Abstract
The goal of this study was to define the role of p38alpha MAP kinase in VEGF-induced vascular permeability increase. Activation of p38 is correlated with increased permeability in endothelial cells treated with VEGF or high glucose and in retinas of diabetic animal models. We have shown previously that p38 inhibitors preserve endothelial barrier function and block VEGF-induced GSK/beta-catenin signaling. Here, we present data demonstrating that adenoviral vector delivery of a dominant negative p38alpha mutant blocks this signaling pathway and preserves barrier function. This p38alpha mutant was altered on its ATP-binding site, which eliminates its kinase activity. Bovine retinal endothelial (BRE) cells were transduced with recombinant adenovirus containing the p38alpha mutants or empty vector. Successful transduction was confirmed by expression of GFP and p38 increase. Blockade of p38 activity by p38alpha mutant was demonstrated by inhibition of VEGF-induced phosphorylation of a p38 target, MAP kinase activated protein kinase 2 (MK-2). The mutant also prevented VEGF-induced GSK phosphorylation and beta-catenin cytosolic accumulation and nuclear translocation as shown by cell fractionation and Western blotting. Quantitative real-time PCR demonstrated that this mutant inhibited VEGF-induced uPAR gene expression. Importantly, this same mutant also strongly abrogated VEGF-induced endothelial barrier breakdown as determined by measuring transcellular electrical resistance and tracer flux through endothelial cell monolayer. This study indicates a critical role of p38alpha in VEGF-induced permeability and offers a new strategy for developing potent and specific therapies for treatment of retinal diseases associated with vascular barrier dysfunction.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 101-108 |
| Number of pages | 8 |
| Journal | Experimental eye research |
| Volume | 100 |
| DOIs | |
| State | Published - Jul 1 2012 |
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Keywords
- Beta-catenin
- Endothelial permeability
- P38 MAP kinase
- UPAR
- VEGF
ASJC Scopus subject areas
- Ophthalmology
- Sensory Systems
- Cellular and Molecular Neuroscience
Cite this
Blockade of VEGF-induced GSK/β-catenin signaling, uPAR expression and increased permeability by dominant negative p38α. / Yang, Jinling; Caldwell, Ruth B.; Behzadian, M. Ali.
In: Experimental eye research, Vol. 100, 01.07.2012, p. 101-108.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Blockade of VEGF-induced GSK/β-catenin signaling, uPAR expression and increased permeability by dominant negative p38α
AU - Yang, Jinling
AU - Caldwell, Ruth B.
AU - Behzadian, M. Ali
PY - 2012/7/1
Y1 - 2012/7/1
N2 - The goal of this study was to define the role of p38alpha MAP kinase in VEGF-induced vascular permeability increase. Activation of p38 is correlated with increased permeability in endothelial cells treated with VEGF or high glucose and in retinas of diabetic animal models. We have shown previously that p38 inhibitors preserve endothelial barrier function and block VEGF-induced GSK/beta-catenin signaling. Here, we present data demonstrating that adenoviral vector delivery of a dominant negative p38alpha mutant blocks this signaling pathway and preserves barrier function. This p38alpha mutant was altered on its ATP-binding site, which eliminates its kinase activity. Bovine retinal endothelial (BRE) cells were transduced with recombinant adenovirus containing the p38alpha mutants or empty vector. Successful transduction was confirmed by expression of GFP and p38 increase. Blockade of p38 activity by p38alpha mutant was demonstrated by inhibition of VEGF-induced phosphorylation of a p38 target, MAP kinase activated protein kinase 2 (MK-2). The mutant also prevented VEGF-induced GSK phosphorylation and beta-catenin cytosolic accumulation and nuclear translocation as shown by cell fractionation and Western blotting. Quantitative real-time PCR demonstrated that this mutant inhibited VEGF-induced uPAR gene expression. Importantly, this same mutant also strongly abrogated VEGF-induced endothelial barrier breakdown as determined by measuring transcellular electrical resistance and tracer flux through endothelial cell monolayer. This study indicates a critical role of p38alpha in VEGF-induced permeability and offers a new strategy for developing potent and specific therapies for treatment of retinal diseases associated with vascular barrier dysfunction.
AB - The goal of this study was to define the role of p38alpha MAP kinase in VEGF-induced vascular permeability increase. Activation of p38 is correlated with increased permeability in endothelial cells treated with VEGF or high glucose and in retinas of diabetic animal models. We have shown previously that p38 inhibitors preserve endothelial barrier function and block VEGF-induced GSK/beta-catenin signaling. Here, we present data demonstrating that adenoviral vector delivery of a dominant negative p38alpha mutant blocks this signaling pathway and preserves barrier function. This p38alpha mutant was altered on its ATP-binding site, which eliminates its kinase activity. Bovine retinal endothelial (BRE) cells were transduced with recombinant adenovirus containing the p38alpha mutants or empty vector. Successful transduction was confirmed by expression of GFP and p38 increase. Blockade of p38 activity by p38alpha mutant was demonstrated by inhibition of VEGF-induced phosphorylation of a p38 target, MAP kinase activated protein kinase 2 (MK-2). The mutant also prevented VEGF-induced GSK phosphorylation and beta-catenin cytosolic accumulation and nuclear translocation as shown by cell fractionation and Western blotting. Quantitative real-time PCR demonstrated that this mutant inhibited VEGF-induced uPAR gene expression. Importantly, this same mutant also strongly abrogated VEGF-induced endothelial barrier breakdown as determined by measuring transcellular electrical resistance and tracer flux through endothelial cell monolayer. This study indicates a critical role of p38alpha in VEGF-induced permeability and offers a new strategy for developing potent and specific therapies for treatment of retinal diseases associated with vascular barrier dysfunction.
KW - Beta-catenin
KW - Endothelial permeability
KW - P38 MAP kinase
KW - UPAR
KW - VEGF
UR - http://www.scopus.com/inward/record.url?scp=84862653106&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84862653106&partnerID=8YFLogxK
U2 - 10.1016/j.exer.2012.03.011
DO - 10.1016/j.exer.2012.03.011
M3 - Article
C2 - 22564969
AN - SCOPUS:84862653106
VL - 100
SP - 101
EP - 108
JO - Experimental Eye Research
JF - Experimental Eye Research
SN - 0014-4835
ER -