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Hyperhomocysteinemia contributes to vascular dysfunction and risks of cardiovascular diseases. Stromal cell-derived factor-1 (SDF-1), a chemokine expressed by endothelial cells (ECs), is highly expressed in advanced atherosclerotic lesions. The interplays among homocysteine, chemokines and shear stress in regulating vascular endothelial function are not clearly understood.
To investigate the mechanisms for modulations of EC SDF-1 expression by homocysteine and shear stress.
Homocysteine stimulation induced dose- and time-dependent SDF-1 expression and phosphorylation of mitogen-activated protein kinases (MAPKs) ERK, JNK, and p38. By using specific inhibitors, small interfering RNA (siRNA), and dominant negative mutants, we demonstrated that activation of JNK pathway is critical for the homocysteine-induced SDF-1 expression. Transcription factor ELISA and chromatin immunoprecipitation assays showed that homocysteine increased Sp1- and AP-1-DNA binding activities in ECs. Inhibition of Sp-1 and AP-1 activations by specific siRNA blocked the homocysteine-induced SDF-1 promoter activity and expression. Preshearing of ECs for 1-4 h at 20 dyn/cm2 inhibited the homocysteine-induced JNK phosphorylation, Sp1 and AP-1 activation, and SDF-1 expression. The homocysteine-induced SDF-1 expression was suppressed by nitric oxide (NO) donor. Inhibitor or siRNA for endothelial NO synthase (eNOS) abolished the shear-inhibition of SDF-1 expression.
Our findings serve to elucidate the molecular mechanisms underlying the homocysteine induction of SDF-1 expression in ECs and the shear stress protection against this induction.
Vascular endothelial cells (ECs) are constantly exposed to fluid shear stress, the nature and magnitude of which play a significant role in the homeostasis of blood vessels. The preferential development of atherosclerosis at arterial branches and curvatures, where local flow is disturbed, suggests a role of shear stress in atherogenesis. Adhesion of circulating monocytes and T-lymphocytes to ECs and their subsequent migration across the EC monolayer are early events in atherogenesis.1 Physiological levels of shear stress can modulate cellular signaling and EC function in ways that are protective against atherogenesis.2,3 A number of pathophysiologically relevant genes, such as adhesion molecules,4 growth factors,5 and chemokines,6 have been shown to be regulated by shear stress.
Chemokines play a significant role in atherosclerotic lesion development, primarily by inducing the transendothelial migration of leukocytes. Furthermore, chemokines may stimulate smooth muscle cell (SMC) proliferation and migration from media to intima, as well as angiogenesis, thus influencing plaque formation, progression and rupture.7 Chemokines are divided into CXC, CC, C, or CX3C based on their structural properties and primary amino acid sequence.8 Stromal cell-derived factor-1 (SDF-1), a member of the CXC family, is a strong chemoattractant in EPCs, lymphocytes, and monocytes9,10 and implicated in atherogenesis.11 SDF-1 has also been shown to induce platelet activation and aggregation via CXCR4 expressed on platelets, suggesting a link with atherothrombosis.12 SDF-1 expression can be regulated by extracellular factors derived from inflammatory or injured vascular tissues. The molecular mechanisms involved in the up-regulation of SDF-1 expression in pathophysiological conditions such as hyperhomocysteinemia is still unknown.
Homocysteine is a sulfur-containing amino acid formed during the metabolic demethylation of methionine. Elevated blood levels of homocysteine are related to higher risks of coronary heart disease, stroke and peripheral vascular disease.13 Homocysteine may promote vascular damage and atherothrombosis via several mechanisms, including release of proinflammatory mediators, induction of endothelial dysfunction, and activation of apoptotic pathways in vascular cells.14 The relationship between the plasma levels of homocysteine and SDF-1, the mechanisms underlying homocysteine-induced SDF-1 expression, and the role of shear stress in the modulation of homocysteine-induced gene expression still remain unclear.
We aimed to establish the relationship between plasma homocysteine and SDF-1 levels in human blood and the interplay between shear stress and homocysteine stimulation in modulating endothelial gene expression by analyzing the effect of shear stress on homocysteine-induced SDF-1 expression in human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs). We found that the induction of SDF-1 expression by homocysteine was mediated via phosphorylation of JNK and activation of the transcription factors activator protein 1 (AP-1) and Sp1. Furthermore, fluid shear stress attenuated the homocysteine-induced SDF-1 expressions at mRNA and protein levels, and ECs subjected to shear stress suppress the homocysteine-induced JNK phosphorylation and transcription factor activation. In addition, homocysteine-induced SDF-1 expression was modulated by nitric oxide (NO): An NO donor suppressed homocysteine-induced SDF-1 expression, and inhibition of endothelial NO synthase (eNOS) attenuated shear stress-inhibition of SDF-1 expression. These findings on the mechanisms of suppression of homocysteine-induced responses in ECs by shear stress provide new insights into the pathophysiology underlying the atheroresistancy of straight segments of vascular tree.
