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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 10, 2005 as doi:10.1096/fj.04-2219fje. |
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* Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata-IRCCS, Rome, Italy;
Laboratorio di Biologia Vascolare e Terapia Genica, Centro Cardiologico Fondazione "I. Monzino"-IRCCS, Milan, Italy; and
Institute of Cardiology, Catholic University, Ospedale Gemelli, Rome, Italy
3Correspondence: Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata-IRCCS, Via Monti di Creta 104, Rome 00167, Italy. E-mail: m.napolitano{at}idi.it
SPECIFIC AIMS
Laminar shear stress (LSS) represents a major atheroprotective stimulus, as it inhibits endothelial cell death and has antioxidant and antithrombotic activities. As the underlying mechanisms are still poorly characterized, we aimed at investigating the role of chemokine receptors in LSS-mediated biological activities on endothelial cells (ECs).
PRINCIPAL FINDINGS
1. Laminar shear stress strongly inhibited CXCR4 expression on human primary endothelial cells
Chemokine receptor expression in response to 15 dynes/cm2/s–1 LSS was evaluated on human umbilical vein endothelial cells (HUVEC) by RNase protection assay (RPA). The CXCR4 message was strongly and rapidly down-regulated by LSS treatment at 0.5–16 h at all time points (up to 93.6%±0.5) (Fig. 1
A, C, D) whereas mRNA levels of other chemokine receptors expressed by ECs (i.e., CCR2 and CCR8) were not affected (Fig. 1B
). We demonstrated that LSS-induced CXCR4 down-regulation occurred at the transcriptional but not at the post-transcriptional level (not shown). In contrast, CXCR4 message levels were unmodified at 2 and 5 h of treatment and only slightly decreased (20.7±0.6%) at 16 h in respect to static control cells (Fig. 1E
) after exposure of ECs to 4 dynes/cm2/s–1 (low) shear stress. SDF-1 mRNA levels were not modulated by 2–16 h of LSS treatment (Fig. 1F
).
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2. Laminar shear stress treatment inhibited SDF-1 mediated chemotaxis of HUVECs
We next investigated whether the CXCR4 down-regulation induced by LSS that we observed at the protein level (not shown) may be of functional significance and evaluated the responsiveness of LSS-treated HUVECs to the CXCR4 specific agonist SDF-1. HUVECs were subjected to LSS for 16 h or kept in static conditions and analyzed for their ability to migrate in response to SDF-1 in a 48-well microchamber assay. SDF-1-directed chemotaxis after LSS was totally inhibited at SDF-1 concentration of 10–500 ng/mL (not shown). HUVECs’ chemotaxis in response to the CCR8-restricted chemokine I-309/CCL1, whose receptor expression in HUVECs was not regulated by LSS, was not significantly affected (not shown).
3. CXCR4 overexpression induced caspase-dependent and SDF-1-inducible ECs apoptosis. CXCR4 overexpression inhibited LSS-induced protection from apoptosis on ECs
CXC Chemokine receptor (CXCR) 4 signaling has been reported to modulate cell chemotaxis, survival, and apoptosis. As gp120 was shown to induce cellular apoptosis in ECs partly via CXCR4, we hypothesized that SDF-1-induced CXCR4 signaling in HUVECs may be proapoptotic (Fig. 2
A) and that LSS-triggered CXCR4 down-regulation may contribute to cell survival in response to LSS.
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We found that CXCR4 overexpression significantly inhibited the antiapoptotic effect of LSS (Fig. 2B
) and promoted a caspase-dependent (Fig. 2C, D
) and SDF-1-inducible cellular apoptosis (Fig. 2F
) that was dependent on CXCR4 expression levels (Fig. 2E, F
). Such CXCR4-mediated effect was associated to the impairment of LSS-induced ERK1/2 phosphorylation, a key event in cell survival (not shown). These results suggest that CXCR4 down-regulation may be a mediator of the antiapoptotic effect of LSS.
