Published online before print February 28, 2008, doi: 10.1161/CIRCRESAHA.108.171082
© 2008 American Heart Association, Inc.
Molecular Medicine |
Homocysteine Inhibits Arterial Endothelial Cell Growth Through Transcriptional Downregulation of Fibroblast Growth Factor-2 Involving G Protein and DNA Methylation
From the Departments of Internal Medicine (P.-Y.C., C.-M.L., Y.-T.L.) and Biochemistry and Molecular Biology (S.-C.L., Y.-J.C., W.-H.H.), National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei; Graduate Institute of Cell and Molecular Biology (S.-F.C.), Taipei Medical University, Taipei; the Department of Biochemistry and Molecular Biology (W.S.L.L.), Program in Genes and Development, University of Texas M.D. Anderson Cancer Center, Houston; and the Department of Medicine (T.A.D., C.-H.C.), Baylor College of Medicine, Houston, Tex.
Correspondence to Yuan-Teh Lee, MD, PhD, No. 7, Chung-Shan South Rd, Taipei 100, Taiwan. E-mail ytlee{at}ha.mc.ntu.edu.tw
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Homocysteine (Hcy) contributes to atherogenesis and angiostasis by altering the phenotype of arterial endothelial cells (ECs). The present study was aimed at elucidating potential mechanisms by which Hcy can slow EC proliferation and induce EC apoptosis, thereby disrupting endothelial integrity. Given the strong mitogenic and antiapoptotic properties of fibroblast growth factor (FGF)2, we examined whether Hcy can modulate its expression. In cultured human coronary and bovine aortic ECs, Hcy exerted time- and concentration-dependent (0 to 500 µmol/L) reduction of the mRNA and protein levels of FGF2, whereas vascular endothelial growth factor expression was not affected until Hcy reached a proapoptotic 500 µmol/L. By testing a panel of signal transduction inhibitors, we found that the Hcy-induced downregulation of FGF2 was specifically attenuated by pertussis toxin, an inhibitor of Gi protein signaling. Hcy induced cell cycle arrest at the G1/S transition and increased TUNEL-positive apoptotic cells in a graded manner. These effects were effectively counteracted by exogenous FGF2. Reporter gene assays showed that Hcy downregulated FGF2 by transcriptional repression of the gene promoter encompassed in a CpG dinucleotide-rich island. This region was heavily methylated at the cytosine residues by Hcy despite decreased methylation potential (S-adenosylmethionine to S-adenosylhomocysteine ratio). Normal levels of FGF2 transcription were restored to ECs simultaneously exposed to Hcy and 5-aza-deoxycytidine. We conclude that homocysteine disrupts the growth and survival of ECs through a G protein–mediated pathway associated with altered promoter DNA methylation and the transcriptional repression of FGF2.
Key Words: homocysteine • endothelial cells • growth factors • transcriptional regulation • DNA methylation
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Occlusive vascular diseases profoundly contribute to morbidity and mortality in industrialized populations, prompting exhaustive approaches to identifying and characterizing potential etiologic factors. One potential factor, an elevated plasma homocysteine (Hcy) concentration, arises from the abnormal metabolism of methionine from dietary protein. Nearly 40 years ago, a series of case studies led to the proposal that despite different metabolic deficiencies causing homocystinuria, the common excess metabolite homocysteine induced premature atherosclerosis and fatal thrombosis.1,2 Controversy remains over whether hyperhomocysteinemia (HHcy) is an independent or a conditional risk factor or simply a marker of cardiovascular disease.3–6 Mild to moderate HHcy has been detected in 12% to 47% of patients exhibiting coronary, cerebral, and peripheral arterial diseases.3 Abundant clinical and epidemiological studies have associated HHcy with heightened risks of arterial diseases, yet not all prospective studies have reached this conclusion. Recent metaanalyses have indicated that increases in plasma Hcy concentration of merely 3 to 5 µmol/L can raise the risk for myocardial infarction, stroke, or venous thromboembolism by 10% to 27%.7–9 Likewise, some, yet not all, clinical trials testing Hcy-lowering strategies have found a diminished recurrence of cardiovascular events such as restenosis, stroke, or venous thromboembolism.10–12
Atherosclerosis/angiostasis partially stems from the injury or phenotypic alteration of endothelial cells (ECs), the cells in the frontline against vascular disturbance. The histological examination of hyperhomocysteinemic (HHcyc) rats revealed an increased recruitment of monocytes to aortic endothelium accompanied by elevated immunostaining for monocyte chemoattractant protein-1, vascular cell adhesion molecule-1, and E-selectin.13 Studies in humans and several transgenic murine models of HHcy have uncovered widespread arterial endothelial dysfunction in cerebral, mesenteric, and cremasteric arterioles.14–17 Endothelial dysfunction in HHcyc mice has been associated with a decreased bioavailability of endothelial NO through diminished eNOS activity; however, the induction of oxidative stress does not consistently represent a Hcy-specific mechanism of endothelial damage.16,17 Recently, more and more data have accumulated that inhibition of angiogenesis in HHcyc subjects can be attributed to EC apoptosis and impaired angiogenic response involving EC proliferation, migration, and tube formation.18,19 Although the angiostatic effect of Hcy has been suggested to arise from the decreased expression of angiogenic growth factors, the underlying mechanism has not been thoroughly characterized.20