Materials and the procedures of subjects; measurement of plasma homocysteine; SDF-1 enzyme-linked immunosorbent assay (ELISA); endothelial cell culture; shear stress experiment; real-time quantitative PCR; western blot analysis; reporter gene construct, siRNA, transfection, and luciferase assays; adenoviral Infection; Sp1 and AP-1 transcription factor assays (TF ELISA assays); chromatin immunoprecipitation assay (ChIP); and statistical analysis are provided in Online Supplemental Document.
The mean plasma homocysteine concentration was significantly higher in high-risk patients (30.94 ± 4.13 μmol/L) than normal volunteers (14.08 ± 1.02 μmol/L). Linear regression shows a positive correlation between plasma homocysteine and SDF-1 levels in patients (Figure 1A, r = 0.81, p < 0.0001) and volunteers (Figure 1B, r = 0.51, p < 0.001). Linear regression of data combined the two groups was showed in Figure S1 (r = 0.79, p < 0.0001). We divided all subjects (patients + normal volunteers) according to their plasma homocysteine levels into normal (< 15 μmol/L, n=24, 10 from patients), mild (16-30 μmol/L, n=36, 11 from patients), and intermediate (31-100 μmol/L, n=12, 11 from patients) hyperhomocysteinemia.15 Plasma SDF-1 level was significantly higher in subjects with intermediate hyperhomocysteinemia than normal or mild hyperhomocysteinemia (Figure 1C), indicating that higher levels of plasma homocysteine were associated with higher SDF-1.
HUVECs and HAECs were stimulated with homocysteine at 100 μM for the times indicated, or different doses (0-500 μM) for 4 h (for mRNA expression by real-time PCR) and 12 h (for protein secretion by ELISA). In ECs, the SDF-1 mRNA level began to increase after 1 h of homocysteine stimulation and reached highest level at 4 h; thereafter it gradually reduced to a level that is not significantly different from the static control in HUVECs at 12 hr, but was still elevated at this time in HAECs (Figure 2A). SDF-1 protein in the conditioned medium also increased after stimulation (Figure 2C). The inductions of SDF-1 mRNA expression and protein secretion by homocysteine were dose-dependent (Figures 2B and 2D).
The viability of ECs was over 94% when cultured for 24 h with 20-200 μM homocysteine, and 87% with 500 μM homocysteine (data not shown).
Members of the MAPK superfamily are known to regulate EC gene expression and cellular functions.16 The phosphorylation of ERK, JNK, and p38 in HUVECs increased rapidly after homocysteine stimulation, reaching maximal levels at 2 min for ERK and JNK, and 5 min for p38 (Figure 3A). After transient increases, phosphorylation decreased to nearly basal levels (Figure S2 shows that JNK phosphorylation decreased to control at 60 min, but ERK and p38 phosphorylations were still elevated at 120 min).
HUVECs were incubated with specific inhibitors for ERK (PD98059; 30 μM), JNK (SP600125; 20 μM), or p38 (SB203580; 10 μM) for 1 h before and during stimulation with homocysteine. The homocysteine-induced SDF-1 mRNA expression (Figure 3B) and protein secretion (Figure 3C) were significantly inhibited by SP600125, but not by PD98059 and SB203580. The homocysteine-induced SDF-1 mRNA expression was also inhibited by transfection with JNK-specific siRNA or infection with Ad-DN-JNK, but not by ERK- or p38-specific siRNA (100 μmol/mL each), nor with Ad-DN-ERK or Ad-DN-p38 (Figure 3D). The effectiveness of these treatments was validated: ERK-, JNK-, and p38-specific siRNA (compared with control siRNA) caused a 75% reduction in ERK, JNK, and p38 protein expressions, respectively (data not shown). The DN-ERK, DN-JNK and DN-p38 caused at least 80% inhibitions in homocysteine-induced ERK, JNK and p38 phosphorylation (compared with control Ad-GFP) (data not shown).
