Eotaxin (CCL11) is an eosinophil-specific chemo attractant which has been found to be highly expressed at sites of vascular pathology.13
Eotaxin selectively attracts eosinophils by activating CCR3 receptors.22
However, it is not clear whether eotaxin could contribute to the progression of atherosclerosis. The present study, for the first time, reports three novel findings: 1). eotaxin increases the paracellular permeability in HCAECs; 2). eotaxin decreases the expression of tight junction proteins involved in the regulation of endothelial barrier functions; and 3). eotaxin may mediate its effects through oxidative stress and activation of the p38 MAPK, Stat3 and NF-κB signaling pathways.
In this study, we used a Costar transwell permeability model system to study the effect of eotaxin on paracellular permeability in HCAECs. This model has been used successfully in this laboratory to analyze the effects of ritanovir,20
on endothelial permeability in vitro
. We selected eotaxin concentrations ranging from 50 to 200 ng/mL based on the plasma eotaxin levels in patients with coronary artery disease.22
Our results show that eotaxin treatment for 24 h significantly increased endothelial permeability in HCAECs in a concentration-dependent manner. To rule out the possibility that the permeability increase is not due to the leakage through confluent HCAECs, monolayer was grown to confluence, stained with Calcein AM and checked under fluorescent microscope to make sure that the monolayer was confluent (data not shown). Heat-inactivated (HI) eotaxin did not show any effects on endothelial permeability indicating its specific effect, but not potential contamination of endotoxin. The endothelial apoptotic cascade is an important underlying mechanism of capillary leakage.23,24
In this study, we have shown that eotaxin did not affect apoptosis of HCAECs in the experimental condition.
Chemokines and chemokine receptors have emerged as important factors involved in the mobilization and function of leukocytes. Chemokine receptors are expressed on a wide range of leukocytes, as well as on endothelial cells, neurons, and possibly other cell types where they are involved in signaling events that can lead to eosinophil and mast cell degranulation, T cell activation, lymphocyte homing, chemotaxis, and mitogenic effects as well as hematopoiesis.25
In addition, overexpression of eotaxin and its receptor, CCR3, in human atherosclerosis have also been reported.11
. In our study, we found that CCR3 receptor is constitutively expressed in HCAECs; while eotaxin treatment did not further increase the expression of CCR3 receptors. CCR3 expression was also observed in human atherosclerotic arteries. Pretreatment of HCAECs with anti-CCR3 antibody significantly reduced eotaxin-induced permeability increase. These findings suggest that CCR3 may play an important role in eotaxin-mediated endothelial permeability increase in HCAECs.
Intercellular junctional structures mediate adhesion and communication between adjoining endothelial cells and comprise of tight junction molecules including transmembrane proteins such as occludin, claudin and JAM-1 and intracellular proteins such as ZO-1 and cingulin as well as adherens junction proteins including transmembrane protein VE-cadherin and intracellular protein β-catenin. Tight junctions serve the major functional purpose of providing a “barrier” and a “fence” within the membrane by regulating paracellular permeability and maintaining cell polarity. In this study, we show that eotaxin significantly decreases the expression of tight junction proteins ZO-1, occludin and claudin-1 in a concentration-dependent manner at both mRNA and protein levels. However, eotaxin does not decrease the expression of VE-cadherin and JAM-1. These data indicate that down-regulation of tight junction proteins may be the key mechanism involved in the paracellular permeability increase in eotaxin-treated HCAECs.
ROS including superoxide anion, hydrogen peroxide, hydroxyl radical, and peroxynitrite play critical roles in cardiovascular disease.26
They could directly cause vascular damages, and could also act as signaling molecules for the gene expression in response to proinflammatory stimuli.27-29
ROS cause endothelial barrier dysfunction through alterations in the cytoskeleton and extracellular matrix.30
ROS are known to quench NO.30
NO synthesis inhibition can potentiate agonist-induced increases in vascular permeability or increase basal microvascular permeability via an alteration of endothelial actin cytoskeleton.31
In the present study, we showed a significant decrease of cellular GSH levels in eotaxin-treated cells, indicating that eotaxin may increase ROS production in HCAECs. Co-treatment of HCAECs with eotaxin and antioxidant MnTBAP substantially increase GSH levels, suggesting that MnTBAP may inhibit eotaxin-induced oxidative stress in HCAECs. To further elucidate the role of ROS in eotaxin-mediated permeability increase, we treated the cells with antimycin A, an inhibitor of electron transport in mitochondria and have been used as a ROS generator in biological systems. We found that antimycin A can induce endothelial permeability and ROS production in HCAECs. To further confirm the involvement of ROS in eotaxin-mediated hyper-permeability, HCAECs were treated with either antioxidant ginkgolide B32
or commonly used antioxidant MnTBAP, a cell permeable SOD mimetic, which effectively blocked the eotaxin-induced increase in vascular permeability and decrease in the expression of tight junction proteins, respectively. The natural antioxidant gingokolide B, a traditional Chinese herb from the plant Gingko Biloba
, has less toxicity and side effects. Antioxidants are believed to counteract ROS and reduce the incidence of coronary artery disease.33
Thus, the current study suggest that eotaxin-induced oxidative stress may be one of the molecular mechanisms involved in the damage of endothelial barrier function and the use of antioxidants could be a novel strategy in the treatment of patients with high incidence of cardiovascular disease.
MAPKs play an important role in mediating cellular functions in response to many extracellular stimuli. There are three important MAPKs including extracellular signal regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 in the cell. In the present study, we show that eotaxin can activate p38 in HCAECs. MAPK p38 activation was at 45 min of eotaxin treatment and there was no activation of p38 at 2 hours after eotaxin treatment. We previously reported that quick activation of MAPK p38 (within 5 to 10 min) was observed during lysophosphatidylcholine-induced increase of monolayer cell permeability in HCAECs.21
Sidestream cigarette smoke could also activate p38 within 60 min and induce endothelial permeability increase in human pulmonary endothelial cells.34
More importantly, specific p38 inhibitor SB203580 effectively blocked eotaxin-induced permeability increase in HCAECs. Blocking ROS generation also inhibited eotaxin-induced phosphorylation of p38, which indicates ROS acts as upstream effector of p38 under the stimulation of eotaxin. Possibly transient expression of p38 initiates the events leading to dysregulation of barrier function through the activation of downstream signal transduction pathways. Indeed, IL-6 can increase endothelial permeability through activation of transcriptional factor Stat3.35
Activation of transcription factor NF-κB has also been indicated for the TNF-α-permeability increase in HCAECs.18
We found activation of Stat3 and NF-κB under the stimulation of eotaxin in HCAEC. It is possible that Stat3 and NF-κB may regulate the expression of tight junction proteins. However, a direct link between Stat3 and NF-κB activation and repression of tight junction proteins has not been determined in this study.
In summary, the present study demonstrates that eotaxin can increase vascular permeability in HCAECs. The underlying molecular mechanisms may involve down-regulation of tight junction proteins, increase of oxidative stress and activation of MAPK p38, Stat3 and NF-κB. This study provides a new understanding of biological functions of eotaxin on the vascular system. Reducing oxidative stress or inhibiting p38 activation may be new strategies for inhibiting the detrimental effects of eotaxin on the vascular system, thereby preventing cardiovascular disease.