The specific conversion of Hcy to S-adenosyl Hcy (SAH) represents another mechanism capable of perturbing EC phenotype.21,22 Altered methylation patterns associated with increased levels of SAH have been reported to modulate protein function and the transcriptional control of GC-rich promoters.23,24 In addition, Hcy conversion to SAH was associated with decreases in p21ras carboxymethylation, extracellular signal-regulated kinase 
-mediated signaling and cyclin A expression.22,25,26 Altogether, ECs treated with clinically relevant concentrations of Hcy exhibited decreased DNA synthesis and delayed progression through the cell cycle at the G1/S transition.22
We previously demonstrated that copper-oxidized LDL (oxLDL) and L5, an electronegative LDL isolated from hypercholesterolemic or type 2 diabetic human plasma, can inhibit EC proliferation and angiogenesis.27,28 These angiostatic effects of oxLDL and L5 were accompanied by the downregulation of fibroblast growth factor (FGF)2 and inhibited by FGF2 supplementation.29–32 In contrast, vascular endothelial growth factor (VEGF) failed to attenuate oxLDL-induced angiostasis.29 We also found that a pertussis toxin–sensitive G protein pathway was involved in the oxLDL-mediated downregulation of FGF2.31 The ability of Hcy to inhibit EC proliferation and angiogenesis resembled that of our modified LDLs. On the basis of this finding, we confirmed the effects of clinically significant Hcy concentrations on EC growth and survival before mechanistic studies investigating whether Hcy can alter the expression of FGF2 or VEGF through specific regulatory pathways.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The chemicals and reagents used in this study and all experimental techniques, including cell cultures and treatment protocols, inhibitors of signal transduction pathways, DNA synthesis, FGF2 and VEGF ELISA, cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, fluorescence-activated cell-sorting analysis, TUNEL assays, real-time PCR, cell transfection and reporter gene assays, liquid chromatography–tandem mass spectrometry (MS/MS) measurement for S-adenosylmethionine (SAM) and SAH and bisulfite genomic DNA sequencing are described in the online data supplement, available at http://circres.ahajournals.org.
Statistical Analysis
The significance of the differences between group means was assessed by a modified t test (Bonferroni test) for multiple comparisons. An ANOVA followed by Scheffé’s test for significance were used to compare concentration- and time-dependent responses. A probability value of <0.05 was considered significant. Results were expressed as means±SEM values.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Hcy Decreased DNA Synthesis and Cell Viability
The exposure of bovine aortic ECs (BAECs) to Hcy for 24 hours decreased DNA synthesis in a concentration-dependent manner (Figure 1A). Compared with PBS control, 50 and 100 µmol/L Hcy reduced 3H-thymidine incorporation by 20% and 30%, respectively, without increasing the number of dead cells. Similarly, MTT assay revealed that treatment with 100 µmol/L Hcy decreased cell viability by 40% (Figure 1A). At concentrations >100 µmol/L, Hcy not only decreased DNA synthesis by 70% but also greatly reduced cell viability and markedly increased dead cell population (Figure 1A). Consistent with the inhibitory effect on 3H-thymidine incorporation, Hcy markedly decreased cell viability in a time- and concentration-dependent manner, whereas cysteine had no effect (Figure 1B). When Hcy was removed from the medium at 24 hours, cell viability was restored gradually through 96 hours (Figure I in the online data supplement).
|
), cell viability (
), and cell death (
) were assessed. Values are means±SEM (n=3). *P<0.05, **P<0.01 vs corresponding untreated controls. B, Time-dependent effects. BAECs (5x104) were incubated with PBS (
) or 100 µmol/L Hcy (
), or 100 µmol/L cysteine (
) for 0 to 96 hours, followed by cell viability assay. Values are means±SEM (n=3). *P<0.05 vs PBS control.
Hcy Reduced FGF2 Expression in BAECs and Human Coronary Arterial ECs
Because FGF2 is a potent mitogenic and antiapoptotic protein, we examined whether Hcy may affect its endogenous expression. As assayed by ELISA, Hcy decreased intracellular FGF2 in a concentration-dependent (25 to 500 µmol/L at 24 hours) and time-dependent (24 to 96 hours at 100 µmol/L) manner in BAECs (Figure 2A, top graphs). These concentration- and time-dependent effects of Hcy on FGF2 reduction were species independent because human coronary arterial ECs (HCAECs) had similar response to Hcy (Figure 2A, bottom graphs). FGF2 mRNA levels were determined by real-time PCR (Figure 2B). In both BAECs and HCAECs treated with 100 µmol/L Hcy for 24 hours, FGF2, but not FGFR1, mRNA was decreased by 40% relative to PBS control, compatible with the extent of FGF2 protein reduction assayed by ELISA. Cysteine and methionine, 2 important sulfur-containing amino acids derived from Hcy metabolism, had no effect.
