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Received 21 Jun 2011; first review completed 28 Jun 2011; accepted in final form 11 Aug 2011ABSTRACT—Sepsis and septic shock are associated with cardiac depression. Cardiovascular instability is a major causeof death in patients with sepsis. Focal adhesion kinase (FAK) is a potential mediator of cardiomyocyte responses tooxidative and mechanical stress. Myocardial collagen deposition can affect cardiac compliance and contractility. The aimof the present study was to determine whether the silencing of FAK is protective against endotoxemia-induced alterationsof cardiac structure and function. In male Wistar rats, endotoxemia was induced by intraperitoneal injection oflipopolysaccharide (10 mg/kg). Cardiac morphometry and function were studied in vivo by left ventricular catheterizationand histology. Intravenous injection of small interfering RNA targeting FAK was used to silence myocardial expression ofthe kinase. The hearts of lipopolysaccharide-injected rats showed collagen deposition, increased matrix metalloproteinase2 activity, and myocyte hypertrophy, as well as reduced 24-h +dP/dt and jdP/dt, together with hypotension, increased leftventricular end-diastolic pressure, and elevated levels of FAK (phosphorylated and unphosphorylated). Focal adhesionkinase silencing reduced the expression and activation of the kinase in cardiac tissue, as well as protecting against theincreased collagen deposition, greater matrix metalloproteinase 2 activity, and reduced cardiac contractility that occurduring endotoxemia. In conclusion, FAK is activated in endotoxemia, playing a role in cardiac remodeling and in theimpairment of cardiac function. This kinase represents a potential therapeutic target for the protection of cardiac function inpatients with sepsis.KEYWORDS—Focal adhesion protein-tyrosine kinases, cytokines, inflammation, sepsis, shock septic

Signal transduction through the surface molecule CD40 is critical for cellular activation in immunoinflammatory states such as sepsis. The mechanisms regulating this pathway are not completely understood. Because CD40 displays potentially regulatory cysteine residues and CD40 is probably exposed to NO in the inflammatory milieu, we hypothesized that S-nitrosylation, the interaction of NO with cysteines residues, acts as a post-translational modification on CD40, coregulating the signaling activity and, therefore, the level of cellular activation. As assessed by the biotin switch and the reduction/chemiluminescence S-nitrosylation detection techniques, CD40 was found to be S-nitrosylated endogenously and upon exposure to NO donors in both human and murine macrophages. S-nitrosylation of CD40 was associated with milder activation by its ligand (CD40L), leading to reduced in vitro cytokine (IL-1beta, IL-12, and TNF-alpha) production, which was reversed in the presence of inhibitors of NO synthesis. S-nitrosylated CD40 was found in resting RAW 246.7 macrophages and BALB/c mice peritoneal macrophages, turning into the denitrosylated state upon in vitro or systemic exposure, respectively, to LPS. Moreover, monocytes from patients with sepsis displayed denitrosylated CD40 in contrast to the CD40 S-nitrosylation measured in healthy individuals. Finally, in an attempt to explain how S-nitrosylation regulates CD40 activation, we demonstrate that NO affects the redistribution of CD40 on the cell surface, which is a requirement for optimal signal transduction. Our results support a novel post-translational regulatory mechanism in which the CD40 signal may be, at least in part, dependent on cellular activation-induced receptor denitrosylation.

Mice expressing human cholesteryl ester transfer protein (huCETP) are more resistant to Escherichia coli bacterial wall LPS because death rates 5 days after intraperitoneal inoculation of LPS were higher in wild-type than in huCETP+/+ mice, whereas all huCETP+/+ mice remained alive. After LPS inoculation, plasma concentrations of TNF-alpha and IL-6 increased less in huCETP+/+ than in wild-type mice. LPS in vitro elicited lower TNF-alpha production by CETP expressing than by wild-type macrophages. In addition, TNF-alpha production by RAW 264.7 murine macrophages increased on incubation with LPS but decreased in a dose-dependent manner when human CETP was added to the medium. Human CETP in vitro enhanced the LPS binding to plasma high-density lipoprotein/low-density lipoprotein. The liver uptake of intravenous infused 14C-LPS from Salmonella typhimurium was greater in huCETP+/+ than in wild-type mice. Present data indicate for the first time that CETP is an endogenous component involved in the first line of defense against an exacerbated production of proinflammatory mediators.

Hypertonic saline solution (HS solution, NaCl 7.5%) has shown to restore hemodynamic parameters in hemorrhagic shock and to decrease the inflammation triggered by ischemia-reperfusion injury (I-R). Therefore, our objective was to investigate the effects of HS solution on the mechanisms involved in I-R, in an experimental model of controlled hemorrhagic shock. Wistar rats (280-350 g) were submitted to controlled bleeding, keeping the mean arterial pressure around 40 mmHg, for 1 h. After that, rats were randomized and treated with HS solution (4 mL/kg) or normal saline (34 mL/kg). There were no differences in hemodynamic parameters between both groups for at least 2 h after shock. No difference either was observed in reactive oxygen species generation (measured indirectly by malondialdehyde concentration) or cytokines (interleukins 6 and 10) production (measured by enzyme-linked immunosorbent assay). Quantitative analysis of lung tissue showed a smaller neutrophil infiltration in animals that received HS solution. Moreover, the animals in the HS group showed an increased expression of heat shock protein 70. Therefore, we concluded that treatment of hemorrhagic shock with HS solution can decrease pulmonary inflammation and increase cellular protection by up-regulating heat shock protein 70 expression.