Methionine is the precursor of homocysteine, a sulfur amino acid intermediate in the methylation and transsulfuration pathways. Elevated plasma homocysteine (hyperhomocysteinemia) is associated with occlusive vascular disease. Whether homocysteine per se or a coincident metabolic abnormality causes vascular disease is still an open question. Animals with genetic hyperhomocysteinemia have so far not displayed atheromatous lesions. However, when methionine-rich diets are used to induce hyperhomocysteinemia, vascular pathology is often observed. Such studies have not distinguished the effects of excess dietary methionine from those of hyperhomocysteinemia. We fed apolipoprotein E-deficient mice with experimental diets designed to achieve three conditions: (i) high methionine intake with normal blood homocysteine; (ii) high methionine intake with B vitamin deficiency and hyperhomocysteinemia; and (iii) normal methionine intake with B vitamin deficiency and hyperhomocysteinemia. Mice fed methionine-rich diets had significant atheromatous pathology in the aortic arch even with normal plasma homocysteine levels, whereas mice fed B vitamin-deficient diets developed severe hyperhomocysteinemia without any increase in vascular pathology. Our findings suggest that moderate increases in methionine intake are atherogenic in susceptible mice. Although homocysteine may contribute to the effect of methionine, high plasma homocysteine was not independently atherogenic in this model. Some product of excess methionine metabolism rather than high plasma homocysteine per se may underlie the association of homocysteine with vascular disease. Methionine is the precursor of homocysteine, a sulfur amino acid intermediate in the methylation and transsulfuration pathways. Homocysteine, a non-protein-forming sulfur amino acid, was first implicated as a cause of occlusive vascular disease by K. S. McCully, who noted the high prevalence of early arteriosclerotic and thromboembolic disease in patients with congenital homocysteinuria (1). Since then, a growing body of epidemiological evidence has shown a strong association of elevated plasma homocysteine with vascular disease in the general population (2-7). The association remains strong after adjustment for major determinants of homocysteine, such as age and renal function, folate, vitamin B 12 , and vitamin B 6 , suggesting that homocysteine is an independent risk factor for occlusive vascular disease. Together with the known association of inborn errors of homocysteine metabolism and premature vascular disease in humans, these data provide the basis for a compelling, if still controversial (8-11), hypothesis that elevated blood homocysteine is a cause of vascular disease. This hypothesis has engendered great interest because of the possibility that lowering blood homocysteine through nutritional interventions might prove to be a safe and effective means of reducing the associated risk of disease. However, despite a variety of theoretically plausible mechanisms proposed to underlie this ass...

Full activity of fibroblast growth factors (FGFs) requires their internalization in addition to the interaction with cell surface receptors. Recent studies have suggested that the transmembrane proteoglycan syndecan-4 functions as a FGF2 receptor. In this study we investigated the molecular basis of syndecan endocytosis and its role in FGF2 internalization in endothelial cells. We found that syndecan-4 uptake, induced either by treatment with FGF2 or by antibody clustering, requires the integrity of plasma membrane lipid rafts for its initiation, occurs in a non-clathrin-, non-dynamin-dependent manner and involves Rac1, which is activated by syndecan-4 clustering. FGF2 was internalized in a complex with syndecan-4 in 70 kDa dextran-containing endocytic vesicles. FGF2 and syndecan-4 but not dextran endocytosis were blocked by the dominant negative Rac1 while amiloride and the dominant-negative Cdc42 blocked internalization of dextran in addition to FGF2 and syndecan-4. Taken together, these results demonstrate that FGF2 endocytosis requires syndecan-4 clustering-dependent activation of Rac1 and the intact CDC42-dependent macropinocytic pathway.

Background-CD4ϩ and CD8 ϩ T lymphocytes are key regulators of postischemic neovascularization. T-cell activation is promoted by 2 major costimulatory signalings, the B7/CD28 and CD40 -CD40 ligand pathways. Interestingly, CD28 interactions with the structurally related ligands B7-1 and B7-2 are also required for the generation and homeostasis of CD4 ϩ CD25 ϩ regulatory T cells (Treg cells), which play a critical role in the suppression of immune responses and the control of T-cell homeostasis. We hypothesized that Treg cell activation may modulate the immunoinflammatory response to ischemic injury, leading to alteration of postischemic vessel growth. Methods and Results-Ischemia was induced by right femoral artery ligation in CD28-, B7-1/2-, or CD40-deficient mice (nϭ10 per group). CD40 deficiency led to a significant reduction in the postischemic inflammatory response and vessel growth. In contrast, at day 21 after ischemia, angiographic score, foot perfusion, and capillary density were increased by 2.0-, 1.2-, and 1.8-fold, respectively, in CD28-deficient mice, which showed a profound reduction in the number of Treg cells compared with controls. Similarly, disruption of B7-1/2 signaling or anti-CD25 treatment and subsequent Treg deletion significantly enhanced postischemic neovascularization. These effects were associated with enhanced accumulation of CD3-positive T cells and Mac-3-positive macrophages in the ischemic leg. Conversely, treatment of CD28 Ϫ/Ϫ mice with the nonmitogenic anti-CD3 monoclonal antibody enhanced the number of endogenous Treg cells and led to a significant reduction of the postischemic inflammatory response and neovascularization. Finally, coadministration of Treg cells and CD28 Ϫ/Ϫ splenocytes in Rag1 Ϫ/Ϫ mice with hindlimb ischemia abrogated the CD28 Ϫ/Ϫ splenocyte-induced activation of the inflammatory response and neovascularization.