|
In comparison with FGF2, the intracellular VEGF protein levels were not changed until Hcy reached a proapoptotic concentration of 500 µmol/L at 24 hours in BAECs (supplemental Figure IIA) and HCAECs (supplemental Figure IIB). There was no reduction in intracellular VEGF through 96 hours with 100 µmol/L Hcy. In contrast to FGF2 mRNA, which was reduced by 100 µmol/L Hcy, VEGF mRNA levels were not affected at this physiological concentration. When Hcy was raised to 500 µmol/L, both FGF2 and VEGF mRNA were remarkably reduced as a result of cell death (supplemental Figure IIC).
Involvement of G Protein Signaling in Hcy-Induced FGF2 Downregulation
The signal transduction pathways of Hcy-mediated FGF2 downregulation were investigated using pharmacological inhibitors (Figure 2C). At 24 hours Hcy (100 µmol/L) decreased intracellular FGF2 protein by 
40% compared with PBS. This inhibitory effect was remarkably blocked in cells incubated with the G protein inhibitor pertussis toxin (100 ng/mL) for 18 hours before Hcy exposure. In contrast, pretreating the cells with the MEK/MAPK inhibitor PD-98059 (20 µmol/L) for 18 hours or the protein kinase C inhibitor Ro-31 to 8220 (3.5 µmol/L) for 1 hour failed to counteract the inhibitory effects of Hcy on FGF2 protein levels. Blocking the cGMP- and cAMP-dependent protein kinase with HA-1004 (10 µmol/L) for 18 hours or chelating the intracellular Ca2+ with BAPTA-AM (16 µmol/L) for 1 hour also failed to attenuate the effect of Hcy.
Effects of FGF2 on Hcy-Induced Cell Cycle Arrest and Apoptosis
Given that Hcy can inhibit the expression of FGF2 in ECs, we examined whether providing these growth factors could counteract the effects of Hcy on DNA synthesis and cell cycle progression. In the absence of Hcy, both FGF2 and VEGF-165 (50 ng/mL each) significantly increased DNA replication. With 100 µmol/L Hcy, however, only FGF2 completely restored normal levels of DNA synthesis (Figure 3A). Thus, Hcy did not impair the ability of exogenous FGF2 to promote DNA replication. Conversely, in the presence of Hcy, VEGF-165 did not significantly increase DNA replication. The exposure of BAECs to 100 µmol/L Hcy significantly increased the percentage of cells retained in the G1 phase of the cell cycle (from 60% to 76%; P<0.05; n=3) (Figure 3B and supplemental Table II), with a concomitant decrease in cells entering the S phase and no effect on the G2/M transition. As anticipated, the simultaneous treatment of BAECs with FGF2 prevented Hcy-induced G1 arrest, whereas VEGF-165 was ineffective. This could reflect the noninvolvement of VEGF in this effect of Hcy (supplemental Figure II).
|
The effects of FGF2 and VEGF on Hcy-induced EC apoptosis were evaluated using TUNEL assay (Figure 4). BAECs treated with graded concentrations of Hcy for 24 hours revealed a dose-dependent increase in the percentage of TUNEL-positive cells. At 100 µmol/L, Hcy had no significant effect on EC apoptosis compared with PBS control (7.2±5.3% vs 2.0±1.1%; n=3). At 200 and 500 µmol/L, the TUNEL-positive cells represented 38.2±11.4% (P<0.05; n=3) and 78.2±13.1% (P<0.01; n=3), respectively, compared with PBS control. When FGF2 was added to BAECs cultured in 500 µmol/L Hcy, the TUNEL-positive cells decreased markedly (24.7±12.8% vs 78.2±13.1%; P<0.01; n=3). In comparison, VEGF moderately attenuated the apoptosis-inducing activity of high-dose Hcy (45.6±9.3% vs 78.2±13.1%; P<0.05; n=3).
|
P<0.05,
Transcriptional Regulation of FGF2
Human FGF2 genomic DNA fragments were generated by PCR and inserted into the firefly luciferase vector pGL3. The promoter activities of these constructs were evaluated in HCAECs in the presence or absence of 100 µmol/L Hcy (Figure 5). Compared with pGL3-basic, the –968/+43 construct increased luciferase activity by 40-fold. Deletion from –968 to –127 resulted in an additional 3-fold increase, suggesting the removal of negative regulatory elements in this region. Further deletion from –126 to –101 modestly reduced promoter activity. However, further deletion to –34 resulted in a >80% decrease in FGF2 promoter activity, indicating that a basal promoter was located between –100 and –35. The addition of 100 µmol/L Hcy, but not cysteine or methionine, reduced reporter activities by 30% to 40% from constructs spanning –968/+43, –126/+43, and –100/+43 but not in –34/+43 or +24/+179 lacking crucial promoter sequences. These results indicate a basal promoter driving FGF2 transcription that can be repressed by the treatment of HCAECs with Hcy.
