Modifications to the basement membrane protein laminin using glycolaldehyde and A2E: a model for aging in Bruch's membrane.

In a variety of retinal diseases, including age-related macular degeneration (AMD); basement membranes are susceptible to alterations in structure and function. Chemical modifications to basement membrane proteins may deleteriously affect Bruch's membrane leading to the development of AMD. The purpose of this study was to investigate modifications from glycolaldehyde and A2E, which are present in the retinal pigment epithelium (RPE), on the membrane like protein fragment, laminin, as a model for aging of Bruch's membrane in age related eye diseases. Laminin was allowed to react with either glycolaldehyde or A2E during irradiation of A2E and then tryptically digested before analysis with electrospray ionization mass spectrometry (ESI-MS). Modifications to laminin occurred preferentially on lysine or arginine residues. The A2E modified laminin fragments are consistent with additions of A2E derived aldehydes resulting from cleavages closest to the pyridinium ring in A2E and oxidized A2E. These results provide evidence that A2E and advanced glycation endproducts (AGE) may be involved in modifications to essential basement membrane proteins leading to deleterious changes in the retinal pigment epithelium extracellular matrix (RPE-ECM) environment. These preliminary experiments are essential for the identification of these modifications in vivo.

Extracellular matrix alterations in conventional renal cell carcinomas by tissue microarray profiling influenced by the persistent, long-term, low-dose ionizing radiation exposure in humans.

The present study was carried out in order to examine molecular alterations of extracellular matrix (ECM), associated with cell-cell communication in conventional (clear-cell) renal cell carcinomas (cRCCs) influenced by persistent long-term, low-dose ionizing radiation (IR) exposure to patients living more than 19 years after the Chernobyl accident in Cesium 137 (137Cs)-contaminated areas of Ukraine. The ECM major components such as fibronectin, laminin, E-cadherin/beta-catenin complexes and p53 tumor suppressor gene protein, and transforming growth factor beta 1 (TGF-beta1) were immunohistochemically (IHC) evaluated in cRCCs from 59 Ukrainian patients, which represented 18 patients living in non-contaminated areas and 41 patients from 137Cs-contaminated areas. In contrast, a control group of 19 Spanish patients with analogue tumors were also investigated. For IHC evaluation, a tissue microarray technique was used. Decrease or loss and abnormal distribution of fibronectin, laminin, E-cadherin/beta-catenin complexes accompanied by elevated levels of p53 and TGF-beta1 were detected in the Ukrainian cRCCs from 137Cs-contaminated areas with statistically significant differences. Thus, our study suggests that chronic long-term, low-dose IR exposure might result in global remodeling of ECM components of the cRCCs with disruption in peri-epithelial stroma and epithelial basement membranes.

Decrease in laminin content and protein excretion rate after five sixths nephrectomy and low-dose irradiation in the rat.

The effect of low-dose irradiation on laminin distribution and urine protein excretion in the remnant rat kidney has been studied. The rat remnant kidney formed after 5/6 nephrectomy is an experimental model of chronic renal failure. In the remnant kidney, focal segmental glomerulosclerosis is developed characterized by focal or segmental sclerosis in glomeruli, alterations in the tubules and thickening of the glomerular basement membrane. Low dose irradiation has been presumed to suppress sclerotic processes. In this study 24 male Wistar rats were subdivided into the nephrectomized group, nephrectomized and irradiated groups (1 or 3 Grey), and healthy control group. Animals were sacrificed at 2, 4 and 8 weeks after beginning the experiment. Laminin immunohistochemical staining was found along the tubular and glomerular basement membranes in all experimental groups, but with varying intensity. Laminin content in the basement membranes was decreased in early stages (week 2), especially after irradiation followed by increase during the later stages with relatively high levels at the end of the experiment (week 8). Irradiation at a dose of 3 Grey decreased protein excretion compared to the nephrectomized rats at all stages, while 1 Grey dose was ineffective. Based on decreased proteinuria we conclude that moderate low-dose irradiation has beneficial effects on the rat remnant kidney and that laminin in basement membranes is probably not the most crucial component in regulating membrane permeability.

Radiation quality and tissue-specific microenvironments following exposure to 1 GeV/amu Fe.

This paper summarizes quantitative in vivo laminin immunofluorescence analysis of mammary glands and skin epithelial structures from mice exposed to 1 GeV/amu Fe ions. Digital confocal microscopic images were quantified and linked to the rough "core-penumbra" Fe track physical description. Comparison to gamma-ray sparsely ionizing radiation suggested the core of the Fe track being responsible for a biological response only seen with energetic Fe particles. Conclusions for modeling in vivo responses to radiation were then implied.

Quantitative image analysis of laminin immunoreactivity in skin basement membrane irradiated with 1 GeV/nucleon iron particles.

