RGS2 is a primary terminator of ß2-adrenergic receptor-mediated G(i) signaling.

Two major ß-adrenergic receptor (ßAR) subtypes, ß(1)AR and ß(2)AR, are expressed in mammalian heart with ß(1)AR coupling to G(s) and ß(2)AR dually coupling to G(s) and G(i) proteins. In many types of chronic heart failure, myocardial contractile response to both ß(1)AR and ß(2)AR stimulation is severely impaired. The dysfunction of ßAR signaling in failing hearts is largely attributable to an increase in G(i) signaling, because disruption of the G(i) signaling restores myocardial contractile response to ß(1)AR as well as ß(2)AR stimulation. However, the mechanism terminating the ß(2)AR-G(i) signaling remains elusive, while it has been shown activation of the G(i) 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 ß(2)AR-G(i) signaling. Specifically, prolonged absence of agonist stimulation for 24h impairs the ß(2)AR-G(i) signaling, resulting in enhanced ß(2)AR- but not ß(1)AR-mediated contractile response in cultured adult mouse cardiomyocytes. Increased ß(2)AR contractile response is accompanied by a selective upregulation of RGS2 in the absence of alterations in other major cardiac RGS proteins (RGS3-5) or G(s), G(i) or ßAR subtypes. Administration of a ßAR agonist, isoproterenol (ISO, 1.0 nM), prevents RGS2 upregulation and restores the ß(2)AR-G(i) signaling in cultured cells. Furthermore, RGS2 ablation, similar to ßAR agonist stimulation, sustains the ß(2)AR-G(i) signaling in cultured cells, whereas adenoviral overexpression of RGS2 suppresses agonist-activated ß(2)AR-G(i) signaling in cardiomyocytes and HEK293 cells. These findings not only define RGS2 as a novel negative regulator of the ß(2)AR-G(i) signaling but also provide a potential novel target for the treatment of chronic heart failure.

Design, generation, and testing of mammalian expression modules that tag membrane proteins.

The expression of mammalian membrane proteins in laboratory cell lines allows their biological functions to be characterized and carefully dissected. However, it is often difficult to design and generate effective antibodies for membrane proteins in the desired studies. As a result, expressed membrane proteins cannot be detected or characterized via common biochemical approaches such as western blotting, immunoprecipitation, or immunohistochemical analysis, and their cellular behaviors cannot be sufficiently investigated. To circumvent such roadblocks, we designed and generated two sets of expression modules that consist of sequences encoding for three essential components: (1) a signal peptide from human receptor for advanced glycation end products that targets the intended protein to the endoplasmic reticulum for cell surface expression; (2) an antigenic epitope tag that elicits specific antibody recognition; and (3) a series of restriction sites that facilitate subcloning of the target membrane protein. The modules were designed with the flexibility to change the epitope tag to suit the specific tagging needs. The modules were subcloned into expression vectors, and were successfully tested with both Type I and Type III human membrane proteins: the receptor for advanced glycation end products, the Toll-like receptor 4, and the angiotensin II receptor 1. These expressed membrane proteins are readily detected by western blotting, and are immunoprecipitated by antibodies to their relative epitope tags. Immunohistochemical and biochemical analyses also show that the expressed proteins are located at cell surface, and maintain their modifications and biological functions. Thus, the designed modules serve as an effective tool that facilitates biochemical studies of membrane proteins.

Stereochemistry of an agonist determines coupling preference of beta2-adrenoceptor to different G proteins in cardiomyocytes.

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 beta(2)-adrenoceptor agonists activate both G(s) and G(i) proteins, fenoterol, a full agonist of beta(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 beta(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.

Chemokine RANTES is upregulated in monocytes from patients with hyperhomocysteinemia.

AIM: To investigate the changes in plasma level of the chemokine RANTES (regulated upon activation, normal T cells expressed and secreted) and the responsiveness of lipopolysaccharide (LPS)-induced RANTES secretion from monocytes in patients with hyperhomocysteinemia (HHcy). METHODS: The plasma levels of homocysteine (Hcy), folate, and RANTES were measured in 38 control patients with normal Hcy levels and 40 patients with HHcy and the mRNA synthesis of RANTES in isolated human monocytes was determined by RNase protection assays. RESULTS: The plasma level of RANTES was elevated in HHcy patients compared with controls (median 5.3 vs 3.5 ng/mL, P<0.01). LPS-induced RANTES production from monocytes of HHcy patients was also increased significantly. In addition, Hcy directly increased the mRNA level of RANTES in isolated normal human monocytes in a time- and dose-dependent manner. CONCLUSION: Upregulated RANTES from monocytes in HHcy patients may be involved in the atherogenesis of HHcy-induced atherosclerosis.

Endostatin binds biglycan and LDL and interferes with LDL retention to the subendothelial matrix during atherosclerosis.

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 alpha 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.

Folic acid reverses hyper-responsiveness of LPS-induced chemokine secretion from monocytes in patients with hyperhomocysteinemia.

Our previous study demonstrated that homocysteine (Hcy) mediated the expression and secretion of MCP-1 and IL-8 in human monocytes. In the present study, we investigated whether the responsiveness of isolated monocytes to lipopolysaccharide (LPS)-induced chemokine secretion was enhanced in patients with hyperhomocysteinemia (HHcy), and if so, whether this enhanced response could be inhibited by folic acid treatment. We studied 38 control subjects and 40 patients with HHcy. The results showed that MCP-1 secretion from isolated monocytes in response to low-dose LPS in patients with HHcy was significantly higher than that in controls. After patients with HHcy underwent low-dose folic acid treatment (0.8 mg/d) for 6 months, plasma Hcy levels were decreased and the hyper-responsiveness of MCP-1 and IL-8 secreted by isolated monocytes was significantly reversed. Furthermore, folic acid treatment at high concentrations (5 microM) significantly reduced the elevated levels of reactive oxygen species, NADPH oxidase activity and chemokines in response to Hcy in cultured human monocytes. HHcy may contribute to atherogenesis through enhancing the responsiveness of monocytes to inflammatory stimuli and promoting leukocyte recruitment into atherosclerotic plaque. In addition to lowering the plasma levels of Hcy, low-dose folic acid treatment exerts beneficial effects on patients with HHcy by inhibiting pro-inflammatory responses such as chemokine secretion from human monocytes.