|
Methylation Status of FGF2 Gene
An analysis of the human FGF2 gene identified an 1877-bp CpG island spanning from –532 in the 5'-flanking region and extending through exon 1 into the first intron. This portion of the human FGF2 gene encompasses the Hcy-responsive basal promoter, as well as clusters of CpG dinucleotides predicted to be methylation sensitive (supplemental Figure IIIA). To investigate whether methylation participates in Hcy-induced FGF2 downregulation, the methylation status of CpG dinucleotides in the FGF2 promoter was characterized by bisulfite genomic DNA sequencing in Hcy-treated HCAECs (supplemental Figure IIIB). Thirty-one CpG dinucleotides, numbers 1 to 31, in the FGF2 5'-flanking region were analyzed using 2 pairs of primers (CpG primer 1S/1AS; CpG primer 2S/2AS) designed to amplify the FGF2 promoter region. None of the 31 cytosine residues was methylated in the PBS control cells. In contrast, cytosine residue numbers 9 to 28 were methylated in the presence of 100 µmol/L Hcy, and increasing Hcy to 500 µmol/L resulted in methylation of all 31 cytosine residues (supplemental Figure IIIB). These data indicate that Hcy promotes methylation of the CpG dinucleotides in the FGF2 promoter and thus represses FGF2 transcriptional activity in ECs.
Reporter gene assay was performed to compare transcriptional activities of FGF2 in HCAECs cultured in the presence of Hcy with or without the methylation inhibitor 5-aza-deoxycytidine (5-aza-dC). As shown in Figure 6A, the promoter activities of –969/+43 and –126/+43 constructs containing an Hcy-response element were reduced by Hcy; addition of 5-aza-dC significantly attenuated the Hcy effect. Cellular FGF2 mRNA levels in the presence of Hcy with or without 5-aza-dC were compared across HCAECs by real-time PCR (Figure 6B). Consistent with reporter gene assays, Hcy alone resulted in a 40% reduction in FGF2 mRNA. The addition of 5-aza-dC to Hcy-treated HCAECs increased cellular FGF2 mRNA to normal levels. These results indicate that DNA methylation is an important mechanism mediating FGF2 transcription and cell proliferation in HCAECs.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our data showing that Hcy can specifically downregulate FGF2 expression in arterial ECs reveal a novel mechanism whereby HHcy can be angiostatic. The schematic in Figure 7 outlines a signaling pathway through which Hcy can impair endothelial proliferation/survival by FGF2 suppression. As an important risk factor for atherosclerosis, Hcy can therefore exhibit synergistic EC toxicity with oxLDL or L5 by a shared pathway related to FGF2.27,29–31 The close interplay between Hcy and modified LDL is strengthened by a prior report demonstrating that in human umbilical vein endothelial cells, Hcy enhances the oxidation of human LDL and facilitates its subsequent uptake by macrophages.33 Others have also shown that Hcy can upregulate lectin-like oxLDL receptor-1 in endothelial and mononuclear cells and increase oxLDL toxicities.34 Collectively, our results support cross-talk between Hcy and oxLDL and signify the need to upregulate or introduce FGF2 to counteract their pathological effects on ECs.
|
Both FGF2 and VEGF contribute to angiogenesis by promoting EC proliferation, migration, and tube formation. These processes were inhibited in BAECs exposed to Hcy at a supraphysiological concentration (5 mmol/L).18 This observation highlighted the need to confirm the effects of Hcy on EC proliferation and viability within a clinically significant Hcy concentration range before mechanistic investigation. HHcy has been defined as a plasma concentration exceeding 14 µmol/L and is considered severe at levels beyond 100 µmol/L, arising from rare metabolic defects of thiol amino acid metabolism. Notwithstanding, mild HHcy (15 to 30 µmol/L) has been associated with increased risks for a variety of occlusive vascular diseases.3,4,6–9 Thus, our experiments were conducted by incubating BAECs or HCAECs with Hcy ranging from 25 to 500 µmol/L. We have shown here that the treatment of ECs with Hcy concentrations characterizing mild to moderate HHcy (25–100 µmol/L) decreased DNA replication without increasing apoptosis. A structural analog of Hcy, cysteine, did not mimic the effects of Hcy on EC viability or FGF2 expression, indicating that the effects of Hcy occurred independently of thiol reactivity. Hcy-induced changes in EC phenotype were accompanied by decreased levels of intracellular FGF2 but not VEGF at more physiological, nonapoptotic concentrations (
100 µmol/L). On the contrary, high-dose Hcy at proapoptotic levels (500 µmol/L) decreased intracellular FGF2 as well as VEGF, consistent with the activation of degradative pathways and cell death (Figures 1 and 7
). These observations offer a potential explanation for the severe damage or denudation of arterial endothelium in homozygous homocystinurics or experimental primates infused with large amounts of Hcy.2,35
Several other derivatives of Hcy, such as Hcy thiolactone, S-nitroso Hcy, or SAH, affect vascular homeostasis. In BAECs and HCAECs treated with 100 µmol/L Hcy, we found reduced levels of FGF2 protein and mRNA. Thus, our study focused on SAH because this metabolite has been shown to affect gene expression at the epigenetic level by shifting the cellular methylation potential.36 In our culture protocol described by Wang et al,22 adenosine and erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) were added with Hcy to favor the increased production of SAH through the enzyme SAH hydrolase. We measured intracellular levels of SAM and SAH in HCAECs by liquid chromatography–MS/MS to better define the relation between cellular methylation potential (expressed as SAM/SAH ratio) and Hcy-mediated cytotoxicities (Table). Indeed, increasing Hcy concentrations were associated with a dose-dependent increase in intracellular SAH but a concomitant decrease in methylation potential (Table), which is correlated with the observed Hcy effects (Figure 1). These culture conditions, with EHNA and adenosine in the medium, were required to observe the adverse effects of 25 to 500 µmol/L Hcy on EC growth and survival and the downregulation of FGF2. Hcy alone (up to 500 µmol/L) did not induce these EC toxicities (data not shown).
