Carbon monoxide activates NF-kappaB via ROS generation and Akt pathways to protect against cell death of hepatocytes

Heme oxygenase overexpression or exogenous carbon monoxide (CO) protects against hepatocyte apoptosis and fulminant hepatitis. The prevention of hepatocyte apoptosis by CO has been shown to require activation of NF-kappaB. The purpose of these investigations was to determine the mechanism of CO-induced hepatocyte NF-kappaB activation and protection against apoptosis. Primary rat or mouse hepatocytes and Hep3B cells were utilized. CO exposure was performed at 250 parts per million. Main outcome measures included cell viability, reactive oxygen species (ROS) generation, and changes in the levels of the intracellular antioxidants glutathione and ascorbate. Western blotting was performed for phospho-Akt, total Akt, and IkappaBalpha. NF-kappaB activation was determined by electrophoretic mobility shift assay and luciferase reporter assays. We found that CO treatment of hepatocytes prevents spontaneous apoptosis and leads to an increase in ROS production in association with Akt phosphorylation and IkappaB degradation. CO did not increase ROS production in respiration-deficient (rho0) Hep3B cells. Both Akt phosphorylation and IkappaB degradation can be inhibited by the addition of antioxidants. Furthermore, CO-induced NF-kappaB activation is reversed by phosphatidylinositol 3-kinase (PI3-K) inhibitor (LY294002) or antioxidants. Additionally, prevention of spontaneous hepatocyte apoptosis by CO is reversed by PI3-K inhibition and antioxidants. In conclusion, these data implicate a survival pathway of CO-induced ROS, Akt phosphorylation, and NF-kappaB activation in cultured hepatocytes. This pathway may prove to be important in maintenance of hepatic function in both physiological and pathophysiological conditions.     

Stress-Based Plaque Vulnerability Index and Assessment for Carotid Atherosclerotic Plaques Using Patient-Specific Vessel Material Properties

Cardiovascular diseases are closely linked to atherosclerotic plaque development and rupture. Assessment of plaque vulnerability is of fundamental significance to cardiovascular research and disease diagnosis, prevention, treatment and management. Magnetic resonance image (MRI) data of carotid atherosclerotic plaques from 8 patients (5 male, 3 female; age: 62-83, mean=71) were acquired at the University of Washington (UW), Seattle by the Vascular Imaging Laboratory (VIL) with written informed consent obtained. Patient-specific vessel material properties were quantified using Cine MRI data for modeling use. 3D thin-layer models were used to obtain plaque stress and strain for plaque assessment. A stress-based plaque vulnerability index (SPVI) was proposed to combine mechanical analysis, plaque morphology and composition for more complete carotid plaque vulnerability assessment. The five intervals (unit: kPa) [0, 46.8), [46.8, 80), [80, 92), [92, 103), and [103, +∞) from in vivo material models were used for SPVI values of 0, 1, 2, 3 and 4, respectively. The optimized agreement rate was 85.19%. The use of patient-specific material properties in plaque models could potentially improve the accuracy of model stress/strain calculations. SPVI has the potential to improve the current image-based screening and plaque vulnerability assessment schemes.     

Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide.

Although IL-1 is an important modulator of chondrocyte metabolism, the postreceptor events triggered by IL-1 remain obscure. The present study shows that IL-1 induces the biosynthesis of nitric oxide (.N = O) by articular chondrocytes. Synthesis of .N = O is also induced by LPS. Other inflammatory mediators such as IFN-gamma, fibroblast growth factor, and TNF-alpha fail to provoke the production of .N = O, but they increase the potency of IL-1. A combination of IL-1, LPS, and TNF-alpha was shown to induce maximal production of 355 +/- 51 nmol/10(6) cells/72 h of nitrite (NO2-), which was measured as a stable end-product of .N = O generation. The biosynthesis of .N = O requires an induction period of approximately 6 h and continues for at least 72 h. Inhibition of .N = O production with the competitive inhibitor NG-monomethyl-L-arginine (NMA) leads to a suppression of gelatinase and PGE2 synthesis by chondrocytes activated with IL-1 alone. In contrast, NMA enhances the synthesis of both gelatinase and PGE2 after activation with a combination of IL-1, LPS, and TNF-alpha. An increase of PGE2 synthesis from 42.0 +/- 21.0 to 174.0 +/- 33.5 ng/10(6) cells/72 h resulted from the addition of NMA when these stimulatory agents were combined. Exposure of IL-1 and fibroblast growth factor-stimulated chondrocytes to authentic, exogenous .N = O led to an increase of PGE2 synthesis from 5.6 +/- 1.7 of untreated cells to 15.8 +/- 6.8 ng/10(6) of .N = O treated cells within the 1st h. This was followed by a suppression of PGE2 synthesis within the next 2 h.     

Inducible cytosolic enzyme activity for the production of nitrogen oxides from L-arginine in hepatocytes

The in vivo conditions needed for the induction of nitrogen oxide synthesis by hepatocytes were determined. Hepatocytes obtained from rats injected with killed Corynebacterium parvum spontaneously produced NO2(-)+NO3- in culture and were found to contain cytosolic enzyme activity for nitrogen oxide synthesis. The enzyme activity required both L-arginine and NADPH, and was not found in hepatocytes obtained from normal rats or rats injected with lipopolysaccharide (LPS) alone. In contrast, nonparenchymal cells were stimulated to synthesize NO2(-)+NO3- by LPS. These results show the presence of inducible cytosolic enzyme activity for nitrogen oxide synthesis in hepatocytes, which is distinct from nonparenchymal cell NO. synthesis.