig. 5). Since neutrophils were incubated in the adhesion assays for 30 min with activated HCAEC, stimulation of neutrophil adhesion by ET-1[1-32] can be attributed primarily to the effects of this peptide on neutrophils. Indeed, much longer periods of culture of HCAEC with ET-1[1-32] were required to detect slight increases in expression of E-selectin and ICAM-1 on HCAEC (18).

Bottom Line: The neutrophil responses to ET-1[1-32] were mediated via activation of ET(A)receptors through activation of the Ras/Raf-1/MEK/ERK signaling pathway.These results suggest a novel role for gelatinase A and B in the regulation of neutrophil functions and their interactions with endothelial cells.Here we describe the methods in detail as they relate to our previously published work.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, University of Alberta. Medical Sciences Building 3-08, Edmonton Alberta, Canada T6G 2S2. Canada.Research Center, Maisonneuve-Rosemont Hospital and Department of Medicine, University of Montréal. Montréal Québec, Canada H1T 2M4. Canada.Mass Spectrometry Facility, Department of Chemistry, University of Alberta. Edmonton Alberta, Canada T6G 2S2. Canada.Perinatal Research Center, University of Alberta. Edmonton Alberta, Canada T6G 2S2. Canada. janos.g.filep@umontreal.ca

ABSTRACT
Recent evidence indicates novel role for matrix metalloproteinases (MMPs), in particular gelatinase A (MMP-2), in the regulation of vascular biology that are unrelated to their well-known proteolytic breakdown of matrix proteins. We have previously reported that MMP-2 can modulate vascular reactivity by cleavage of the Gly32-Leu33 bound in big endothelin-1 (ET-1) yielding a novel vasoactive peptide ET-1[1-32]. These studies were conducted to investigate whether gelatinolytic MMPs could affect neutrophil-endothelial cell attachment. ET-1[1-32] produced by MMP-2 up-regulated CD11b/CD18 expression on human neutrophils, thereby promoted their adhesion to cultured endothelial cells. ET-1[1-32] evoked release of gelatinase B (MMP-9), which in turn cleaved big ET-1 to yield ET-1[1-32], thus revealing a self-amplifying loop for ET-1[1-32] generation. ET-1[1-32] was rather resistant to cleavage by neutrophil proteases and further metabolism of ET-1[1-32] was not a prerequisite for its biological actions on neutrophils. The neutrophil responses to ET-1[1-32] were mediated via activation of ET(A)receptors through activation of the Ras/Raf-1/MEK/ERK signaling pathway. These results suggest a novel role for gelatinase A and B in the regulation of neutrophil functions and their interactions with endothelial cells. Here we describe the methods in detail as they relate to our previously published work.

No MeSH data available.


Related in: MedlinePlus