Direct antiangiogenic actions of cadmium on human vascular endothelial cells.

The vascular endothelium is a primary target of cadmium (Cd) toxicity, but little is known regarding a potential mechanism whereby Cd may inhibit angiogenesis. Recent findings showing that Cd can disrupt cadherin-mediated cell-cell adhesion suggested that Cd might inhibit angiogenesis by altering the function of VE-cadherin, a molecule that is essential for angiogenesis. To address this issue, endothelial cells (ECs) were exposed to Cd in the presence of serum and subjected to angiogenesis-related cell migration and tube formation assays. Initial examination of cytotoxicity showed that ECs are rather resistant to the acute cytotoxic effects of Cd even at concentrations up to 1 mM. However, 10 microM Cd decreased migration of ECs. Cd concentrations of 500 nM and greater significantly reduced organization of microvascular ECs into tubes. These antiangiogenic effects were evident even when ECs were preincubated with Cd and then washed to remove free Cd, indicating that Cd acted directly on the cells rather than on the extracellular matrix. Immunolocalization studies showed that Cd caused the redistribution of VE-cadherin from cell to cell contacts. These findings indicate that Cd acts in an angiostatic manner on ECs, and that this effect may involve alterations in the localization and function of VE-cadherin.

Kidney injury molecule-1 is an early biomarker of cadmium nephrotoxicity.

Cadmium (Cd) exposure results in injury to the proximal tubule characterized by polyuria and proteinuria. Kidney injury molecule-1 (Kim-1) is a transmembrane glycoprotein not normally detected in the mature kidney, but is upregulated and shed into the urine following nephrotoxic injury. In this study, we determine if Kim-1 might be a useful early biomarker of Cd nephrotoxicity. Male Sprague-Dawley rats were given daily injections of Cd for up to 12 weeks. Weekly urine samples were analyzed for Kim-1, protein, creatinine, metallothionein, and Clara cell protein CC-16. Significant levels of Kim-1 were detected in the urine by 6 weeks and continued to increase throughout the treatment period. This appearance of Kim-1 occurred 4-5 weeks before the onset of proteinuria, and 1-3 weeks before the appearance of metallothionein and CC-16. Higher doses of Cd gave rise to higher Kim-1 excretion. Reverse transcriptase-polymerase chain reaction (RT-PCR) expression analysis showed that Kim-1 transcript levels were increased after 6 weeks at the low dose of Cd. Immunohistochemical analysis showed that Kim-1 was present in proximal tubule cells of the Cd-treated rats. Our results suggest that Kim-1 may be a useful biomarker of early stages of Cd-induced proximal tubule injury.

Long term results after transpupillary thermotherapy in eyes with occult choroidal neovascularisation associated with age related macular degeneration: a prospective trial.

AIM: To evaluate long term results after transpupillary thermotherapy (TTT) in eyes with exudative age related macular degeneration. METHODS: In a prospective clinical study eyes with occult or predominantly occult choroidal neovascularisation and no pretreatment were scheduled to have a TTT with a power of 630 mW. Visual acuity for far and near distances as well as contrast sensitivity were evaluated 6, 12, and 24 months postoperatively and statistically analysed. RESULTS: 47 eyes fulfilled the inclusion criteria. Overall, 70% of the patients showed an improved (14%) or had unchanged (56%) ETDRS vision after 24 months. Reading vision was stabilised (51%) or better (5%) in 56% of the eyes at this time. However, the increasing number of eyes with severe deterioration resulted in a significant decrease of both parameters over time (p = 0.0002 and p = 0.0003, respectively). Contrast sensitivity could be maintained (70%) or improved (9%) in 79%. Statistical analyses indicated a trend but no significant decrease over time (p = 0.056). CONCLUSION: Although in the majority of patients far and near distance acuity could be stabilised on average a significant decrease over time after TTT was observed. Statistical comparison of months 12 and 24 showed no further deterioration.

Effect of a short-term in vitro exposure to the marine toxin domoic acid on viability, tumor necrosis factor-alpha, matrix metalloproteinase-9 and superoxide anion release by rat neonatal microglia.

