To study HC-mediated vascular effects in vivo, we induced HHcy in mice deficient in apoE, in whom hypercholesterolemia and atherosclerosis spontaneously develop (15
). Mice were fed standard rodent chow (diet A); a diet enriched in methionine, but substantially depleted in folate, vitamins B6, and B12 (diet B); or a diet enriched in methionine and the vitamins folate, B6, and B12 (diet C). After 8 weeks on the diet, mice fed methionine-enriched diet B demonstrated an approximately 19-fold increase in mean plasma level of HC compared with control mice receiving diet A (47.3 ± 3.2 vs. 2.5 ± 0.3 μM, respectively; P
< 0.01) (Table ). Importantly, these levels of HC were pathophysiologically relevant (2
). For example, in a series of elderly persons studied by the Framingham investigators, levels of plasma HC ranged from 4.13 to 219.84 μM (2
Induction of HHcy in apoE-null mice: effect on lipids, glucose, and vitamin levels
Induction of HHcy was associated with an approximately twofold increase in mean atherosclerotic lesion area at the aortic sinus in mice fed diet B compared with those mice fed control diet A (Figure , b and f versus a and e, respectively, and Figure i) after 8 weeks. HHcy mice displayed increased numbers of complex atherosclerotic lesions at the aortic sinus, defined as the presence of fibrous caps, cholesterol clefts, or necrosis, compared with controls (Figure j). Atherosclerotic lesions in HHcy mice were characterized by increased numbers of macrophages (Mac-3 antigen) (see Figure d) and smooth muscle cells (data not shown) compared with mice fed diet A. The effects of HHcy were not due to alteration in lipid or glucose metabolism, as levels of cholesterol, triglyceride, glucose, and glycosylated hemoglobin (a measure of extended glycemic control) were unchanged compared with mice fed control chow (Table ). Furthermore, separation of plasma lipoprotein components by fast-pressure liquid chromatography (FPLC) revealed no differences in lipid size or profile (data not shown). Importantly, accelerated atherosclerosis observed in mice fed diet B was not due to diminished levels of folate, vitamins B6 and B12, as mice fed diet D (basal levels of methionine, but decreased levels of folate, vitamins B6/B12) did not display accelerated atherosclerotic lesion area or complexity compared with mice fed diet A (Figure , d and h versus a and e, respectively, and Figure , i–j).
Figure 1 Induction of HHcy accelerates atherosclerotic lesion area and complexity. Male apoE-null mice, 4 weeks of age, were fed (a, e) diet A, n = 30; (b, f) diet B, n = 30; (c, g) diet C, n = 30, or (d, h) diet D, n = 10, for (more ...)
Figure 5 Induction of HHcy enhances expression of TF. (a) HUVECs were exposed to the indicated concentration of BSA, L-HC, or L-cysteine for 8 hours. Cells were harvested and prepared for immunoblotting using goat anti-rat TF IgG (0.5 μg/ml). AP < (more ...)
Because these findings suggested that induction of HHcy by dietary enrichment in methionine (diet B), and not solely dietary depletion of folate and vitamins B6/B12 (diet D), accelerated atherogenesis, we focused our studies on mice fed diets A and B. To explore the molecular consequences of HHcy in the vasculature, we sought evidence for activation of NF-κB, as this transcription factor has been linked to modulation of the proinflammatory response (26
). Previous studies in vitro demonstrated that incubation of vascular smooth muscle cells (VSMCs) with L
-HC (500 μM) resulted in enhanced nuclear translocation of NF-κB (28
). Consistent with these observations, exposure of HUVECs to L
-HC (100 μM) resulted in an approximately 2.4-fold increase in activation of NF-κB, compared with ECs treated with BSA or L
-cysteine (100 μM) (Figure a, lanes 2, 1, and 3, respectively). To test these concepts in vivo, we prepared nuclear extracts from aortae and kidney of mice receiving diet B or A. Compared with mice fed diet A, animals fed methionine-enriched chow (diet B) displayed an approximately tenfold increase in nuclear translocation of NF-κB as measured by electrophoretic mobility-shift assay (EMSA) performed on nuclear extracts derived from aorta (Figure b, lanes 4 and 5, respectively), and an approximately 4.5-fold increase in activation of NF-κB in nuclear extracts prepared from kidney (Figure b, lanes 1 and 2, respectively).