4. SDF-1 treatment and CXCR4 overexpression in HUVECs induce MCP-1 and IL-8 mRNA expression. CXCR4 overexpression impaired the LSS-induced inhibition of MCP-1 expression
Chemokine receptors and their ligands, such as CCR2/MCP-1 and CXCR2/IL-8, have been shown to play an important role in atherosclerotic lesion development. The inhibition of MCP-1 expression by steady laminar SS (>5 h treatment) in HUVECs has been hypothesized to contribute to the atheroprotective effect of LSS.
In our study, CXCR4-overexpressing ECs enhanced MCP-1 and IL-8 mRNA expression as measured by RNase protection assay (RPA) (not shown). Similarly, SDF-1 treatment of HUVEC for 2 and 5 h increased MCP-1 and IL-8 mRNA expression (not shown). LSS-induced inhibition of MCP-1 message was impaired in CXCR4-expressing cells (not shown).
5. CXCR4 protein was abundantly expressed by the atherosclerotic plaque endothelium of human carotid arteries while barely expressed by minimally diseased carotid arteries
Under the conditions of the present study, CXCR4 expression was maximal in static control cells, which mimic the low absent flow that can be found at sites of atherosclerotic lesion development and strongly down-regulated in ECs exposed to LSS. We therefore analyzed CXCR4 expression in human atherosclerotic carotid arteries vs. minimally diseased carotid arteries obtained from patients undergoing endoarterectomy.
CXCR4 was abundantly expressed by the plaque endothelium while its expression was low in minimally diseased endothelium (not shown).
CONCLUSIONS AND SIGNIFICANCE
The endothelial lining of the vasculature plays an important role in sensing blood flow perturbations leading to fine-tuning of gene expression, which in turn regulates blood flow and endothelial cell function. LSS inhibits endothelial cell proliferation and cellular apoptosis induced by growth factor depletion, tumor necrosis factor (TNF), or hydrogen peroxide exposure. The antiapoptotic effect of laminar shear stress is mediated, in part, by the up-regulation of superoxide dismutase and activation of nitric oxide synthase and, ultimately, by nitric oxide (NO) production leading to the inhibition of caspase activation.
Atherosclerotic lesions are preferentially found in areas with low or turbulent shear stress while areas exposed to steady LSS are protected by plaque formation. A large body of evidence suggests that endothelial apoptosis contributes to the development of atherosclerotic lesions in areas of low or turbulent flow with a prevalent occurrence of apoptosis in the downstream part of the plaque. Endothelial apoptosis is proadhesive and promotes smooth cell migration from the media to the intima, an important event in intimal thickening. Further, it stimulates plaque erosion leading to thrombosis and to the establishment of acute coronary syndromes.
Our findings suggest that CXCR4-mediated signals may trigger apoptosis in primary endothelial cells provided sufficient levels of CXCR4 are present. Our data suggest that LSS keeps CXCR4 expression on the endothelium at lower levels than in endothelial cells subjected to low/absent shear stress. This may help regulate endothelial cell survival and integrity, thus contributing to protection toward the development of atherosclerotic lesions.
MCP-1 down-regulation exerted by constant levels of laminar shear stress is hypothesized to play a role in maintaining atherosclerotic lesion-free areas under steady flow. CXCR4 signaling induced an up-regulation of MCP-1 and IL-8 mRNA levels in endothelial cells and impaired LSS-induced inhibition of MCP-1 message.
In agreement with our hypothesis that CXCR4 expression may play a role in atherosclerotic plaque development, we found high levels of CXCR4 expression in endothelial cells from human carotid artery atherosclerotic lesions. In contrast, CXCR4 expression was low in minimally diseased carotid artery endothelium.
The results of the present study suggest that the antiatherogenic effect of laminar shear stress may be mediated, in part, through the down-regulation of CXCR4 (Fig. 3
), thereby affecting the expression of proatherogenic chemokines and the integrity of the endothelial barrier.
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FOOTNOTES
1 These authors contributed equally to this work. 
2 Present address: Laboratory of Cardiovascular Sciences, NIA, NIH, Baltimore MD 21224, USA.
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2219fje;
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