We previously reported that laminin immunoreactivity in mouse mammary epithelium is altered shortly after whole-body irradiation with 0.8 Gy from 600 MeV/nucleon iron ions but is unaffected after exposure to sparsely ionizing radiation. This observation led us to propose that the effect could be due to protein damage from the high ionization density of the ion tracks. If so, we predicted that it would be evident soon after radiation exposure in basement membranes of other tissues and would depend on ion fluence. To test this hypothesis, we used immunofluorescence, confocal laser scanning microscopy, and image segmentation techniques to quantify changes in the basement membrane of mouse skin epidermis. At 1 h after exposure to 1 GeV/nucleon iron ions with doses from 0.03 to 1.6 Gy, neither the visual appearance nor the mean pixel intensity of laminin in the basement membrane of mouse dorsal skin epidermis was altered compared to sham-irradiated tissue. This result does not support the hypothesis that particle traversal directly affects laminin protein integrity. However, the mean pixel intensity of laminin immunoreactivity was significantly decreased in epidermal basement membrane at 48 and 96 h after exposure to 0.8 Gy 1 GeV/nucleon iron ions. We confirmed this effect with two additional antibodies raised against affinity-purified laminin 1 and the E3 fragment of the long-arm of laminin 1. In contrast, collagen type IV, another component of the basement membrane, was unaffected. Our studies demonstrate quantitatively that densely ionizing radiation elicits changes in skin microenvironments distinct from those induced by sparsely ionizing radiation. Such effects may might contribute to the carcinogenic potential of densely ionizing radiation by altering cellular signaling cascades mediated by cell-extracellular matrix interactions.

Development of chicken embryos exposed to an intermittent horizontal sinusoidal 50 Hz magnetic field.

The effects of intermittent exposure (2 h on/22 h off) to a 200 microT horizontal, sinusoidally oscillating (50 Hz) magnetic field were studied in 210 fertilized chicken eggs. Two hundred ten control eggs (sham-exposed) were incubated in the same chamber as the experimental eggs. Chick embryos were examined for developmental anomalies and maturity stage after 48 h of incubation. Immunohistochemical analysis of extracellular membrane components (laminin, fibronectin, and type IV collagen) were conducted on day 7 and histological examinations for malformations of brain, liver, and heart, on days 7, 12, and 18 of incubation. Furthermore, egg fertility and egg weights were evaluated on days 2, 7, 12, and 18. The investigation also measured the body weight of chickens for 90 days from hatching and included histological analysis of body organs. Each variable was investigated blind. Statistical comparison between exposed and sham-exposed values did not show significant differences in any of the variables investigated. Thus, it appears that the exposure of embryos to an intermittent 200 microT magnetic field at 50 Hz does not cause developmental anomalies, changes in maturity stage, alterations in distribution of extracellular membrane components, or malformations in the brain, liver, or heart. Moreover, there were no differences in body weight, morphology, or histology of central nervous system, liver, heart, or testis in 90-day-old chickens hatched from exposed in comparison to sham-exposed eggs.

Radiation-induced transforming growth factor beta and subsequent extracellular matrix reorganization in murine mammary gland.

Little is known about radiation-induced protein expression in vivo nor has the relationship between early molecular events and subsequent tissue repair, fibrosis, or carcinogenesis been fully appraised. In this study, expression of proteins involved in tissue remodeling was examined in mammary gland immediately and shortly after ionizing radiation exposure. Using indirect immunofluorescence, selected antigens were followed as a function of time after 0, 5, or 10 Gy of whole body gamma-radiation in the mammary gland of adult female BALB/c mice. Rapid induction of transforming growth factor beta (TGF beta) immunoreactivity was observed at 1 h post radiation. Extracellular and intracellular TGF beta increased in the periepithelial stromal sheath as evidenced by immunoreactivity with antibodies CC(1-30) and LC(1-30), respectively. Furthermore, both extracellular and intracellular TGF beta were unexpectedly expressed in the previously negative adipose stroma. Elevated expression persisted for 7 days after irradiation. Thus an early response to radiation exposure is the induction of TGF beta, which mediates myriad events during tissue repair, growth, and extracellular matrix production. The distribution of extracellular matrix proteins was examined as a function of time post radiation exposure. Collagen III immunoreactivity decreased in the periepithelial stroma at day 1. In contrast, at day 3 collagen III was newly evident in the adipose stroma, and periepithelial collagen III had increased in both abundance and intensity. By day 7 collagen III expression in the adipose stroma had resolved but was enhanced in the periepithelial stroma. Over this same period stromal collagen I immunoreactivity surrounding the epithelium became diffuse and possibly diminished. Fibronectin, laminin, and collagen IV localization were unchanged over the time course. I postulate that radiation-induced TGF beta may mediate the remodeling of the stromal extracellular matrix in the irradiated mammary gland.

Growth cone guidance by substrate-bound laminin pathways is correlated with neuron-to-pathway adhesivity.

