Cigarette smoking is a major independent risk factor for cardiovascular disease. While the association between chronic smoking and cardiovascular disease is well established, the underlying mechanisms are incompletely understood, partly due to the lack of adequate in vivo animal models. Here, we report a mouse model of chronic smoking-induced cardiovascular pathology. Male C57BL/6J mice were exposed to whole body mainstream cigarette smoke (CS) using a SCIREQ "InExpose" smoking system (48 min/day, 5 days/wk) for 16 or 32 wk. Age-matched, air-exposed mice served as nonsmoking controls. Blood pressure was measured, and cardiac MRI was performed. In vitro vascular ring and isolated heart experiments were performed to measure vascular reactivity and cardiac function. Blood from control and smoking mice was studied for the nitric oxide (NO) decay rate and reactive oxygen species (ROS) generation. With 32 wk of CS exposure, mice had significantly less body weight gain and markedly higher blood pressure. At 32 wk of CS exposure, ACh-induced vasorelaxation was significantly shifted to the right and downward, left ventricular mass was significantly larger along with an increased heart-to-body weight ratio, in vitro cardiac function tended to be impaired with high afterload, white blood cells had significantly higher ROS generation, and the blood NO decay rate was significantly faster. Thus, smoking led to blunted weight gain, hypertension, endothelial dysfunction, leukocyte activation with ROS generation, decreased NO bioavailability, and mild cardiac hypertrophy in mice that were not otherwise predisposed to disease. This mouse model is a useful tool to enable further elucidation of the molecular and cellular mechanisms of smoking-induced cardiovascular diseases.

In endothelium, NO is derived from endothelial NO synthase (eNOS)-mediated L-arginine oxidation. Endogenous guanidinomethylated arginines (MAs), including asymmetric dimethylarginine (ADMA) and N G -methyl-L-arginine (L-NMMA), are released in cells upon protein degradation and are competitive inhibitors of eNOS. However, it is unknown whether intracellular MA concentrations reach levels sufficient to regulate endothelial NO production. Therefore, the dose-dependent effects of ADMA and L-NMMA on eNOS function were determined. Kinetic studies demonstrated that the K m for L-arginine is 3.14 M with a V max of 0.14 mol mg The biological significance of guanidino-methylated arginine derivatives has been known since the inhibitory actions of N G -methyl-L-arginine (L-NMMA) 3 on macrophage induced cytotoxicity were first demonstrated. It was subsequently realized that these effects were mediated through inhibition of NO release (1). NO has been demonstrated as a critical effector molecule in the maintenance of vascular function (2-4). In the vasculature, NO is derived from the oxidation of L-Arg, catalyzed by the constitutively expressed enzyme, eNOS (5-7). This endothelial-derived NO diffuses from the vascular endothelium into the smooth muscle cell layer where it activates soluble guanylate cyclase leading to smooth muscle relaxation (2-4). In addition to its role in the maintenance of vascular tone, NO helps to maintain the anti-atherogenic character of the normal vascular wall. NO, in concert with various cell signaling molecules, has been demonstrated to maintain smooth muscle cell quiescence and as such, counteracts pro-proliferative agents, specifically those involved in the propagation of athero-proliferative disorders (8 -14). As such, eNOS dysfunction is an early symptom of vascular disease and is manifested through insufficient NO bioavailability. Several potential causes of NO deficiency in disease settings have been proposed. Among these, high circulating levels of the endogenous methylarginine NOS inhibitor asymmetric dimethylarginine (ADMA) has been hypothesized to be of particular importance (15)(16)(17)(18)(19)(20)(21). In neurons and the brain, it has been shown that the methyl arginine L-NMMA is also present, however, the levels of this methylarginine have not been previously considered in studies evaluating vascular dysfunction (22).ADMA and L-NMMA are endogenous inhibitors of NOS and are derived from the proteolysis of methylated arginine residues on various proteins. The methylation is carried out by a group of enzymes referred to as protein-arginine methyltransferase (23). Subsequent proteolysis of proteins containing methylarginine groups leads to the release of free methylarginine into the cytoplasm, and if sufficient levels are reached NO production from NOS would be inhibited (24, 25). To date, six different isoforms of the enzyme have been identified with each

FcγR-mediated phagocytosis of IgG-coated particles is a complex process involving the activation of multiple signaling enzymes and is regulated by the inositol phosphatases PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP-1 (Src homology [SH2] domain-containing inositol phosphatase). In a recent study we have demonstrated that SHIP-2, an inositol phosphatase with high-level homology to SHIP-1, is involved in FcγR signaling. However, it is not known whether SHIP-2 plays a role in modulating phagocytosis. In this study we have analyzed the role of SHIP-2 in FcγR-mediated phagocytosis using independent cell models that allow for manipulation of SHIP-2 function without influencing the highly homologous SHIP-1. We present evidence that SHIP-2 translocates to the site of phagocytosis and down-regulates FcγR-mediated phagocytosis. Our data indicate that SHIP-2 must contain both the N-terminal SH2 domain and the C-terminal proline-rich domain to mediate its inhibitory effect. The effect of SHIP-2 is independent of SHIP-1, as overexpression of dominant-negative SHIP-2 in SHIP-1-deficient primary macrophages resulted in enhanced phagocytic efficiency. Likewise, specific knockdown of SHIP-2 expression using siRNA resulted in enhanced phagocytosis. Finally, analysis of the molecular mechanism of SHIP-2 down-regulation of phagocytosis revealed that SHIP-2 down-regulates upstream activation of Rac. Thus, we conclude that SHIP-2 is a novel negative regulator of FcγR-mediated phagocytosis independent of SHIP-1. (Blood. 2006;107:813-820)

Microindentation is an investigational tool often used to determine hardness and other derived material properties of the material bone. This study explored the variation of microindentation hardness results with five independent variables. The variables were: applied mass, dwell time, drying time, time between indentation and measurement, and distance between the center of an indentation and the edge of other indentations and pores. These variables were selected because they represented a reasonable range of specimen investigational steps. We also investigated the cross sections of typical indentation residual impressions to determine the degree of material pile-up at the edges of the impressions. We found that microindentation hardness varied with applied mass and with distance between the indentation and neighboring indentations and pores but not with the other variables. Our recommended minimum applied mass is 0.10 kg versus a previously published value of 0.05 kg. We also found no discernable material pile-up at the residual impression edges, in contrast to reports of others.