Signal pathways underlying homocysteine-induced production of MCP-1 and IL-8 in cultured human whole blood.

AIM: To elucidate the mechanisms underlying homocysteine (Hcy)-induced chemokine production. METHODS: Human whole blood was pretreated with inhibitors of calmodulin (CaM), protein kinase C (PKC), protein tyrosine kinase (PTK), mitogen-activated protein kinase (MAPK), and NF-kappaB and activators of PPARgamma for 60 min followed by incubation with Hcy 100 micromol/L for 32 h. The levels of mitogen chemokine protein (MCP)-1 and interleukin-8 (IL-8) were determined by enzyme-linked immunosorbant assay (ELISA). RESULTS: Inhibitors of PKC (calphostin C, 50-500 nmol/L and RO-31-8220, 10-100 nmol/L), CaM (W7, 28-280 micromol/L), ERK1/2 MAPK (PD 98059, 2-20 micromol/L), p38 MAPK (SB 203580, 0.6-6 micromol/L), JNK MAPK (curcumin, 2-10 micromol/L), and NF-kappaB (PDTC, 10-100 nmol/L) markedly reduced Hcy 100 micromol/L-induced production of MCP-1 and IL-8 in human cultured whole blood, but the inhibitors of PTK (genistein, 2.6-26 micromol/L and tyrphostin, 0.5-5 micromol/L) had no obvious effect on MCP-1 and IL-8 production. PPARgamma activators (ciglitazone 30 micromol/L and troglitazone 10 micromol/L) depressed the Hcy-induced MCP-1 production but not IL-8 production in the cultured whole blood. CONCLUSION: Hcy-induced MCP-1 and IL-8 production is mediated by activated signaling pathways such as PKC, CaM, MAPK, and NF-kappaB. Our results not only provide clues for the signal transduction pathways mediating Hcy-induced chemokine production, but also offer a plausible explanation for a pathogenic role of hyperhomocysteinemia in these diseases.

Homocysteine induces production of monocyte chemoattractant protein-1 and interleukin-8 in cultured human whole blood.

AIM: To investigate whether increased plasma L-homocysteine (Hcy) level could promote monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) in cultured whole blood. METHODS: Human whole blood or different type of peripheral blood cells from health volunteers were incubated with Hcy and/or the inhibitors. MCP-1 and IL-8 level were measured by ELISA assay. RESULTS: Hcy 10-1000 micromol/L induced production of MCP-1 and IL-8 in cultured human whole blood (P<0.05). The major cellular source of these chemokines comed from monocytes. Meanwhile,Hcy also promoted the upregulation of MPO level even at the 10 micromol/L in the cultured whole blood. The intracellular ROS, particular the OH. radicals, play extremely important role in the Hcy-induced MCP-1 and IL-8 production. CONCLUSION: Increased Hcy level in plasma (hyperhomocysteinemia) induced MCP-1 and IL-8 secretion in cultured human whole blood, especially in monocytes via oxidative stress mechanism.

Loss of collagen XVIII enhances neovascularization and vascular permeability in atherosclerosis.

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(-/-)) mice into the atherosclerosis-prone apolipoprotein E-deficient (ApoE(-/-)) strain. After 6 months on a cholesterol diet, aortas from ApoE(-/-);Col18a1(-/-) and ApoE(-/-);Col18a1(+/-) 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(-/-) 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.

Homocysteine mediated expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human monocytes.

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 micromol/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-kappaB or the activator of peroxisome proliferator-activated receptor gamma (PPARgamma) 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-kappaB in a PPARgamma activator-sensitive manner. Thus, activation of PPARgamma may become a therapeutic target for preventing Hcy-induced proatherogenic effects.

Oxidant stress mechanism of homocysteine potentiating Con A-induced proliferation in murine splenic T lymphocytes.

OBJECTIVE: An elevated plasma homocysteine (Hcy) level is considered an independent risk factor for atherosclerosis. However, the mechanisms by which hyperhomocysteinemia induces atherosclerosis are only partially understood. The effect of Hcy on T lymphocyte proliferation and its mechanisms were examined in normal and hyperhomocysteinemia ApoE-knockout mice. METHODS: The mouse splenic T-cells were treated with Hcy, related compounds and/or antioxidants in the presence or absence of Concanavalin A (Con A). DNA synthesis, cell apoptosis, interleukin-2 level and production of reactive oxygen species (ROS) were measured. RESULTS: Hcy (0.3-3.0 mM) and related compounds with thiol (-SH), such as cysteine and glutathione significantly potentiated Con A-induced proliferation and partially inhibited apoptosis in T lymphocytes, but it had no direct effect on resting T lymphocyte. ApoE-knockout mice with hyperhomocysteinemia (the level of plasma Hcy was 20.3+/-2.9 vs. 2.6+/-0.6 microM in control group, P<0.05) had a significant promotion of T-cell proliferation in response to Con A. Hcy (0.3-3.0 mM) also increased the intracellular ROS. Radical scavengers reduced Hcy effect. CONCLUSIONS: 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.