Abstract
Barrier function and shape changes of endothelial cells (EC) are regulated by phosphorylation/dephosphorylation of key signaling and contractile elements. EC contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. EC dysfunction elicited by thrombin was found to correlate with actin microfilament redistribution. It is known that calcineurin (Cn) is involved in thrombin-induced EC dysfunction because inhibition of Cn potentiates PKC activity and the phosphorylation state of EC myosin light chain is also affected by Cn activity. Immunofluorescent detection of Cn catalytic subunit (CnA) isoforms coexpressed with GFP was visualized on paraformaldehyde (PFA) fixed bovine pulmonary artery endothelial cells (BPAEC). Actin microfilaments were stained with Texas Red-phalloidin. Cytotoxic effects of transfections or treatments and the efficiency of transfections were assessed by flow cytometry. Treatment of BPAEC with Cn inhibitors (cyclosporin A and FK506) hindered recovery of the cells from thrombin-induced EC dysfunction. Inhibition of Cn in the absence of thrombin had no effect on cytoskeletal actin filaments. We detected attenuated thrombin-induced stress fiber formation and changes in cell shape only when cells were transfected with constitutively active CnA and not with various CnA isoforms. Flow cytometry (FCM) analysis has proved that cytotoxic effect of treatments is negligible. We observed that Cn is involved in the recovery from thrombininduced EC dysfunction. Inhibition of Cn caused prolonged contractile effect, while overexpression of constitutively active CnA resulted in reduced thrombin-induced stress fiber formation.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 405-411 |
| Number of pages | 7 |
| Journal | Cytometry Part A |
| Volume | 75 |
| Issue number | 5 |
| DOIs | |
| State | Published - May 1 2009 |
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Keywords
- Barrier function
- Endothelium
- Immunofluorescence
- Isoforms of catalytic subunit of calcineurin
- Stress fibers
ASJC Scopus subject areas
- Pathology and Forensic Medicine
- Histology
- Cell Biology
Cite this
Role of calcineurin in thrombin-mediated endothelial cell contraction. / Kolozsvári, Bernadett; Szíjgyártó, Zsolt; Bai, Péter; Gergely, Pál; Verin, Alexander Dmitriyevich; Garcia, Joe G.N.; Bakó, Éva.
In: Cytometry Part A, Vol. 75, No. 5, 01.05.2009, p. 405-411.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Role of calcineurin in thrombin-mediated endothelial cell contraction
AU - Kolozsvári, Bernadett
AU - Szíjgyártó, Zsolt
AU - Bai, Péter
AU - Gergely, Pál
AU - Verin, Alexander Dmitriyevich
AU - Garcia, Joe G.N.
AU - Bakó, Éva
PY - 2009/5/1
Y1 - 2009/5/1
N2 - Barrier function and shape changes of endothelial cells (EC) are regulated by phosphorylation/dephosphorylation of key signaling and contractile elements. EC contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. EC dysfunction elicited by thrombin was found to correlate with actin microfilament redistribution. It is known that calcineurin (Cn) is involved in thrombin-induced EC dysfunction because inhibition of Cn potentiates PKC activity and the phosphorylation state of EC myosin light chain is also affected by Cn activity. Immunofluorescent detection of Cn catalytic subunit (CnA) isoforms coexpressed with GFP was visualized on paraformaldehyde (PFA) fixed bovine pulmonary artery endothelial cells (BPAEC). Actin microfilaments were stained with Texas Red-phalloidin. Cytotoxic effects of transfections or treatments and the efficiency of transfections were assessed by flow cytometry. Treatment of BPAEC with Cn inhibitors (cyclosporin A and FK506) hindered recovery of the cells from thrombin-induced EC dysfunction. Inhibition of Cn in the absence of thrombin had no effect on cytoskeletal actin filaments. We detected attenuated thrombin-induced stress fiber formation and changes in cell shape only when cells were transfected with constitutively active CnA and not with various CnA isoforms. Flow cytometry (FCM) analysis has proved that cytotoxic effect of treatments is negligible. We observed that Cn is involved in the recovery from thrombininduced EC dysfunction. Inhibition of Cn caused prolonged contractile effect, while overexpression of constitutively active CnA resulted in reduced thrombin-induced stress fiber formation.
AB - Barrier function and shape changes of endothelial cells (EC) are regulated by phosphorylation/dephosphorylation of key signaling and contractile elements. EC contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. EC dysfunction elicited by thrombin was found to correlate with actin microfilament redistribution. It is known that calcineurin (Cn) is involved in thrombin-induced EC dysfunction because inhibition of Cn potentiates PKC activity and the phosphorylation state of EC myosin light chain is also affected by Cn activity. Immunofluorescent detection of Cn catalytic subunit (CnA) isoforms coexpressed with GFP was visualized on paraformaldehyde (PFA) fixed bovine pulmonary artery endothelial cells (BPAEC). Actin microfilaments were stained with Texas Red-phalloidin. Cytotoxic effects of transfections or treatments and the efficiency of transfections were assessed by flow cytometry. Treatment of BPAEC with Cn inhibitors (cyclosporin A and FK506) hindered recovery of the cells from thrombin-induced EC dysfunction. Inhibition of Cn in the absence of thrombin had no effect on cytoskeletal actin filaments. We detected attenuated thrombin-induced stress fiber formation and changes in cell shape only when cells were transfected with constitutively active CnA and not with various CnA isoforms. Flow cytometry (FCM) analysis has proved that cytotoxic effect of treatments is negligible. We observed that Cn is involved in the recovery from thrombininduced EC dysfunction. Inhibition of Cn caused prolonged contractile effect, while overexpression of constitutively active CnA resulted in reduced thrombin-induced stress fiber formation.
KW - Barrier function
KW - Endothelium
KW - Immunofluorescence
KW - Isoforms of catalytic subunit of calcineurin
KW - Stress fibers
UR - http://www.scopus.com/inward/record.url?scp=65749110425&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=65749110425&partnerID=8YFLogxK
U2 - 10.1002/cyto.a.20707
DO - 10.1002/cyto.a.20707
M3 - Article
VL - 75
SP - 405
EP - 411
JO - Cytometry. Part A : the journal of the International Society for Analytical Cytology
JF - Cytometry. Part A : the journal of the International Society for Analytical Cytology
SN - 1552-4922
IS - 5
ER -