|
We have shown here that Hcy downregulated FGF2 through the transcriptional repression of its promoter. Hcy reportedly modulates the expression of multiple genes in different cell types through changes in transcription factor activity37 or DNA methylation.25 This prompted our analysis of the Hcy-responsive basal promoter identified here from –100 to –34 using the TRANSFAC database. Although searches failed to reveal NF-
B sites, we identified GC boxes with CpG islands that can be regulated by methylation.38 In cultured cells, Hcy has been shown to induce gene-specific DNA hypomethylation regulating the expression of Hcy-induced endoplasmic reticulum protein (HERP) and an imprinted gene H19.39,40 However, some reports indicate that hypomethylation can be followed by the global hypermethylation of DNA through mechanisms not yet understood.39 In fact, leukocytic DNA from an SAH hydrolase-deficient individual paradoxically has been found to be hypermethylated.41 Our discovery that Hcy-induced FGF2 downregulation could be attenuated by 5-aza-dC, a well-known demethylating reagent,42 also supported the paradoxical hypermethylation of the FGF2 promoter as illustrated in Figure 7. Among the genes previously shown to be upregulated in Hcy-treated human umbilical vein endothelial cells, the bifunctional enzyme NAD-dependent methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (NMDMC) has been identified with the ability to increase the production of 5-methyltetrahydrofolate required for Hcy remethylation.43 Thus, ECs may be able to adjust to elevated levels of SAH by upregulating enzymes capable of compensatory remethylation reactions promoting DNA hypermethylation.
Our findings present important clinical implications because angiogenesis plays a crucial role in different physiological and pathological processes such as embryonic development, wound repair, tumor growth, and atherosclerosis. FGF2 and VEGF are not necessarily functionally redundant during angiogenesis accompanying atherosclerosis, such as plaque neovascularization and collateral arteriogenesis.44,45 In the present study, it is obvious that FGF2 is more susceptible to Hcy than VEGF in terms of gene expression: FGF2 was suppressed at as low as 100 µmol/L, but VEGF reduction did not occur until Hcy reached a proapoptotic concentration of 500 µmol/L. This explains why VEGF failed to attenuate cell cycle arrest but could ameliorate apoptosis when Hcy concentrations were raised from antiproliferative to antisurvival levels (Figure 7). Moreover, our findings that Hcy-mediated programmed cell death could be effectively ameliorated by either FGF2 or VEGF also support the involvement of two distinct apoptotic pathways in ECs as previously reported.46 Consistent with these results, it is well known that Hcy activates the unfolded protein response and induces apoptosis through a Fas-mediated extrinsic pathway, which could presumably be prevented by VEGF.47,48 In contrast, FGF2 protects against EC apoptosis through an intrinsic pathway independent of VEGF signaling.46 Altogether, these reports suggest a complex interplay between FGF2 and VEGF signaling in Hcy metabolism that can incur endothelial phenotypic changes.
In summary, compensatory arteriogenesis has been shown to be orchestrated by either FGF2 or VEGF. oxLDL, L5, or Hcy may inhibit this process by repressing FGF2 expression in ECs through a species-independent manner.27–31 Understanding the mechanisms by which Hcy and modified LDL, including oxLDL and L5, can inhibit angiogenesis may suggest novel therapeutic strategies to upregulate angiogenesis, thereby promoting the perfusion of ischemic tissues, such as for collateral arteriogenesis or optimal embryonic development, or to downregulate angiogenesis and attenuate cancer.
|
Acknowledgments |
|---|
Sources of Funding
This study was supported by National Science Council grants NSC 91-2320-B-002-185, NSC 93-2314-B-002-125, NSC 94-2320-B-002-121 and NSC 95-2320-B-002-116 (to P.-Y.C., S.-C.L., and Y.-T.L.); National Taiwan University Hospital grant NTUH92A14, Taipei, Taiwan, grants NTUH92A14, NTUH93A02, NTUH95S342, and NTUH96S643 (to P.-Y.C., S.-C.L., and Y.-T.L.); American Diabetes Association research grant 1-04-RA-13 (to C.-H.C.); and NIH training grant T32 HL07812 for postdoctoral fellowship (to T.A.D.).
