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 · kg ⁻¹ , IP, every 48 hours), with or without cotreatment with the HO inhibitor tin protoporphyrin-IX (15 mg · kg ⁻¹ , 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 ≈25% 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 μmol/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.

Ions and water transported across the endothelium lining the blood–brain barrier contribute to the fluid secreted into the brain and are important in maintaining appropriate volume and ionic composition of brain interstitial fluid. Changes in this secretion process may occur after stroke. The present study identifies at transcript and protein level ion transporters involved in the movement of key ions and examines how levels of certain of these alter following oxidative stress. Immunohistochemistry provides evidence for Cl⁻/HCO₃ ⁻ exchanger, AE2, and Na⁺, HCO₃ ⁻ cotransporters, NBCe1 and NBCn1, on brain microvessels. mRNA analysis by RT-PCR reveals expression of these transporters in cultured rat brain microvascular endothelial cells (both primary and immortalized GPNT cells) and also Na⁺/H⁺ exchangers, NHE1 (primary and immortalized) and NHE2 (primary cells only). Knock-down using siRNA in immortalized GPNT cells identifies AE2 as responsible for much of the Cl⁻/HCO₃ ⁻ exchange following extracellular chloride removal and NHE1 as the transporter that accounts for most of the Na⁺/H⁺ exchange following intracellular acidification. Transcript levels of both AE2 and NHE1 are increased following hypoxia/reoxygenation. Further work is now required to determine the localization of the bicarbonate transporters to luminal or abluminal membranes of the endothelial cells as well as to identify and localize additional transport mechanisms that must exist for K⁺ and Cl⁻.

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 TNFα, LCN-2, and Interleukin 1β 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.

NADPH oxidase is involved in cell signaling, regulating proliferation of vascular cells, especially in endothelium. The Nox4 catalytic subunit has a major role in endothelial cells, but growth arrest of cultured endothelial cells following serum deprivation paradoxically increases mRNA for Nox4. We investigated the relationships between Nox4 mRNA stability and protein expression in human microvascular endothelial cells. Serum starvation increased the steady-state level of Nox4 mRNA but paradoxically diminished Nox4 protein expression. mRNA transcription in the absence of serum is maintained by the p38MAP kinase pathway, for inhibition of p38MAP kinase reduced both Nox4 mRNA and Nox4 promoter activity. In serum-starved cells, reintroduction of serum increased Nox4 protein levels within 30 min and up to 24 h. In contrast, the mRNA decreased equally rapidly after serum stimulation. Inhibition of Nox4 translation by cycloheximide blocked serum-induced mRNA degradation and Nox4 protein synthesis, and actinomycin-D also delayed Nox4 mRNA decay. Therefore, Nox4 mRNA level falls after serum stimulation because of a translation-initiated mRNA destabilization program. Clearly Nox4 mRNA is regulated at both transcriptional and post-transcriptional levels, and the steady state level of Nox4 mRNA does not accurately reflect Nox4 protein abundance and functions, with implications for regulation of cell proliferation and survival.