Figure 2 HHcy enhances activation of NF-κB as seen using EMSA. (a) HUVECs were exposed to BSA, L-HC, or L-cysteine (100 μM) for 8 hours. Nuclear extracts were prepared and subjected to EMSA. AP < 0.05 vs. lanes 1 and 3. In lane 4, a 100-fold (more ...)
Since activation of NF-κB is linked to enhanced expression of genes centrally involved in atherogenesis (29
), we explored whether induction of HHcy was associated with increased vascular inflammation. As previous studies suggested that the transcriptional/translational control of expression of VCAM-1, important in the binding of inflammatory mononuclear cells to the vessel wall, is mediated at least in part by activation of NF-κB (30
), we assessed levels of this adhesion molecule in vivo. Mice fed methionine-enriched diet B displayed an approximately 3.7-fold increase in expression of VCAM-1 antigen in aortic tissue compared with mice fed diet A (Figure a).
Figure 3 Induction of HHcy enhances vascular expression of VCAM-1 and RAGE. (a–d) VCAM-1. ApoE-null mice were fed the indicated diet for 8 weeks. Aortae were removed, and lysates were prepared. Lysate protein (10 μg) was subjected to immunoblotting (more ...)
Immunohistochemistry revealed that VCAM-1 was expressed in the atherosclerotic lesions of mice fed diet A and B (Figure , b and c).
In addition, previous studies suggested that the promoter of the gene encoding RAGE (32
), a multiligand signal-transduction receptor of the immunoglobulin superfamily linked to propagation of proinflammatory phenomena (24
), possessed functional binding elements for NF-κB (33
). In vitro, incubation of HUVEC with L
-HC (100 μM) resulted in an approximately 2.8-fold increase in expression of RAGE antigen by immunoblotting compared with cells treated with BSA or L
-cysteine (Figure e). In vivo,
aortic tissue retrieved from mice fed diet B displayed an approximately 4.5-fold increase in expression of RAGE compared with those mice receiving control diet A (Figure f). Immunohistochemistry revealed that the expression of RAGE was increased in the atherosclerotic lesions of mice fed diet B versus diet A (Figure , h and g, respectively).
EN-RAGEs, members of the S100/calgranulin family of proinflammatory cytokines (34
) and signal-transducing ligands of RAGE, have been identified in the atherosclerotic plaques of chow-fed apoE–null mice (36
). In the presence of HHcy, mice fed diet B displayed an approximately 3.7-fold increase in expression of EN-RAGE antigen in the aorta compared with controls, using immunoblotting (Figure a). Immunohistochemistry revealed that EN-RAGE was expressed in the atherosclerotic lesions in a manner increased in mice fed diet B compared with diet A (Figure , c and b, respectively). Further evidence for HC-mediated global cellular activation was demonstrated by the observation that levels of plasma TNF-α were increased 6.1-fold in HHcy mice fed diet B versus those fed control diet A (Figure e).
Figure 4 Induction of HHcy enhances vascular inflammation and expression and activity of MMP-9 and increases elastolysis in aortic tissue from apoE-null mice. ApoE-null mice were fed the indicated diet for 8 weeks. (a–d) EN-RAGE. In a, aortae were removed, (more ...)
In view of these findings suggesting heightened proinflammatory responses in HHcy vasculature, we assessed levels and activity of tissue-destructive enzymes such as MMPs because previous studies suggested that MMP protein and activity, present in the atherosclerotic plaque, might promote instability and rupture of vascular lesions (37
). Compared with mice fed diet A, HHcy mice demonstrated an approximately fivefold increase in levels of MMP-9 antigen by immunoblotting (Figure f). Importantly, zymography studies revealed increased activity of MMP-9 in aortic tissue obtained from mice with HHcy (diet B) compared with those fed normal chow (Figure g). Consistent with these observations, examination of elastic fibers underlying atherosclerotic lesions at the aortic sinus stained by van Giessen’s method revealed significantly increased elastolysis in mice receiving diet B compared with those mice fed chow (Figure h).
