The production of proinflammatory factors in vascular cells play an important role in atherogenesis.19,20
SDF-1 is a potent chemokine that recruits mononuclear cells to the inflamed tissues,10,11
and has been shown to be highly expressed in human atherosclerotic plaques.12
Moreover, increased levels of blood SDF-1 have been associated with stable coronary disease,21
and SDF-1 is likely to be involved in neointimal hyperplasia or thrombus formation after injury.22
The ability of homocysteine to stimulate SDF-1 gene expression in ECs may lead to the elevation of SDF-1 in the circulation during hyperhomocysteinemia. The mechanism by which homocysteine regulates SDF-1 gene expression of ECs, however, remains unclear. The novel findings of the present study are (Figure S7
): 1) plasma homocysteine is positively correlated with SDF-1 in both high-risk patients and normal volunteers, 2) homocysteine stimulates SDF-1 mRNA expression and protein secretion in ECs, 3) homocysteine-induced SDF-1 expression in ECs is mediated via JNK phosphorylation and Sp1, AP-1 activation, 4) shear stress attenuates the homocysteine-induced SDF-1 expression, and 5) the effect of homocysteine and shear stress on EC SDF-1 expression is mediated by NO.
The results of this study demonstrate that homocysteine not only promotes the secretion of SDF-1, but also induces their gene transcription and expression in human ECs. Analysis of the human SDF-1 promoter activity with different plasmid constructs revealed that AP-1 and Sp1 function as the cis
-element for homocysteine responsiveness via JNK phosphorylation. SDF-1 promoter has different binding sites for various transcriptional factors.17
Previous studies have shown that Sp123
can be activated through the JNK pathway in ECs. Regulation of gene expression through the use of combinations of different transcription factors such as Sp1 and AP-1 has been reported,25,26
and JNK is involved in the phosphorylation and activation of c-Jun.27
In this study, we performed luciferase assays to show that Sp1 and AP-1 cooperate to activate the human SDF-1 promoter and we used TF ELISA and ChIP assays to demonstrate that the regulation of SDF-1 gene expression in ECs was mediated by increased Sp1- and AP-1-DNA binding activities following JNK phosphorylation. Previous study has shown that Sp1 induces a conformational change in the DNA that may contribute to the activation of AP-1 binding.28
The molecular details of Sp1 and AP-1 cooperation in activating the SDF-1 promoter need further investigations. Based on our results, we propose a possible signal transduction pathway in ECs in which homocysteine induces JNK phosphorylation, which activates Sp1 and AP-1, thus resulting in SDF-1 transcriptional activation.
Hyperhomocysteinemia has been defined as a plasma concentration of homocysteine exceeding 15 μmol/L, and is considered severe at levels beyond 100 μmol/L.15
Severe hyperhomocysteinemia produced by genetic disorders usually has plasma homocysteine elevated over than 100 μmol/L. When untreated, affected individuals have a 50% chance of developing a major vascular disease.18
Mild hyperhomocysteinemia is quite prevalent in the general population (plasma homocysteine 15-50 μmol/L), but also has been shown to be associated with increased risks for cardiovascular diseases.29,30
Previous studies have demonstrated that homocysteine might induce the expression of chemokines, e.g., monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) in human monocytes and aortic ECs,31,32
and MCP-1 in human vascular SMCs.33
Homocysteine not only promotes the secretion of MCP-1 and IL-8 in human monocytes, but also enhances the responsiveness of monocytes to MCP-1 in hyperhomocysteinemia patients.31
The present findings of a positive correlation of plasma homocysteine with SDF-1 levels and the elevation of SDF-1 in patients group suggest that SDF-1 may play an important role in the pathogenesis of cardiovascular diseases. Furthermore, our results on human EC cultures showed that treatment with homocysteine concentrations characterizing mild to severe hyperhomocysteinemia (20-500 μmol/L) has stimulatory effects on SDF-1 expression. Therefore, hyperhomocysteinemia might provide an effective stimulus for SDF-1 accumulation in the arterial wall, thus promoting the recruitment of leukocytes, and contributing to proinflammatory responses.
