RESUMEN
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 h 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 against the membrane. The grafts were analyzed by quantitative polymerase chain reaction (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 (2 h), transient, increased oxidative stress was observed. Elevated expression of the antioxidants α1-microglobulin and heme oxygenase-1 was seen in the mitochondria-rich inner segments after 48 h 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 the oxidative environment may play a key role in their rapid demise. These findings shed new light on pathological reactions in biomechanically related conditions in vivo.
Asunto(s)
alfa-Globulinas/genética , Proteínas Portadoras/genética , Mitocondrias/fisiología , Estrés Oxidativo , alfa-Globulinas/metabolismo , Animales , Proteínas Portadoras/metabolismo , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Reacción en Cadena en Tiempo Real de la Polimerasa , PorcinosRESUMEN
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.
RESUMEN
PURPOSE: To explore oxidative stress and the radical scavenger α(1)-microglobulin (A1M) in the vitreous body of human eyes with primary rhegmatogenous retinal detachment (RRD). METHODS: Levels of carbonyl groups, a marker of oxidative stress, and A1M were measured by ELISA and RIA in 14 vitreous samples derived from patients suffering from RRD, and compared with 14 samples from macula hole (MH) patients. Carbonyl group and A1M levels in RRD samples were statistically related to detachment characteristics. Analysis of total protein level, SDS-PAGE, and Western blotting of A1M was also performed. In a separate experiment, mRNA expression of A1M was measured by RT-PCR in rat retina explants. RESULTS: Levels of carbonyl groups and A1M varied widely in RRD vitreous samples, but were significantly higher in samples derived from eyes with large detachment area and macula-off status, while the presence of vitreous hemorrhage did not show any significant correlation. Compared with MH samples, RRD samples displayed significantly higher levels of A1M, whereas changes in total protein levels and carbonyl groups were not significant. Novel forms of A1M, not previously seen in plasma, were found in the vitreous body by Western blotting. Furthermore, A1M expression was seen in rat retina explants and was upregulated after 24 h of culturing. CONCLUSION: Oxidative stress is a prominent feature of human eyes with primary RRD, and is directly related to detachment severity. Affected eyes can launch a protective response in the form of the radical scavenger A1M possibly derived from the retina. The results thus indicate potential therapeutic cell loss prevention in RRD by employing the endogeneous radical scavenger A1M.
Asunto(s)
Biomarcadores/metabolismo , Depuradores de Radicales Libres/metabolismo , Estrés Oxidativo , Desprendimiento de Retina/metabolismo , Cuerpo Vítreo/metabolismo , alfa-Macroglobulinas/metabolismo , Western Blotting , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , Radioinmunoensayo , Reacción en Cadena en Tiempo Real de la Polimerasa , Desprendimiento de Retina/cirugía , Vitrectomía , Desprendimiento del Vítreo/metabolismo , Desprendimiento del Vítreo/cirugía , alfa-Macroglobulinas/genéticaRESUMEN
During bleeding the skin is subjected to oxidative insults from free heme and radicals, generated from extracellular hemoglobin. The lipocalin α(1)-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 (K(d)â=â0.96×10(-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.