Substrate-bound laminin pathways prepared by the method of Hammarback et al. [J.A. Hammarback, S.L. Palm, L.T. Furcht, and P.C. Letourneau (1985). J. Neurosci. Res. 13, 213-220] guided peripheral nervous system neurites (dissociated dorsal root ganglia and sympathetic ganglia) and central nervous system neurites (dissociated spinal cord and brain). Guidance of individual growth cones by 7- to 10-micron-wide laminin pathways was observed using time-lapse video microscopy. Fibronectin pathways, produced by the method used for laminin pathways, did not guide neurites. The guidance effect of laminin pathways was quantified and found to correlate with the concentration of laminin initially applied to the substratum. The concentration of laminin initially applied to the substratum also correlated with increased adhesivity of dorsal root ganglia (DRG) neurons to laminin constituting the pathways relative to uv-irradiated laminin that borders the pathways. The guidance effect of laminin pathways was blocked by anti-laminin antibodies or by laminin but not by anti-fibronectin antibodies. This study demonstrates that guidance of DRG neurites by laminin occurs at the growth cone in a manner consistent with the hypothesis of guidance by differential neuron-to-substratum adhesivity.

The irradiation effects on the cytoskeletons of C3H/He mouse mammary tumor cells and vascular basement membrane in relation to vascular invasion: a model of intraoperative radiotherapy.

We investigated the short-term effects of a single high-dose radiation upon transplanted MM46 tumor cells in mice by means of immunohistochemistry and electron microscopy. The irradiation induced: 1) giant cell formation from the 3rd day, 2) arrest of tumor cell mitosis in prophase and metaphase due to the disorganization of the mitotic spindles, 3) changes in immunoreactivity of laminin and cytoskeletons, and 4) multilayering of the vascular basal lamina and perivascular fibrosis. The above findings suggest a decrease in tumor cell compliance, growth and invasiveness and the potentiation of defensive host responses against vascular invasion after irradiation. The analysis of the temporal sequences of the events indicates that the time lapse between the optimal host response, tumor growth and invasion constitutes a critical period.

Cell-cell matrix interactions in induced lung injury. IV. Quantitative alterations in pulmonary fibronectin and laminin following X irradiation.

Male LAF/1 mice were locally irradiated at doses of 5, 9, and 13 Gy and compared with untreated and sham-irradiated animals. Lungs were subsequently examined at times of 1, 4, 13, 28, 41, and 63 weeks postirradiation (PI) for alterations in pulmonary fibronectin (Fbn) and laminin (Lam) as a consequence of the irradiation. Thoroughly perfused lungs dissected clear of major airways were homogenized and fractionated by centrifugation into two fractions, soluble (supernate) and insoluble (pellet). Each fraction was analyzed by nonequilibrium competitive enzyme-linked immunoassay (ELISA) for Fbn and Lam normalized to mg protein. The results show a dose-related increase in soluble Fbn demonstrable at 1 week PI and approaching seven times control values by 28 weeks for doses of 13 Gy. Thereafter amounts decrease steadily to 63 weeks. Insoluble Fbn remains at or near control values through 13 weeks, increases in a dose-related fashion almost fivefold by 41 weeks for doses of 13 Gy, and then decreases by 63 weeks. Soluble Lam increased slightly during the duration of the study, returning to normal by 63 weeks. Insoluble Lam shows a dose-dependent increase demonstrable at 4 weeks PI which continues through 63 weeks. Interactions between these alterations in Fbn and Lam and previously reported changes in basal laminar proteoglycans may, in concert with other cellular and extracellular components, relate to the initiation and/or maintenance of radiation-induced pulmonary fibrosis.

Determination of the molecular weight of neuronectin, a conditioned medium-derived, substrate-binding neurite-extension factor: comparison with laminin using radiation-inactivation analysis.

Neurite outgrowth from a wide variety of peripheral neurons is stimulated and may be directed in culture by a substrate-binding factor(s) derived from medium conditioned over numerous types of cells. This factor, or family of factors, which we shall call neuronectin by reason of its ability to serve as an attachment molecule for neurons, has been studied by target-size analysis using radiation inactivation. The radiation-inactivation method has the unique advantage of providing a means for determining the actual functional size of a biologically active molecule irrespective of its state of purification. By this method, the functional size of the major neurite outgrowth-promoting activity (neuronectin) from mouse heart cell conditioned medium has been found to be 350,000 Da. While neuronectin has not yet been purified, determination of the actual functional size provides a framework within which possible models must fit. Thus, although neurite outgrowth-promoting activity in this system is found to be associated with a complex containing laminin, fibronectin, heparan sulfate proteoglycan, and other extracellular matrix molecules, the total size of the functional molecule or molecular complex serving as the major source of activity is limited to 350,000 Da. Consequently, our results suggest that neuronectin from mouse heart cell-conditioned medium is different from laminin (Mr approximately 900,000), a molecule that also exhibits neurite-promoting activity. In addition to the difference in molecular size, neuronectin and laminin differ in that laminin, unlike neuronectin, gives rise to toxic or inhibitory products when exposed to high-energy radiation.