Abstract-Our previous studies suggest that heme oxygenase (HO)-1 induction and/or subsequent bilirubin generation in endothelial cells may suppress superoxide generation of from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. In this study, we examined the consequence of HO-1 induction in vivo on NADPH oxidase activity. Three doses of hemin (25 mg ⅐ kg Ϫ1, IP, every 48 hours), with or without cotreatment with the HO inhibitor tin protoporphyrin-IX (15 mg ⅐ kg Ϫ1, IP), were given to apolipoprotein E-deficient mice, which display vascular oxidative stress. Hemin treatment increased HO-1 expression and activity in aorta (undetectable at baseline) and kidney (by 3-fold) and significantly reduced both NADPH oxidase activity (by Ϸ25% to 50%) and superoxide generation in situ. The increase in HO-1 activity and inhibition of NADPH oxidase activity by hemin were reversed by tin protoporphyrin-IX and were not associated with changes in Nox2 or Nox4 protein levels. Hemin also reduced plasma F 2 -isoprostane levels by 23%. The inhibition of NADPH oxidase activity by hemin in the aorta was mimicked by bilirubin in vitro (0.01 to 1 mol/L). Bilirubin also concentration-dependently reduced NADPH oxidase-dependent superoxide production stimulated by angiotensin II in rat vascular smooth muscle cells and by phorbol 12-myristate 13-acetate in human neutrophil-like HL-60 cells. HO-1 overexpression by plasmid-mediated gene transfer in rat vascular smooth muscle cells decreased NADPH-stimulated superoxide production. Thus, systemic expression of HO-1 suppresses NADPH oxidase activity by mechanisms at least partly mediated by the bile pigment bilirubin, thereby reducing oxidative stress. Key Words: bilirubin Ⅲ heme oxygenase-1 Ⅲ NADPH oxidase Ⅲ oxidative stress Ⅲ reactive oxygen species R educed nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase expressed in nonphagocytic cells has a critical role in influencing the redox balance in vasculature. This enzyme consists of the membrane-bound Nox and p22phox subunits, the cytosolic p47phox and p67phox subunits, and a small GTPase, Rac. Activation of this enzyme results in a multicomponent electron transfer machinery that carries single electron from NADPH to molecular oxygen, yielding a superoxide anion. 1 Excess superoxide generation in the vasculature decreases the availability of endothelial NO, resulting in endothelial dysfunction. 2 Superoxide and derivative reactive oxygen species (ROS) promote lipid peroxidation, foam cell formation, and expression of proinflammatory molecules in vascular cells. 3,4 Mounting evidence indicates that NADPH oxidase is involved in vascular oxidative stress and dysfunction in a variety of conditions, such as hyperlipidemia, diabetes mellitus, atherosclerosis, and hypertension. 5,6 Increased oxidative stress in vascular tissues stimulates the expression of heme oxygenase (HO)-1, the inducible form that is the rate-limiting enzyme in heme degradation and production of the bile pigment bilirubin. 7 HO-1 induction has potent pro...
Fluid secretion across the blood-brain barrier, critical for maintaining the correct fluid balance in the brain, entails net secretion of HCO 3 − , which is brought about by the combined activities of ion transporters situated in brain microvessels. These same transporters will concomitantly influence intracellular pH (pH i ). To analyse the transporters that may be involved in the maintenance of pH i and hence secretion of HCO 3 − , we have loaded primary cultured endothelial cells derived from rat brain microvessels with the pH indicator BCECF and suspended them in standard NaCl solutions buffered with Hepes or Hepes plus 5% CO 2 /HCO 3 − . pH i in the standard solutions showed a slow acidification over at least 30 min, the rate being less in the presence of HCO 3 − than in its absence. However, after accounting for the difference in buffering, the net rates of acid loading with and without HCO 3 − were similar. In the nominal absence of HCO 3 − the rate of acid loading was increased equally by removal of external Na + or by inhibition of Na + /H + exchange by ethylisopropylamiloride (EIPA). By contrast, in the presence of HCO 3 − the increase in the rate of acid loading when Na + was removed was much larger and the rate was then also significantly greater than the rate observed in the absence of both Na + and HCO 3 − . Removal of Cl − in the presence of HCO 3 − produced an alkalinization followed by a resumption of the slow acid gain. Removal of Na + following removal of Cl − increased the rate of acid gain. In the presence of HCO 3 − and initial presence of Na + and Cl − , DIDS inhibited the changes in pH i produced by removal of either Na + or Cl − . These are the expected results if these cells possess an AE-like Cl − /HCO 3 − exchanger, a 'channel-like' permeability allowing slow influx of acid (or efflux of HCO 3 − ), a NBC-like Cl − -independent Na + −HCO 3 − cotransporter, and a NHE-like Na + /H + exchanger. The in vitro rates of HCO 3 − loading via the Na + −HCO 3 − cotransporter could, if the transporter is located on the apical, blood-facing side of the cells, account for the net secretion of HCO 3 − into the brain.
Tissue engineering and cell implantation therapies are gaining popularity because of their potential to repair and regenerate tissues and organs. To investigate the role of inflammatory cytokines in new tissue development in engineered tissues, we have characterized the nature and timing of cell populations forming new adipose tissue in a mouse tissue engineering chamber (TEC) and characterized the gene and protein expression of cytokines in the newly developing tissues. EGFP-labeled bone marrow transplant mice and MacGreen mice were implanted with TEC for periods ranging from 0.5 days to 6 weeks. Tissues were collected at various time points and assessed for cytokine expression through ELISA and mRNA analysis or labeled for specific cell populations in the TEC. Macrophage-derived factors, such as monocyte chemotactic protein-1 (MCP-1), appear to induce adipogenesis by recruiting macrophages and bone marrow-derived precursor cells to the TEC at early time points, with a second wave of nonbone marrow-derived progenitors. Gene expression analysis suggests that TNFa, LCN-2, and Interleukin 1b are important in early stages of neo-adipogenesis. Increasing platelet-derived growth factor and vascular endothelial cell growth factor expression at early time points correlates with preadipocyte proliferation and induction of angiogenesis. This study provides new information about key elements that are involved in early development of new adipose tissue.
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