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Heme oxygenase-1 (HO-1) cleaves the porphyrin ring of heme into carbon monoxide, Fe2+, and biliverdin, which is then converted into bilirubin. Heme-derived Fe2+ induces the expression of the iron-sequestering protein ferritin and activates the ATPase Fe2+-secreting pump, which decrease intracellular free Fe2+ content. Based on the antioxidant effect of bilirubin and that of decreased free cellular Fe2+, we questioned whether HO-1 would modulate the expression of proinflammatory genes associated with endothelial cell (EC) activation. We tested this hypothesis specifically for the genes E-selectin (CD62), ICAM-1 (CD54), and VCAM-1 (CD106). We found that HO-1 overexpression in EC inhibited TNF-α-mediated E-selectin and VCAM-1, but not ICAM-1 expression, as tested at the RNA and protein level. Heme-driven HO-1 expression had similar effects to those of overexpressed HO-1. In addition, HO-1 inhibited the activation of NF-κB, a transcription factor required for TNF-α-mediated up-regulation of these genes in EC. Bilirubin and/or Fe2+ chelation mimicked the effects of HO-1, whereas biliverdin or carbon monoxide did not. In conclusion, HO-1 inhibits the expression of proinflammatory genes associated with EC activation via a mechanism that is associated with the inhibition of NF-κB activation. This effect of HO-1 is mediated by bilirubin and/or by a decrease of free intracellular Fe2+ but probably not by biliverdin or carbon monoxide.

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The influence of applied stress on the measurement of hardness and elastic modulus using nanoindentation methods has been experimentally investigated using special specimens of aluminum alloy 8009 to which controlled stresses could be applied by bending. When analyzed according to standard methods, the nanoindentation data reveal changes in hardness with stress similar to those observed in conventional hardness tests. However, the same analysis shows that the elastic modulus changes with stress by as much as 10%, thus suggesting that the analysis procedure is somehow deficient. Comparison of the real indentation contact areas measured optically to those determined from the nanoindentation data shows that the apparent stress dependence of the modulus results from an underestimation of the contact area by the nanoindentation analysis procedures.

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The elastic properties of several microstructural components of dry human vertebrae (T-12 and L-1) and tibiae have been investigated in the longitudinal and transverse directions using nanoindentation. The largest Young's modulus was that for the interstitial lamellae in the longitudinal direction (25.7 +/- 1.7 GPa). This was followed in decreasing order by osteons in the longitudinal direction (22.4 +/- 1.2 GPa), trabeculae in the longitudinal direction (19.4 +/- 2.3 GPa), an average over osteons and interstitial lamellae in the transverse direction [16.6 +/- 1.1 GPa (it was difficult to microstructurally distinguish osteons from interstitial lamellae in the transverse direction)], and trabeculae in the transverse direction (15.0 +/- 2.5 GPa). An ANOVA statistical analysis revealed that the values all are significantly different (p < 0.05). Since the elastic moduli in the longitudinal direction are all greater than in the transverse, measurable elastic anisotropies exist in the components. The hardnesses also varied among the microstructural components in the range 0.52-0.74 GPa.

Biliverdin, a product of heme oxygenase-1 (HO-1) enzymatic action, is converted into bilirubin, which has been considered a waste product in the past. We now show that administration of biliverdin has a salutary effect in organ transplantation. A brief course of treatment with biliverdin leads to long-term survival of H-2 incompatible heart allografts. Furthermore, those recipients harboring long-surviving (>100 days) allografts were tolerant to donor antigens indicated by the acceptance of second donor strain hearts but not third-party grafts. Treatment with biliverdin decreased intragraft leukocyte infiltration and inhibited T cell proliferation. Likely related to tolerance induction, biliverdin interferes with T cell signaling by inhibiting activation of nuclear factor of activated T cells (NFAT) and nuclear factor kappaB (NF-kappaB), two transcription factors involved in interleukin-2 (IL-2) transcription and T cell proliferation, as well as suppressing Th1 interferon-gamma (IFN-gamma) production in vitro. These findings support the potential use of biliverdin, a natural product, in transplantation and other T cell mediated immune disorders.

Substrate effects on the measurement of thin film mechanical properties by nanoindentation methods have been studied experimentally using a model soft film on hard substrate system: aluminum on glass. The hardness and elastic modulus of aluminum films with thicknesses of 240, 650, and 1700 nm sputter-deposited on glass were systematically characterized as a function of indenter penetration depth using standard nanoindentation methods. Scanning electron and atomic force microscopy of the hardness impressions revealed that indentation pileup in the aluminum is significantly enhanced by the substrate. The substrate also affects the form of the unloading curve in a manner that has important implications for nanoindentation data analysis procedures. Because of these effects, nanoindentation measurement techniques overestimate the film hardness and elastic modulus by as much as 100% and 50%, respectively, depending on the indentation depth. The largest errors occur at depths approximately equal to the film thickness.