Biology Reference
In-Depth Information
loss of cell-cell contacts is a general cause of endothelial dysfunction upon various
pathological stimuli. Thrombin, a protease produced from inactive prothrombin
by proteolytic cleavage on the surface of injured endothelium, is an inflammation
response mediator. Thrombin is responsible for specific cleavage and activation of
protease-activated receptors (PARs), members of the GPCR superfamily [104]. In
EC, PAR-1, which can couple to G
α
12/13, G
α
q/11 and G
α
i heterotrimeric proteins,
stimulates, in turn, PLC
and RhoA pathways and inhibits adenylyl cyclase
[23, 41, 94]. These pathways can essentially lead to an activation of MLC kinase and
inhibition of MLC phosphatase, stress fiber formation and disassembly of cell-cell
junctions in EC [40, 84, 101]. Another agent compromising endothelial integrity
is bacterial lipopolysaccharide (LPS), a component of gram-negative bacteria cell
wall. LPS is a potent inflammatory agent involved in cell response in bacterial
infections. Bovine pulmonary artery EC (BPAEC) exposed to LPS exhibited a
concentration-dependent decrease in transendothelial electrical resistance, activa-
tion of Src, phosphorylation of the focal adhesion protein paxillin, and reduced
expression of the AJ proteins, VE-cadherin and
β
,PKC
α
β
-catenin [17]. LPS treatment, as
well as stimulation of EC with TGF-
and VEGF, increased endothe-
lial permeability via phosphorylation of MLC 20 and hsp27 and formation of stress
fibers [5, 9, 64].
A number of microtubule (MT)-destabilizing agents were also shown to promote
a loss of endothelial integrity via activation of RhoA/ROK pathway and MLC 20
phosphorylation [10, 100]. In cultured HPAEC, RhoA activation was promoted
by Rho-specific GEF-H1 released from disrupted MT upon nocodazole treat-
ment [8]. Another MT-destabilizer, cytostatic anti-tumor agent, 2-methoxyestradiol,
was also able to activate Rho kinase and p38 MAPK followed by hsp27 and
MLC 20 phosphorylation [13]. These studies done in our laboratory clearly demon-
strated a link between MT disruption and following actin stress fiber formation.
Moreover, the molecular mechanisms of endothelial dysfunction and a critical role
of actin-mediated cytoskeleton rearrangement regulated by RhoA/ROK signaling
suggest that purinoceptor-mediated pathways are potential therapeutical approaches
to restore a barrier function of endothelium. Indeed, data obtained from our labo-
ratory and by others using cellular and animal models of acute endothelial injuries
have confirmed an ability of purinergic agonists to protect endothelial integrity from
various disruptive stimuli. In particular, protective effects of ATP and NECA have
been demonstrated in cellular and mouse models of LPS-induced endothelial injury
[55] and our unpublished observation, Fig. 3.2), in thrombin-treated EC [83] and in
cultured EC under oxidative stress conditions [1].
The protective function is mainly associated with PKA activation and decreased
dephosphorylation of MLC 20 . Under normal conditions, extracellular ATP added
to the cultured HPAEC, enhanced the integrity rapidly increasing transendothelial
electrical resistance [54]. Similar effect was also obtained upon stimulation by other
P2Y purinoceptor agonists: ADP and 2MeSATP [54]. ATP-induced transendothelial
electrical resistance increase was significantly attenuated by PKA inhibitors, H89
and KT5720A, and correlated with VASP phosphorylation, dephosphorylation of
β
1, TNF-
α
Search WWH ::




Custom Search