Abstract-Homocysteine (Hcy) is an independent risk factor for cardiovascular disease. Monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) are major chemokines for leukocyte trafficking and have been identified in atheromatous plaques. MCP-1 and IL-8 have been found to express mainly by macrophages in human lesion. We undertook this study to determine whether Hcy could induce the secretion of chemokines from human monocytes and, if so, to explore the mediating mechanism. We found that clinically relevant levels of Hcy (10 to 1000 mol/L) increased the protein secretion and mRNA expression as well as activity of MCP-1 and IL-8 in cultured primary human monocytes. These effects of Hcy were primarily mediated by reactive oxygen species (ROS) through NAD(P)H oxidase, because Hcy could upregulate the production of ROS and the inhibitors of protein kinase C, calmodulin, free radical scavengers, or NAD(P)H oxidase abolished Hcy-induced ROS production and MCP-1 and IL-8 secretion in these cells. Furthermore, the inhibitors of mitogen-activated protein kinase (p38 and extracellular signal-regulated kinase 1/2) and nuclear factor-B or the activator of peroxisome proliferator-activated receptor ␥ (PPAR␥) significantly decreased Hcy-induced MCP-1 and IL-8 secretion in these cells. These data indicate that pathophysiological levels of Hcy can alter human monocyte function by upregulating MCP-1 and IL-8 expression and secretion via enhanced formation of intracellular ROS originated from NAD(P)H oxidase source via calmodulin or protein kinase C signaling pathways and that Hcy-induced ROS subsequently activates mitogen-activated protein kinase (p38 and ERK1/2) and nuclear factor-B in a PPAR␥ activator-sensitive manner. Thus, activation of PPAR␥ may become a therapeutic target for preventing Hcy-induced proatherogenic effects.

Background-Plaque neovascularization is thought to promote atherosclerosis; however, the mechanisms of its regulation are not understood. Collagen XVIII and its proteolytically released endostatin fragment are abundant proteoglycans in vascular basement membranes and the walls of major blood vessels. We hypothesized that collagen XVIII in the aortic wall inhibits the proliferation and intimal extension of vasa vasorum. Methods and Results-To test our hypothesis, we bred collagen XVIII-knockout (Col18a1 Ϫ/Ϫ ) mice into the atherosclerosis-prone apolipoprotein E-deficient (ApoE Ϫ/Ϫ ) strain. After 6 months on a cholesterol diet, aortas from ApoE Ϫ/Ϫ ; Col18a1 Ϫ/Ϫ and ApoE Ϫ/Ϫ ;Col18a1 ϩ/Ϫ heterozygote mice showed increased atheroma coverage and enhanced lipid accumulation compared with wild-type littermates. We observed more extensive vasa vasorum and intimal neovascularization in knockout but not heterozygote aortas. Endothelial cells sprouting from Col18a1 Ϫ/Ϫ aortas were increased compared with heterozygote and wild-type aortas. In contrast, vascular permeability of large and small blood vessels was enhanced with even heterozygous loss of collagen XVIII but was not suppressed by increasing serum endostatin to wild-type levels. Conclusions-Our results identify a previously unrecognized function for collagen XVIII that maintains vascular permeability. Loss of this basement membrane proteoglycan enhances angiogenesis and vascular permeability during atherosclerosis by distinct gene-dose-dependent mechanisms.

A fundamental question regarding receptor-G protein interaction is whether different agonists can lead a receptor to different intracellular signaling pathways. Our previous studies have demonstrated that although most ␤ 2 -adrenoceptor agonists activate both G s and G i proteins, fenoterol, a full agonist of ␤ 2 -adrenoceptor, selectively activates G s protein. Fenoterol contains two chiral centers and may exist as four stereoisomers. We have synthesized a series of stereoisomers of fenoterol and its derivatives and characterized their receptor binding and pharmacological properties. We tested the hypothesis that the stereochemistry of an agonist determines selectivity of receptor coupling to different G protein(s). We found that the R,R isomers of fenoterol and methoxyfenoterol exhibited more potent effects to increase cardiomyocyte contraction than their S,R isomers. It is noteworthy that although (R,R)-fenoterol and (R,R)-methoxyfenoterol preferentially activate G s signaling, their S,R isomers were able to activate both G s and G i proteins as evidenced by the robust pertussis toxin sensitivities of their effects on cardiomyocyte contraction and on phosphorylation of extracellular signal-regulated kinase 1/2. The differential G protein selectivities of the fenoterol stereoisomers were further confirmed by photoaffinity labeling studies on G s , G i2 , and G i3 proteins. The inefficient G i signaling with the R,R isomers is not caused by the inability of the R,R isomers to trigger the protein kinase A (PKA)-mediated phosphorylation of the ␤ 2 -adrenoceptor, because the R,R isomers also markedly increased phosphorylation of the receptor at serine 262 by PKA. We conclude that in addition to receptor subtype and phosphorylation status, the stereochemistry of a given agonist plays an important role in determining receptor-G protein selectivity and downstream signaling events.