BACKGROUND: The excitatory amino acid domoic acid, a glutamate and kainic acid analog, is the causative agent of amnesic shellfish poisoning in humans. No studies to our knowledge have investigated the potential contribution to short-term neurotoxicity of the brain microglia, a cell type that constitutes circa 10% of the total glial population in the brain. We tested the hypothesis that a short-term in vitro exposure to domoic acid, might lead to the activation of rat neonatal microglia and the concomitant release of the putative neurotoxic mediators tumor necrosis factor-alpha (TNF-alpha), matrix metalloproteinases-2 and-9 (MMP-2 and -9) and superoxide anion (O2-). RESULTS: In vitro, domoic acid [10 microM-1 mM] was significantly neurotoxic to primary cerebellar granule neurons. Although neonatal rat microglia expressed ionotropic glutamate GluR4 receptors, exposure during 6 hours to domoic acid [10 microM-1 mM] had no significant effect on viability. By four hours, LPS (10 ng/mL) stimulated an increase in TNF-alpha mRNA and a 2,233 % increase in TNF-alpha protein In contrast, domoic acid (1 mM) induced a slight rise in TNF-alpha expression and a 53 % increase (p < 0.01) of immunoreactive TNF-alpha protein. Furthermore, though less potent than LPS, a 4-hour treatment with domoic acid (1 mM) yielded a 757% (p < 0.01) increase in MMP-9 release, but had no effect on MMP-2. Finally, while PMA (phorbol 12-myristate 13-acetate) stimulated O2- generation was elevated in 6 hour LPS-primed microglia, a similar pretreatment with domoic acid (1 mM) did not prime O2- release. CONCLUSIONS: To our knowledge this is the first experimental evidence that domoic acid, at in vitro concentrations that are toxic to neuronal cells, can trigger a release of statistically significant amounts of TNF-alpha and MMP-9 by brain microglia. These observations are of considerable pathophysiological significance because domoic acid activates rat microglia several days after in vivo administration.

Interaction of cadmium (Cd(2+)) with a 13-residue polypeptide analog of a putative calcium-binding motif of E-cadherin.

Previous studies from our laboratory have shown that Cd(2+) can selectively damage the tight junctions between epithelial cells in culture. Recently, we have obtained evidence suggesting that this effect may involve the interaction of Cd(2+) with E-cadherin, a Ca(2+)-dependent cell adhesion molecule that is localized at the adhering junctions of epithelial cells. To begin to determine whether or not Cd(2+) might interact directly with the E-cadherin molecule, we studied the binding of Cd(2+) to peptide B, a synthetic, 13-residue polypeptide that corresponds to one of the extracellular Ca(2+) binding regions of mouse E-cadherin (also known as uvomorulin). The binding of Cd(2+) to peptide B was evaluated by using an equilibrium microdialysis technique and the radioactive isotope (109)Cd(2+). The effects of the binding on the conformation of the peptide were evaluated by circular dichroism (CD) spectroscopy. The results showed that Cd(2+) bound to peptide B, with a maximum of one Cd(2+) binding site per molecule and an apparent dissociation constant (K(d)) of 640 microM. The binding of Cd(2+) was reduced in the presence of excess Ca(2+), an effect that was overcome by raising the concentration of Cd(2+). Both Cd(2+) and Ca(2+) caused a shift in the CD spectrum of the peptide. However, the shift produced by Cd(2+) was about 3 times the magnitude of that produced by Ca(2+). These results indicate that Cd(2+) can interact with the Ca(2+) binding site on the peptide B molecule and distort the secondary structure of the peptide. These findings are consistent with the hypothesis that E-cadherin may be a direct molecular target for Cd(2+) toxicity.

Comparison of the cytotoxic effects of cadmium (Cd(2+)) in high and low resistance strains of MDCK cells that express different levels of E-Cadherin.

Previous studies from our laboratory have shown that cadmium (Cd(2+)) can disrupt the adhering and occluding junctions between MDCK cells. Recently, we have obtained evidence to suggest that Cd(2+) produces this effect by interacting with E-cadherin, a Ca(2+)-dependent cell adhesion molecule that is localized at the adhering junctions of epithelial cells. The objective of the present study was to examine the junctional and cytotoxic effects of Cd(2+) in subcloned strains of MDCK cells that express different levels of E-cadherin. One strain (MDCK I) expresses high levels of E-cadherin and develops a transepithelial electrical resistance of more than 800 Omega.cm(2), whereas the other strain (MDCK II) expresses much lower levels of E-cadherin and develops a transepithelial resistance of less than 100 Omega.cm(2). The results showed that exposure to 20 mum Cd(2+) for 2-4 hours caused a pronounced loss of E-cadherin from the cell borders in both strains of cells. In the MDCK I cells, the loss of E-cadherin coincided with a decrease in the transepithelial electrical resistance and the loss of the tight junction-associated proteins, ZO-1 and occludin, from the cell borders. By contrast, the MDCK II cells first exhibited a significant increase in the transepithelial electrical resistance that did not begin to decline until the cells had been exposed for 4-6 hours, a time that coincided with the loss of ZO-1 and occludin from the cell borders. Additional results showed that the MDCK I cells were slightly more sensitive to the lethal effects of Cd(2+) than were the MDCK II cells. These findings indicate that E-cadherin may be an early target for Cd(2+) toxicity in both high and low resistance strains of MDCK cells. However, they also suggest that the disruption of E-cadherin-dependent cell-cell junctions may trigger somewhat different responses in the two cell lines.