Disclosures
None.
|
Footnotes |
|---|
Presented in part at the 2005 American College of Cardiology Scientific Sessions, Orlando, Fla, March 6–9, 2005, and published in abstract form (J Am Coll Cardiol. 2005;45[suppl A]:368A).
*Both authors contributed equally to this work. 
Original received December 26, 2006; resubmission received January 9, 2008; revised resubmission received February 15, 2008; accepted February 20, 2008.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Schimke RN, McKusick VA, Huang T, Pollack AD. Homocystinuria. Studies of 20 families with 38 affected members. JAMA. 1965; 193: 711–719.
2. McCully KS. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am J Pathol. 1969; 56: 111–128.[Medline] [Order article via Infotrieve]
3. Malinow MR, Bostom AG, Krauss RM. Homocyst(e)ine, diet, and cardiovascular diseases: a statement for healthcare professionals from the Nutrition Committee, American Heart Association. Circulation. 1999; 99: 178–182.
4. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, Graham I. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med. 1991; 324: 1149–1155.[Abstract]
5. Kullo IJ, Ballantyne CM. Conditional risk factors for atherosclerosis. Mayo Clin Proc. 2005; 80: 219–230.
6. Stampfer MJ, Malinow MR, Willett WC, Newcomer LM, Upson B, Ullmann D, Tishler PV, Hennekens CH. A prospective study of plasma homocysteine and risk of myocardial infarction in US physicians. JAMA. 1992; 268: 877–881.
7. Homocysteine Studies Collaboration. Homocysteine and risk of ischemic heart disease and stroke: a meta-analysis. JAMA. 2002; 288: 2015–2022.
8. Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ. 2002; 325: 1202.
9. den Heijer M, Lewington S, Clarke R. Homocysteine, MTHFR and risk of venous thrombosis: a meta-analysis of published epidemiological studies. J Thromb Haemost. 2005; 3: 292–299.[CrossRef][Medline] [Order article via Infotrieve]
10. Lange H, Suryapranata H, De LG, Borner C, Dille J, Kallmayer K, Pasalary MN, Scherer E, Dambrink JH. Folate therapy and in-stent restenosis after coronary stenting. N Engl J Med. 2004; 350: 2673–2681.
11. Toole JF, Malinow MR, Chambless LE, Spence JD, Pettigrew LC, Howard VJ, Sides EG, Wang CH, Stampfer M. Lowering homocysteine in patients with ischemic stroke to prevent recurrent stroke, myocardial infarction, and death: the Vitamin Intervention for Stroke Prevention (VISP) randomized controlled trial. JAMA. 2004; 291: 565–575.
12. den Heijer M, Willems H, Blom H, Gerrits W, Cattaneo M, Eichinger S, Rosendaal F, Bos GM. Homocysteine lowering by B vitamins and the secondary prevention of deep-vein thrombosis and pulmonary embolism. A randomised, placebo-controlled, double-blind trial. Blood. 2007; 109: 139–144.
13. Wang G, Woo CWH, Sung FL, Siow YL, O K. Increased monocyte adhesion to aortic endothelium in rats with hyperhomocysteinemia: role of chemokine and adhesion molecules. Arterioscler Thromb Vasc Biol. 2002; 22: 1777–1783.
14. Chao CL, Kuo TL, Lee YT. Effects of methionine-induced hyperhomocysteinemia on endothelium-dependent vasodilation and oxidative status in healthy adults. Circulation. 2000; 101: 485–490.
15. Eberhardt RT, Forgione MA, Cap A, Leopold JA, Rudd MA, Tolliet M, Heyrick S, Stark R, Klings ES, Moldovan NI, Yaghoubi M, Goldschmidt-Clermont PJ, Farber HW, Cohen R, Loscalzo J. Endothelial dysfunction in a murine model of mild hyperhomocyst(e)inemia. J Clin Invest. 2000; 106: 483–491.[Medline] [Order article via Infotrieve]
16. Jiang X, Yang F, Tan H, Liao D, Bryan RM, Randhawa JK, Rumbaut RE, Durante W, Schafer AI, Yang X, Wang H. Hyperhomocysteinemia impairs endothelial function and eNOS activity via PKC activation. Arterioscler Thromb Vasc Biol. 2005; 25: 2515–2521.