The endothelial lining of the vasculature plays an important role in sensing blood flow perturbations leading to the modulation of gene expression in EC. At vessel bifurcations in an arterial tree, disturbed flow accompanied by LSS predisposes ECs to inflammation in which proinflammatory factors are involved; in contrast, laminar shear stress with a clear direction exerts atheroprotective effects.34,35
Our study showed that homocysteine-induced SDF-1 up-regulation was inhibited in ECs subjected to HSS with a clear direction. Pre-exposure of ECs to a high level of shear stress inhibited homocysteine-induced signal transduction and SDF-1 expression. Previous studies demonstrated that shear stress also induced JNK activation in ECs, and that this shear effect was dependent on the specific extracellular matrix.36,37
However, the shear-induced JNK activation seemed to be dependent on cell type and culture conditions. In our experiments, HUVECs cultured on FN or COLI had similar inhibitory effect on homocysteine-induced SDF-1 expression. It is likely that shear stress modulates inflammatory responses by other mechanisms. Berk et al.38
reported that shear stress induced greater extent of ERK activation, and that the ERK activation then repressed JNK activation in HUVECs, indicating that ERK pathway may mediate the shear stress suppression of JNK. Yoshizumi et al.39
suggested that the possible mechanisms for this ERK-induced suppression include the JNK phosphatases and JNK interacting proteins. The tyrosine phosphatase activity of SHP-2 appears to be required for tumor necrotic factor-α (TNF-α)-induced signaling pathways. Inhibition of the phosphatase activity by shear stress represents a presumable mechanism by which shear stress modulates inflammatory factor-induced signal transduction. Therefore, elucidation of the mechanisms in homocysteine- and shear-mediated signal transduction requires further studies on ECs by manipulating activities of other upstream or downstream signaling proteins.
NO has been recognized to be an anti-inflammatory molecule. It is produced by eNOS in response to physiological stimuli such as acetylcholine, thrombin, or shear stress.40
There is evidence in support of a role of NO in the regulating of gene expression in vascular cells. Endogenous NO production inhibited cytokine-induced expression of adhesion molecules as well as leukocyte adhesion.41
Exogenous addition of NO decreased MCP-1 expression in human ECs,42
and also inhibited the expression of MCP-1 in SMCs.43
Previously observations showed that homocysteine decreases NO bioavailability in cultured ECs.18,44
In addition, homocysteine-induced EC apoptosis was inhibited by NO.45
In this study, administration of a SNAP significantly suppressed homocysteine-induced SDF-1 expression. Furthermore, NO produced from eNOS in response to shear stress has been shown to play a crucial role in shear-mediated anti-atherogenic effects.46
Decreased bioavailability of NO may contribute to thrombosis and atherosclerosis, since the prominent effects of EC-derived NO produced in response to shear stress include vessel relaxation and inhibition of cytokine-triggered platelet and monocyte adhesion.46
Our findings on the effects of L-NAME and eNOS siRNA on the shear-induced inhibition in SDF-1 expression in ECs are in concert with the hypothesis that NO may play a role in the shear-mediated suppression of proatherogenic factor-regulated genes. Pre-exposure of ECs to 4 h HSS inhibited homocysteine-induced SDF-1 expression, and this shear effect was blocked by treatment of L-NAME and transfection of eNOS siRNA. Thus, our findings also indicate that EC-derived NO may mediate homocysteine-induced SDF-1 expression in human ECs.
In summary, the present study demonstrates for the first time an increased expression of SDF-1 in ECs and in the circulating blood during hyperhomocysteinemia. This study has identified a unique molecular mechanism of homocysteine-induced JNK phosphorylation, Sp1 and AP-1 activations, and SDF-1 expression in ECs. Our findings provide a molecular basis for the mechanisms underlying the protective function of laminar shear stress against this SDF-1 induction.