The human SDF-1 gene promoter contains multiple transcription factor binding sites, which have been shown to be responsive to different stimuli.17 To elucidate the cis-acting elements in the SDF-1 gene promoter that mediate the homocysteine-induced SDF-1 transcription, luciferase assay was conducted with p-1010-Luc plasmid and several deletion promoters constructs (Figure 4A). Transient transfection of ECs showed that the 5′-flanking region of human SDF-1 (-1010/+122) could drive transcription of a luciferase reporter (Figure 4B). Construction and analyses of 5′-deletion mutants in the -1010/+122 region of SDF-1, which directed maximum luciferase activity, showed that the activity decreased to ~45% following sequence deletion from -430 to -214, to ~75% after further deletion to -111, and was nearly abolished following 5′-deletion to -20 (Figure 4B).
To test whether AP-1 and Sp1 activations are involved in the signal transduction pathway leading to the homocysteine-induced SDF-1 gene expression, we transfected HUVECs with siRNA of Sp1 or c-jun, or incubated them with the specific inhibitor for Sp1 (mithramycin A, 100 nM) for 1 h, prior to stimulation with 50 or 100 μM homocysteine for 4 h. The homocysteine-induced SDF-1 mRNA expression (Figure 4C) and SDF-1 promoter activity (Figure 4D) were significantly reduced by inhibition with mithramycin A, or siRNA of Sp1 or c-jun.
To investigate whether Sp1 and AP-1 bind the SDF-1 promoter region in HUVECs, we performed quantitative analysis for Sp1 and AP-1 binding activities in vitro by using TF ELISA kits (Panomics). Treatment of HUVECs with 100 μM homocysteine caused both Sp1- and AP-1-DNA binding activities to increase at 30 min and remain elevated for at least 2 h (Figures 5A and 5B, respectively).
ChIP analysis was performed by subjecting immunoprecipitated chromosomal DNA with anti-Sp1 antibody to PCR using primers designed to amplify the SDF-1 promoter region harboring the Sp1 binding sites. Sp1 indeed bound to the SDF-1 promoter region containing the Sp1 sites (Figure 5C). Similarly, the DNA sequence including the AP-1 sites was specifically immunoprecipitated with anti-c-jun antibody (Figure 5D). These data suggested that the Sp1 and AP-1 binding sites play critical roles in the regulation of SDF-1 by homocysteine.
To evaluate whether the inhibition of SDF-1 expression by JNK signaling pathway occurred at the transcriptional level, we studied the effect of SP600125, JNK siRNA, and DN-JNK on homocysteine-induced SDF-1 promoter activity with the use of p1010-luc reporter construct that contains -1010 bp of the proximal promoter sequences of human SDF-1 gene (p1010-Luc plasmid). Stimulation with homocysteine increases the luciferase activity significantly in HUVECs over the unstimulated control after normalization by transfection control (Figure 6A). Transfection of ECs with JNK siRNA or DN-JNK resulted in a marked inhibition of the homocysteine-induced SDF-1 promoter activity. However, transfection of cells with siRNA for ERK or p38, DN-ERK and DN-p38 had little inhibitory effect on this homocysteine inducibility (Figure 6A). In HUVECs, transfection with siRNA or promoter constructs under the concentrations used does not cause cytotoxicity based on the cell number and morphology (data not shown).
To explore whether JNK activates the SDF-1 promoter leading to SDF-1 transcription via activation of Sp1 and AP-1, HUVECs were pretreatment with MAPK inhibitors, transfection with JNK siRNA, or infection with Ad-DN-JNK, and followed by homocysteine stimulation, and then the Sp-1 and AP-1 activations were assessed by TF ELISA kits. Homocysteine-induced AP-1 and Sp1-DNA binding activities were significantly inhibited in cells pretreatment with SP600125, JNK siRNA, or Ad-DN-JNK, but not in pretreatment with PD98059, SB203580, control siRNA, or Ad-GFP (Figures 6B and 6C).
Pre-exposure of ECs to HSS at 20 dyn/cm2 for 4 h significantly inhibited homocysteine-induced SDF-1 mRNA expression (Figure 7A), and this effect is shear time-dependent in HUVECs (Figure S3). Low shear stress (LSS) at 0.5 dyn/cm2 did not have such an effect. Both HSS and LSS alone had no significant effect on SDF-1 expression (Figure 7A). In addition, HUVECs cultured on fibronectin (FN) or collagen type I (COLI) and pre-exposed to HSS for 4 h had similar inhibitory effect on homocysteine-induced SDF-1 expression (Figure S4). Pre-exposure of HUVECs to HSS resulted in a marked inhibition of the homocysteine-induced JNK phosphorylation (Figure 7B). Pre-exposure of HUVECs to HSS also resulted in a significant inhibition of the homocysteine-induced SDF-1 promoter activity (Figure S5).