In addition to regulation of inflammatory genes, multiple studies suggested that HHcy is associated with altered levels of factors essential for precise control of hemostatic, thrombotic, and fibrinolytic enzymes (39
). Even minute differences in levels of such factors likely underlie the observed increased incidence of venous and arterial thromboembolism that occurs in human subjects with HHcy. We tested the concept that exposure of endothelium to HC might alter hemostatic balance, since instability of the atherosclerotic plaque is linked, at least in part, to thrombotic events in the vessel wall (40
). Since TF is the key trigger of the procoagulant pathway in vivo, its expression in the setting of HHcy was assessed. In vitro, HUVECs exposed to L
-HC displayed an approximately 5.2-fold increase in TF antigen by immunoblotting compared with HUVECs treated with L
-cysteine (100 μM) or BSA (Figure a, lanes 2, 3, and 1, respectively). Consistent with these observations, immunoblotting of aortic extracts prepared from HHcy mice fed diet B revealed an approximately 7.5-fold increase in levels of procoagulant TF compared with mice fed chow (Figure b). The expression of TF largely colocalized with that of Mac-3, identifying the macrophage as a prevalent source of TF in the vascular lesions of apoE–null mice with HHcy (Figure , g and d, respectively).
Taken together, these findings suggested that induction of HHcy accelerated atherosclerotic lesion formation and complexity and enhanced vascular inflammation, hypercoagulability, and molecular mediators of plaque instability. A central question arising from these observations and epidemiologic investigations in human subjects is whether suppression of HHcy might modulate the course of atherogenesis. To test this, we prepared a diet enriched in methionine, as well as vitamins necessary for the metabolism of HC, folate, and vitamins B6/B12 (diet C). Compared with mice receiving diet B (19-fold increased HC), animals receiving diet C showed only an 7.4-fold increase in HC compared with controls (Table ). The effects of vitamin enrichment in diet C were evident; levels of folate, vitamins B6 and B12 were elevated approximately 6.6-fold, 31.2-fold, 9.5-fold, respectively, compared with levels observed in mice fed diet B (Table ). Ingestion of diet C did not result in altered control of glycemia or levels of total cholesterol or triglyceride compared with mice receiving diet B (Table ). Furthermore, lipid size/profile determined by FPLC did not differ among mice fed diet A, B, or C (data not shown).
We first tested whether reduction of HHcy might modulate atherosclerosis in this model. Mice fed diet C demonstrated only a 40% increase in lesion area compared with a twofold increase observed in mice fed diet B (Figure , c and g and Figure , b and f, respectively; and Figure i). Furthermore, lesions in apoE-null mice fed diet C were limited to fatty streaks; no complex lesions were noted (Figure j).
These observations suggested that suppression of accelerated atherosclerosis in mice fed diet C was associated with diminished vascular activation. To test this concept, we explored the effects of reduced HC levels on putative pathogenic mechanisms underlying the effects of HHcy. Consistent with the premise that HHcy mediated enhanced vascular inflammation, nuclear extracts prepared from mice fed diet C displayed significantly decreased nuclear translocation of NF-κB in the aorta and kidney compared with mice fed diet B (Figure b, lanes 6 and 5 and lanes 3 and 2, respectively).
In parallel with diminished activation of NF-κB in vascular tissue from mice fed diet C, immunoblotting studies revealed that the expression of VCAM-1, RAGE, and EN-RAGE antigens in aortic tissue was significantly diminished compared with that observed in mice fed diet B (Figure , a and f, and Figure a). In addition, levels of plasma TNF-α in mice fed diet C were reduced nearly to levels observed in mice fed diet A (Figure e). Similarly, expression/activity of MMP-9 were reduced in mice fed diet C versus diet B (Figure , f and g). Consistent with this observation, examination of elastic fibers underlying atherosclerotic plaques in lesions from mice fed diet C revealed decreased elastolysis compared with mice with HHcy fed diet B (Figure h).
Lastly, since plaque instability and rupture markedly alter the clinical course of atherosclerosis, we assessed levels of likely contributory molecules in this model. Compared with mice fed diet B, those fed diet C demonstrated an approximately twofold decrease in levels of TF in the aorta (Figure b).