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.

A 3-year field experiment examined the effects of non-flooded mulching cultivation and traditional flooding and four fertilizer N application rates (0, 75, 150 and 225 kg ha À1 for rice and 0, 60,120, and 180 kg N ha À1 for wheat) on grain yield, N uptake, residual soil N min and the net N balance in a rice-wheat rotation on Chengdu flood plain, southwest China. There were significant grain yield responses to N fertilizer. Nitrogen applications of >150 kg ha À1 for rice and >120 kg ha À1 for wheat gave no increase in crop yield but increased crop N uptake and N balance surplus in both water regimes. Average rice grain yield increased by 14% with plastic film mulching and decreased by 16% with wheat straw mulching at lower N inputs compared with traditional flooding. Rice grain yields under SM were comparable to those under PM and TF at higher N inputs. Plastic film mulching of preceding rice did not affect the yield of succeeding wheat but straw mulching had a residual effect on succeeding wheat. As a result, there was 17-18% higher wheat yield under N0 in SM than those in PM and TF. Combined rice and wheat grain yields under plastic mulching was similar to that of flooding and higher than that of straw mulching across N treatments. Soil mineral N (top 60 cm) after the rice harvest ranged from 50 to 65 kg ha À1 and was unaffected by non-flooded mulching cultivation and N rate. After the wheat harvest, soil N min ranged from 66 to 88 kg N ha À1 and increased with increasing fertilizer N rate. High N inputs led to a positive N balance (160-621 kg ha À1 ), but low N inputs resulted in a negative balance (À85 to À360 kg ha À1). Across N treatments, the net N balances of SM were highest among the three cultivations systems, resulting from additional applied wheat straw (79 kg ha À1 ) as mulching materials. There was not clear trend found in net N balance between PM and TF. Results from this study indicate non-flooded mulching cultivation may be utilized as an alternative option for saving water, using efficiently straw and maintaining or improving crop yield in rice-wheat rotation systems. There is the need to evaluate the long-term environmental risks of non-flooded mulching cultivation and improve system productivity (especially with straw mulching) by integrated resource management. #

These data indicate that ROS generated by thiol (-SH) of Hcy auto-oxidation are involved in Hcy effect on Con A-induced T lymphocyte proliferation. These findings suggest a novel mechanism may be involved in chronic inflammatory progression of atherosclerosis with hyperhomocysteinemia.

Two major β-adrenergic receptor (βAR) subtypes, β1AR and β2AR, are expressed in mammalian heart with β1AR coupling to Gs and β2AR dually coupling to Gs and Gi proteins. In many types of chronic heart failure, myocardial contractile response to both β1AR and β2AR stimulation is severely impaired. The dysfunction of βAR signaling in failing hearts is largely attributable to an increase in Gi signaling, because disruption of the Gi signaling restores myocardial contractile response to β1AR as well as β2AR stimulation. However, the mechanism terminating the β2AR-Gi signaling remains elusive, while it has been shown activation of the Gi signaling is dependent on agonist stimulation and subsequent PKA-mediated phosphorylation of the receptor. Here we demonstrate that regulator of G protein signaling 2 (RGS2) is a primary terminator of the β2AR-Gi signaling. Specifically, prolonged absence of agonist stimulation for 24h impairs the β2AR-Gi signaling, resulting in enhanced β2AR- but not β1AR-mediated contractile response in cultured adult mouse cardiomyocytes. Increased β2AR contractile response is accompanied by a selective upregulation of RGS2 in the absence of alterations in other major cardiac RGS proteins (RGS3-5) or Gs, Gi or βAR subtypes. Administration of a βAR agonist, isoproterenol (ISO, 1.0 nM), prevents RGS2 upregulation and restores the β2AR-Gi signaling in cultured cells. Furthermore, RGS2 ablation, similar to βAR agonist stimulation, sustains the β2AR-Gi signaling in cultured cells, whereas adenoviral overexpression of RGS2 suppresses agonist-activated β2AR-Gi signaling in cardiomyocytes and HEK293 cells. These findings not only define RGS2 as a novel negative regulator of the β2AR-Gi signaling, but also provide a potential novel target for the treatment of chronic heart failure.

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Retention of lipoproteins to proteoglycans in the subendothelial matrix (SEM) is an early event in atherosclerosis. We recently reported that collagen XVIII and its proteolytically released fragment endostatin (ES) are differentially depleted in blood vessels affected by atherosclerosis. Loss of collagen XVIII/ES in atherosclerosis-prone mice enhanced plaque neovascularization and increased the vascular permeability to lipids by distinct mechanisms. Impaired endothelial barrier function increased the influx of lipoproteins across the endothelium; however, we hypothesized that enhanced retention might be a second mechanism leading to the increased lipid content in atheromas lacking collagen XVIII. We now demonstrate a novel property of ES that binds both the matrix proteoglycan biglycan and LDL and interferes with LDL retention to biglycan and to SEM. A peptide encompassing the ␣ coil in the ES crystal structure mediates the major blocking effect of ES on LDL retention. ES inhibits the macrophage uptake of biglycan-associated LDL indirectly by interfering with LDL retention to biglycan, but it has no direct effect on the macrophage uptake of native or modified lipoproteins. Thus, loss of ES in advanced atheromas enhances lipoprotein retention in SEM. Our data reveal a third protective role of this vascular basement membrane component during atherosclerosis. Lipid accumulation and macrophage-derived foam cells are prominent features of atherosclerotic plaques. The response-to-retention hypothesis proposes that serum lipoproteins are retained by proteoglycans in the subendothelial matrix (SEM) and subsequently modified and taken up by macrophages to form foam cells that constitute the fatty streak type of atheroma (1-3). Biglycan, a dermatan sulfate proteoglycan in human and mouse atheromas, binds LDL by ionic interactions involving specific basic amino acids in apolipoprotein B-100 and sulfated sugar modifications of biglycan (4-6). LDL association or aggregation with arterial wall proteoglycans increases rates of LDL oxidation and macrophage uptake (7,8). Mice expressing mutant apolipoprotein B-100 produce LDL particles with lower proteoglycan affinity and develop less atherosclerosis (9, 10). Dermatan sulfate and chondroitin sulfate proteoglycans such as biglycan and versican are increased, whereas heparan sulfate proteoglycans are reduced in atheromas compared with normal blood vessels (4, 11). Therefore, relative changes in the matrix composition of atheromas could enhance LDL retention of SEM during atherosclerosis.The aorta is an abundant tissue source of the heparan sulfate proteoglycan collagen XVIII and its proteolytically released endostatin (ES) fragment, which has previously been shown to inhibit angiogenesis in cancer and atherosclerosis models (12)(13)(14)(15). The ES portion of collagen XVIII has no attachment sites for glycosaminoglycans (GAGs) but has high affinity for heparin, which is necessary for its antiangiogenesis functions (16). Our recent data showed that collagen XVIII is differ...