Preshearing of HUVECs at HSS for 4 h significantly inhibited the homocysteine-induced Sp-1- and AP-1-DNA binding activities in vivo by ChIP assays (Figures 7C and 7D) and in vitro by using TF ELISA kits (Figures S6A and S6B). These results provide additional evidence that shear stress plays an important role in the inhibition of the JNK-, Sp1- and AP-1-mediated SDF-1 expression in ECs induced by homocysteine stimulation.
A major effect of hyperhomocysteinemia-induced EC dysfunction appears to be related to decreased bioavailability of EC-derived NO.18 To investigate whether the homocysteine-induced SDF-1 expression is modulated by NO, both HUVECs and HAECs were incubated with different doses of NO donor S-nitroso-N-acetyl-penicillamine (SNAP) for 4 h before and during stimulation with homocysteine. The homocysteine-induced mRNA expression of EC SDF-1 was significantly inhibited by 20-100 ìM SNAP treatment (Figure 8A). Conversely, addition of 100 μM NG-nitro-L-arginine methyl ester (L-NAME) or transfection of eNOS siRNA before exposure to 4 h of HSS abolished the shear-mediated inhibition of SDF-1 expression (Figure 8B). These results indicated that NO plays an important role in the homocysteine-induction and shear-inhibition of SDF-1 expression in ECs.
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 and AP-124 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.
Figure S1. Relationships between plasma levels of homocysteine and SDF-1 in all subjects (n = 72). Black circles: patients recruited from outpatients at high risk for cardiovascular events. White circles: normal volunteers from routine physical examinations.
Figure S2. Stimulation with homocysteine induces HUVECs to increase their phosphoryalation of ERK (A), JNK (B), and p38 (C). HUVECs were kept as controls (CL) or stimulated with 100 μM homocysteine for the times indicated, and the phosphorylations of ERK, JNK, and p38 were determined by using Western blot analysis. Phosphorylated ERK, JNK, and p38 levels are presented as band densities (normalized to total protein levels) relative to CL. The results are mean ± SEM from 3 independent experiments. *P < 0.05 versus control EC (CL).
Figure S3. HUVECs were kept as controls (CL) or presheared at HSS (20 dyn/cm2) for 1 ~ 4 h before homocysteine stimulation. Static ECs were stimulated with homocysteine without preshearing (static). Data are presented folds of control ECs (CL), mean ± SEM. #P < 0.05 versus static ECs (static) with homocysteine stimulation.
Figure S4. HUVECs cultured on FN- or COLI-coated glass were presheared at HSS (20 dyn/cm2) for 4 h (HSS/FN or HSS/COLI) before homocysteine stimulation. Static ECs were ECs cultured on FN- or COLI-coated glass and stimulated with homocysteine without preshearing (static). Data are presented folds of control ECs (CL), mean ± SEM. *P < 0.05 versus CL. #P < 0.05 versus static ECs (static/FN or static/COLI) with homocysteine stimulation.
Figure S5. HUVECs were kept as controls (CL) or presheared at HSS (20 dyn/cm2) or LSS (0.5 dyn/cm2) for 4 h before homocysteine stimulation. Static ECs were stimulated with homocysteine without preshearing (static). The SDF-1 p1010-Luc activity after 4 h homocysteine stimulation was determined by luciferase assay normalized to β-galactosidase. Data are presented folds of control ECs (CL), mean ± SEM. *P < 0.05 versus CL. #P < 0.05 versus static ECs (static) with homocysteine stimulation.
Figure S6. HUVECs were kept as controls (CL) or presheared at HSS (20 dyn/cm2) or LSS (0.5 dyn/cm2) for 4 h before homocysteine stimulation. Static ECs were stimulated with homocysteine without preshearing (static). Sp1 (A) and AP-1 (B) activation after 2 h homocysteine stimulation in HUVECs was performed by TF ELISA assay. Data are presented folds of control ECs (CL), mean ± SEM. *P < 0.05 versus CL. #P < 0.05 versus static ECs (static) with homocysteine stimulation.
Figure S7. Schematic representation of the inhibitory effects of shear stress and the signaling pathway regulating the expressions of SDF-1 in ECs in response to homocysteine stimulation.
Sources of Funding: This work was supported by St. Martin De Porres Hospital (project no. P0703) (M.-L.S.); NIH (NHLBI) Research Grant HL-085159 (S.C); and National Science Council (Taiwan) (grant NSC97-2320-B-415-007-MY3) (C.-N.C.)