17. Dayal S, Arning E, Bottiglieri T, Boger RH, Sigmund CD, Faraci FM, Lentz SR. Cerebral vascular dysfunction mediated by superoxide in hyperhomocysteinemic mice. Stroke. 2004; 35: 1957–1962.
18. Rodriguez-Nieto S, Chavarria T, Martinez-Poveda B, Sanchez-Jimenez F, Rodriguez Quesada A, Medina MA. Anti-angiogenic effects of homocysteine on cultured endothelial cells. Biochem Biophys Res Commun. 2002; 293: 497–500.[CrossRef][Medline] [Order article via Infotrieve]
19. Duan J, Murohara T, Ikeda H, Sasaki K, Shintani S, Akita T, Shimada T, Imaizumi T. Hyperhomocysteinemia impairs angiogenesis in response to hindlimb ischemia. Arterioscler Thromb Vasc Biol. 2000; 20: 2579–2585.
20. Shastry S, Tyagi N, Hayden MR, Tyagi SC. Proteomic analysis of homocysteine inhibition of microvascular endothelial cell angiogenesis. Cell Mol Biol. 2004; 50: 931–937.[Medline] [Order article via Infotrieve]
21. Dayal S, Bottiglieri T, Arning E, Maeda N, Malinow MR, Sigmund CD, Heistad DD, Faraci FM, Lentz SR. Endothelial dysfunction and elevation of S-adenosylhomocysteine in cystathionine beta-synthase deficient mice. Circ Res. 2001; 88: 1203–1209.
22. Wang H, Yoshizumi M, Lai K, Tsai JC, Perrella MA, Haber E, Lee ME. Inhibition of growth and p21ras methylation in vascular endothelial cells by homocysteine but not cysteine. J Biol Chem. 1997; 272: 25380–25385.
23. Clarke S. Protein methylation. Curr Opin Cell Biol. 1993; 5: 977–983.[CrossRef][Medline] [Order article via Infotrieve]
24. Cedar H. DNA methylation and gene activity. Cell. 1988; 53: 3–4.[CrossRef][Medline] [Order article via Infotrieve]
25. Lee ME, Wang H. Homocysteine and hypomethylation: a novel link to vascular disease. Trends Cardiovasc Med. 1999; 9: 49–54.[CrossRef][Medline] [Order article via Infotrieve]
26. Wang H, Jiang X, Yang F, Chapman GB, Durante W, Sibinga NE, Schafer AI. Cyclin A transcriptional suppression is the major mechanism mediating homocysteine-induced endothelial cell growth inhibition. Blood. 2002; 99: 939–945.
27. Chen CH, Jiang T, Yang JH, Jiang W, Lu J, Marathe GK, Pownall HJ, Ballantyne CM, McIntyre TM, Henry PD, Yang CY. Low-density lipoprotein in hypercholesterolemic human plasma induces vascular endothelial cell apoptosis by inhibiting fibroblast growth factor 2 transcription. Circulation. 2003; 107: 2102–2108.
28. Lu J, Jiang W, Yang JH, Chang PY, Walterscheid JP, Chen HH, Marcelli M, Tang D, Liao WSL, Yang CY, Chen CH. Electronegative LDL impairs vascular endothelial cell integrity in diabetes by disrupting FGF2 autoregulation. Diabetes. 2008; 57: 158–166.[CrossRef][Medline] [Order article via Infotrieve]
29. Chen CH, Cartwright J Jr, Li Z, Lou S, Nguyen HH, Gotto AM Jr, Henry PD. Inhibitory effects of hypercholesterolemia and oxidized LDL on angiogenesis-like endothelial growth in rabbit aortic explants: essential role of basic fibroblast growth factor. Arterioscler Thromb Vasc Biol. 1997; 17: 1303–1312.
30. Chen CH, Jiang W, Via DP, Luo S, Li TR, Lee YT, Henry PD. Oxidized low-density lipoprotein inhibits endothelial cell growth by suppressing basic fibroblast growth factor expression. Circulation. 2000; 101: 171–177.
31. Chang PY, Luo S, Jiang T, Lee YT, Lu SC, Henry PD, Chen CH. Oxidized low-density lipoprotein down-regulates endothelial bFGF through a pertussis toxin–sensitive G-protein pathway: mediator role of platelet-activating factor–like phospholipids. Circulation. 2001; 104: 588–593.
32. Chang PY, Lu SC, Su TC, Chou SF, Huang WH, Morrisett JD, Chen CH, Liau CS, Lee YT. Lipoprotein-X reduces LDL atherogenicity in primary biliary cirrhosis by preventing LDL oxidation. J Lipid Res. 2004; 45: 2116–2122.
33. Nakano E, Taiwo FA, Nugent D, Griffiths HR, Aldred S, Paisi M, Kwok M, Bhatt P, Hill MH, Moat S, Powers HJ. Downstream effects on human low density lipoprotein of homocysteine exported from endothelial cells in an in vitro system. J Lipid Res. 2005; 46: 484–493.