Cadmium (Cd2+) disrupts E-cadherin-dependent cell-cell junctions in MDCK cells.

Previous studies from our laboratory have shown that Cd2+ can selectively disrupt E-cadherin-dependent cell-cell junctions in the porcine renal epithelial cell line, LLC-PK1. The objective of the present studies was to determine whether or not Cd2+ could produce similar effects in Madin-Darby canine kidney (MDCK) cells, an immortal epithelial cell line derived from dog kidney. This is an important issue because MDCK cells have been used extensively as a model system to study the basic mechanisms of E-cadherin-dependent cell-cell adhesion. MDCK cells on permeable membrane supports were exposed to Cd2+ by adding CdCl2 to either the apical or the basolateral compartment. The integrity of cell-cell junctions was assessed by morphologic observation of the cells and by monitoring the transepithelial electrical resistance. The results showed that exposure to 10-40 microM Cd2+ for 15 min-4 h caused the cells to separate from each other without detaching from the growing surface. The separation of the cells was accompanied by a marked drop in the transepithelial electrical resistance, a loss of E-cadherin from the cell-cell contacts, and a reorganization of the actin cytoskeleton. These effects were much more pronounced when Cd2+ was added basolaterally than when it was added apically. Moreover, the effects of Cd2+ were qualitatively similar to those observed when the cells were incubated in Ca(2+)-free medium. These results show that Cd2+ can disrupt E-cadherin-dependent cell-cell junctions in MDCK cells, and they indicate that this cell line would be an appropriate model for further mechanistic studies in this area.

Binding of cadmium (Cd2+) to E-CAD1, a calcium-binding polypeptide analog of E-cadherin.

Recent studies have shown that Cd2+ can damage the Ca(2+)-dependent junctions between renal epithelial cells in culture, and preliminary evidence suggests that this effect may involve the interaction of Cd2+ with E-cadherin, a Ca(2+)-dependent cell adhesion molecule that is localized at the adhering junctions of epithelial cells. To determine whether or not Cd2+ might bind directly to the E-cadherin molecule, we studied the binding of Cd2+ to E-CAD1, a recombinant, 145-residue polypeptide that corresponds to one of the extracellular Ca(2+)-binding regions of mouse E-cadherin. By using an equilibrium microdialysis technique, we were able to show that Cd2+ could, in fact, bind to E-CAD1. The binding was saturable, with a maximum of one Cd2+ binding site per E-CAD1 molecule. The apparent dissociation constant (KD) for the binding was about 20 microM, a concentration similar to that which has been shown to disrupt the junctions between epithelial cells. Other results showed that the binding of CD2+ was greatly reduced when excess Ca2+ was included in the dialysis solution. These results suggest that Cd2+ can interact with the Ca2+ binding regions on the E-CAD1 molecule, and they provide additional support for the hypothesis that E-cadherin might be a molecular target for Cd2+ toxicity.

Effects of glutathione depletion on the cytotoxic actions of cadmium in LLC-PK1 cells.

Recent studies have shown that exposure of LLC-PK1 cells to micromolar concentrations of Cd2+ for 1-4 hr causes the disruption of the junctions between the cells, whereas exposure to higher concentrations of Cd2+ for longer periods of time causes more severe toxic effects and cell death. Studies suggesting that glutathione may serve a protective role against Cd2+ toxicity in other tissues and cells led us to examine the effects of glutathione depletion on the cytotoxic actions of Cd2+ in the LLC-PK1 cell line. Confluent cells on Falcon cell culture inserts were depleted of glutathione by exposing them to 250 microM buthionine sulfoximine for 18 hr and then exposed to various concentrations of Cd2+ for up to 24 hr. The integrity of cell-cell junctions was assessed by morphologic observation of the cells and by monitoring the transepithelial electrical resistance. Cell viability was evaluated by monitoring the release of lactate dehydrogenase into the medium. The results showed that depleting the cells of glutathione did not alter the early junction-perturbing effects of Cd2+, but greatly enhanced the lethal effects. In both the glutathione-depleted and the normal cells, junctional changes were evident after as little as 1 hr of Cd2+ exposure. While the normal cells did not begin to die until they had been exposed to Cd2+ for 12-24 hr, the glutathione-depleted cells began to die after only 8 hr of Cd2+ exposure. Additional results showed that Cd2+ exposure had no effect on the total levels of glutathione at the time in which the junctional effects were occurring, but caused a marked decrease in glutathione levels at the time the cells were dying. These results indicate that the early junctional effects of Cd2+ do not result from alterations in intracellular glutathione or sulfhydryl metabolism, whereas the more severe cytotoxic effects and cell death may involve glutathione-sensitive mechanisms.

