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During bleeding the skin is subjected to oxidative insults from free heme and radicals, generated from extracellular hemoglobin. The lipocalin α1-microglobulin (A1M) was recently shown to have reductase properties, reducing heme-proteins and other substrates, and to scavenge heme and radicals. We investigated the expression and localization of A1M in skin and the possible role of A1M in the protection of skin tissue from damage induced by heme and reactive oxygen species. Skin explants, keratinocyte cultures and purified collagen I were exposed to heme, reactive oxygen species, and/or A1M and investigated by biochemical methods and electron microscopy. The results demonstrate that A1M is localized ubiquitously in the dermal and epidermal layers, and that the A1M-gene is expressed in keratinocytes and up-regulated after exposure to heme and reactive oxygen species. A1M inhibited the heme- and reactive oxygen species-induced ultrastructural damage, up-regulation of antioxidation and cell cycle regulatory genes, and protein carbonyl formation in skin and keratinocytes. Finally, A1M bound to purified collagen I (Kd = 0.96×10−6 M) and could inhibit and repair the destruction of collagen fibrils by heme and reactive oxygen species. The results suggest that A1M may have a physiological role in protection of skin cells and matrix against oxidative damage following bleeding.

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α1-microglobulin (A1M) is a 26 kDa plasma and tissue protein with reductase activity and radical- and heme-binding anti-oxidative functions. In addition, exposure of A1M to hemoglobin has been shown to induce proteolytic elimination of a C-terminal tetrapeptide yielding a heme-degrading form, truncated A1M (t-A1M). Myeloperoxidase (MPO), a heme-containing enzyme that catalyzes the production of free radicals and hypochlorite, is released by neutrophils during the inflammatory response to bacterial infections. MPO-induced low density lipoprotein (LDL)-oxidation in blood has been suggested as a causative factor in atherosclerosis. In this study we have hypothesized that A1M interacts with MPO in a similar mode as with hemoglobin, and is a regulator of its activity. The results show that A1M is proteolytically cleaved, with formation of t-A1M, after exposure to MPO, and that t-A1M contains iron and heme-degradation products. The reaction is dependent of pH, time and concentration of substrates and a pH-value around 7 is shown to be optimal for cleavage. Furthermore, A1M inhibits MPO- and hydrogen peroxide-induced oxidation of LDL. The results suggest that A1M may have a role as an inhibitor of the damaging effects of the neutrophil respiratory burst on bystander tissue components.

Purpose: The purpose of this study was to explore the relationship between oxidative stress, antioxidant defense, mitochondrial structure and biomechanical tissue support in the isolated porcine retina. Methods:Full-thickness retinal sheets were isolated from adult porcine eyes. Retinas were cultured for 2 or 48 hours using 1) a previously established low-support explant protocol with photoreceptors positioned against the culture membrane (porous polycarbonate) or 2) a high-support procedure developed by our group, apposing the Müller cell endfeet and inner limiting membrane (ILM) against the membrane. The grafts were analyzed by quantitative PCR, immunohistochemistry, and transmission electron microscopy (TEM), and culture medium was assayed for the cell damage and oxidative stress markers lactate dehydrogenase and protein carbonyls. Results:In explants cultured with physical support to the inner border, cone photoreceptors were preserved and lactate dehydrogenase levels were reduced, although an initial (2h), transient, increased oxidative stress was observed. Elevated expression of the antioxidants A1M (α1-microglobulin) and heme oxygenase-1 were seen in the mitochondria-rich inner segments after 48h compared to low-support counterparts.Housekeeping gene expression suggested a higher degree of structural integrity of mitochondria in high-support explants, and TEM of inner segments confirmed preservation of a normal mitochondrial morphology. Conclusion:Providing retinal explants with inner retinal support leads to mobilization of antioxidant proteins, preservation of mitochondrial function and increased cell viability.Consequently, the failure of low-support retinal cultures to mobilize an adequate response to 2 the oxidative environment may play an key role in their rapid demise. These findings shed new light on pathological reactions in biomechanically related conditions in vivo.