34. Nagase M, Ando K, Nagase T, Kaname S, Sawamura T, Fujita T. Redox-sensitive regulation of LOX-1 gene expression in vascular endothelium. Biochem Biophys Res Commun. 2001; 281: 720–725.[CrossRef][Medline] [Order article via Infotrieve]
35. Harker LA, Ross R, Slichter SJ, Scott CR. Homocysteine-induced arteriosclerosis: the role of endothelial cell injury and platelet response in its genesis. J Clin Invest. 1976; 58: 731–741.[Medline] [Order article via Infotrieve]
36. Hermes M, Osswald H, Mattar J, Kloor D. Influence of an altered methylation potential on mRNA methylation and gene expression in HepG2 cells. Exp Cell Res. 2004; 294: 325–334.[CrossRef][Medline] [Order article via Infotrieve]
37. Au-Yeung KK, Woo CW, Sung FL, Yip JC, Siow YL, O K. Hyperhomocysteinemia activates nuclear factor-kappaB in endothelial cells via oxidative stress. Circ Res. 2004; 94: 28–36.
38. Bird AP. CpG-rich islands and the function of DNA methylation. Nature. 1986; 321: 209–213.[CrossRef][Medline] [Order article via Infotrieve]
39. Lentz B, Bleich S, Beutler S, Schierf B, Schwager K, Reulbach U, Kornhuber J, Bonsch D. Homocysteine regulates expression of Herp by DNA methylation involving the AARE and CREB binding sites. Exp Cell Res. 2006; 312: 4049–4055.[CrossRef][Medline] [Order article via Infotrieve]
40. Devlin AM, Bottiglieri T, Domann FE, Lentz SR. Tissue-specific changes in H19 methylation and expression in mice with hyperhomocysteinemia. J Biol Chem. 2005; 280: 25506–25511.
41. Baric I, Fumic K, Glenn B, Cuk M, Schulze A, Finkelstein JD, James SJ, Mejaski-Bosnjak V, Pazanin L, Pogribny IP, Rados M, Sarnavka V, Scukanec-Spoljar M, Allen RH, Stabler S, Uzelac L, Vugrek O, Wagner C, Zeisel S, Mudd SH. S-adenosylhomocysteine hydrolase deficiency in a human: a genetic disorder of methionine metabolism. Proc Natl Acad Sci U S A. 2004; 101: 4234–4239.
42. Santini V, Kantarjian HM, Issa JP. Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann Intern Med. 2001; 134: 573–586.
43. Kokame K, Kato H, Miyata T. Homocysteine-respondent genes in vascular endothelial cells identified by differential display analysis. J Biol Chem. 1996; 271: 29659–29665.
44. Chen CH, Walterscheid JP. Plaque angiogenesis versus compensatory arteriogenesis in atherosclerosis. Circ Res. 2006; 99: 787–789.
45. Bochkov VN, Philippova M, Oskolkova O, Kadl A, Furnkranz A, Karabeg E, Afonyushkin T, Gruber F, Breuss J, Minchenko A, Mechtcheriakova D, Hohensinner P, Rychli K, Wojta J, Resink T, Erne P, Binder BR, Leitinger N. Oxidized phospholipids stimulate angiogenesis via autocrine mechanisms, implicating a novel role for lipid oxidation in the evolution of atherosclerotic lesions. Circ Res. 2006; 99: 900–908.
46. Alavi A, Hood JD, Frausto R, Stupack DG, Cheresh DA. Role of Raf in vascular protection from distinct apoptotic stimuli. Science. 2003; 301: 94–96.
47. Zhang C, Cai Y, Adachi MT, Oshiro S, Aso T, Kaufman RJ, Kitajima S. Homocysteine induces programmed cell death in human vascular endothelial cells through activation of the unfolded protein response. J Biol Chem. 2001; 276: 35867–35874.
48. Suhara T, Fukuo K, Yasuda O, Tsubakimoto M, Takemura Y, Kawamoto H, Yokoi T, Mogi M, Kaimoto T, Ogihara T. Homocysteine enhances endothelial apoptosis via upregulation of Fas-mediated pathways. Hypertension. 2004; 43: 1208–1213.
Related Article:
Circ. Res. 2008 102: 869-870.
This article has been cited by other articles:
 |
|
 |
|
  A. Zulli, E. Lau, B. P.P. Wijaya, X. Jin, K. Sutarga, G. D. Schwartz, J. Learmont, P. J. Wookey, A. Zinellu, C. Carru, et al. High Dietary Taurine Reduces Apoptosis and Atherosclerosis in the Left Main Coronary Artery: Association With Reduced CCAAT/Enhancer Binding Protein Homologous Protein and Total Plasma Homocysteine but not Lipidemia Hypertension, June 1, 2009; 53(6): 1017 - 1022. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Medina Hyperhomocysteinemia and Occlusive Vascular Disease: An Emergent Role for Fibroblast Growth Factor 2 Circ. Res., April 25, 2008; 102(8): 869 - 870. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||