The cadmium-induced disruption of tight junctions in LLC-PK1 cells does not result from apoptosis.

Exposure of LLC-PK1 cells to low micromolar concentrations of Cd2+ for 1-4 hours causes the disruption of the adhering and occluding junctions between the cells, whereas exposure to higher concentrations of Cd2+ for longer periods of time causes more severe toxic effects and cell death. The objective of the present studies was to determine whether or not the junctional effects of Cd2+ might be a consequence of apoptotic injury. LLC-PK1 cells on cell culture inserts were exposed to either Cd2+ or tumor necrosis factor (TNF-alpha) plus cycloheximide, a treatment that has recently been shown to cause apoptosis in LLC-PK1 cells. The results showed that at the time the Cd2(+)-induced junctional changes were occurring, there was no increase in the number of apoptotic cells or evidence of DNA fragmentation. By contrast, TNF-alpha plus cycloheximide induced changes that were characteristic of apoptosis. These results indicate that the disruption of intercellular junctions by Cd2+ in the LLC-PK1 cell line occurs independently of apoptosis.

Comparison of the cytotoxic effects of cadmium chloride and cadmium-metallothionein in LLC-PK1 cells.

Recent studies have shown that ionic cadmium (Cd2+) can selectively damage the tight junctions between LLC-PK1 cells. The objective of the present studies was to determine if cadmium that is bound to metallothionein (Cd-Mt) can also damage the junctions between these cells. Cells on Falcon Cell Culture Inserts were exposed to Cd2+ or Cd-Mt from the apical and basolateral compartments. The integrity of cell junctions was assessed by monitoring the transepithelial electrical resistance, and cell viability was evaluated by monitoring the release of lactate dehydrogenase into the medium. Exposure to Cd2+ for 1-4 hours caused a pronounced decrease in the transepithelial resistance without affecting cell viability. By contrast, exposure to Cd-Mt had little effect on the electrical resistance until the cells began to die, which did not occur until 24-48 hours of exposure. Additional results showed that the cells accumulated Cd2+ more rapidly than Cd-Mt. These results indicate that Cd-Mt does not damage the junctions between LLC-PK1 cells, but that it can kill the cells after prolonged exposure.

Surface binding and uptake of cadmium (Cd2+) by LLC-PK1 cells on permeable membrane supports.

Recent studies have shown that Cd2+ has relatively specific damaging effects on cell-cell junctions in the renal epithelial cell line, LLC-PK1. The objective of the present studies was to examine the surface binding and uptake of Cd2+ by LLC-PK1 cells in relation to the disruption of cell-cell junctions. LLC-PK1 cells on Falcon Cell Culture Inserts were exposed to CdCl2 containing trace amounts of 109Cd2+ from either the apical or the basolateral compartments, and the accumulation of 109Cd2+ was monitored for up to 8 h. The integrity of cell-cell junctions was assessed by monitoring the transepithelial electrical resistance. The results showed that the cells accumulated 3-4 times more Cd2+ from the basolateral compartment than from the apical compartment. The accumulation of Cd2+ from the basolateral compartment occurred in two phases: a rapid, exponential phase that occurred in 1-2 h and coincided with a decrease in transepithelial resistance, and a slower, linear phase that continued for 6-8 h. The Cd2+ that accumulated during the rapid phase was easily removed by washing the cells in EGTA, indicating that most of it was bound to sites on the cell surface. By contrast, most of the Cd2+ that accumulated during the slower phase could not be removed by EGTA, indicating that it had been taken up by the cells. Additional studies showed that the rapid phase of Cd2+ accumulation was enhanced when Ca2+ was present at low concentrations (0.1 mM), and was greatly reduced when Ca2+ was present at high concentrations (10 mM).(ABSTRACT TRUNCATED AT 250 WORDS)