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Effect of calcium-phosphate crystals on intracellular calcium homeostasis and viability of smooth muscle cell

Sharma, G.; Rietdorf, K.; Proudfoot, D.; McDonald, F. and Bootman, M. (2015). Effect of calcium-phosphate crystals on intracellular calcium homeostasis and viability of smooth muscle cell. In: Proceedings of the Physiological Society, article no. PC282.

URL: http://www.physoc.org/proceedings/abstract/Proc%20...
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Abstract

Smooth muscle cells have a protective role in atherosclerotic plaques by increasing collagen synthesis required for fibrous cap thickening. Formation of calcium phosphate (CaP) crystals occurs in atherosclerosis, vascular inflammation, diabetes, chronic renal disease and ageing. Studies have suggested that CaP crystals trigger smooth muscle cell death, which could lead to atherosclerotic plaque rupture.
In order to understand the effects of CaP crystals on smooth muscle cells, we performed calcium imaging experiments using A7r5 cells loaded with the calcium-sensitive indicator Fura-2. We added different concentrations of CaP crystals to the cells (2.5, 12.5 and 62.5 µg/ml), and recorded the intracellular calcium concentration for 45 minutes. Propidium iodide was added to the extracellular medium to monitor plasma membrane integrity simultaneously with calcium imaging. We observed complex calcium signals (Figure 1) in the A7r5 cells in response to CaP crystals: transient calcium responses or calcium oscillations that did not lead to cell death, or substantial, irreversible, calcium increases that immediately preceded loss of Fura-2 from the cells. The entry of propidium iodide occurred during the irreversible calcium signal, suggesting that the influx of calcium ions from the extracellular medium may contribute to the calcium rise associated with cell death. The onset of calcium signals typically occurred with a delay of >15 minutes after the CaP addition. There was a concentration-dependent effect of CaP on both the latency until the first obvious calcium signal, and also the latency until cell death. Latencies for the first calcium signal were 1435 ± 156 and 1363 ± 50.5 seconds for 2.5 and 12.5 µg/ml CaP. The latency to the first calcium signal was significantly shorter (901 ± 75.9 seconds) following the addition of 62.5 µg/ml CaP (P<0.05, 1-way ANOVA). The latencies for cell death were 1868 ± 82.2 and 1916 ± 52.6 seconds for 2.5 and 12.5 µg/ml CaP and again significantly shorter (1302 ± 51.9 seconds) for 62.5 µg/ml CaP crystals (P<0.001, 1-way ANOVA). However, there was no significant difference in the amplitude of calcium signals evoked by 2.5, 12.5 or 62.5 µg/ml CaP crystals.
CaP crystals caused a concentration-dependent significant increase in the percentage of A7r5 cell death: 13 ± 2.5, 77.9 ± 5.4 and 99 ± 0.95 percent of cells died following addition of 2.5, 12.5 and 62.5 µg/ml CaP crystals, respectively (P<0.001, 1-way ANOVA).
Our data show that CaP crystals have the capacity to kill smooth muscle cells by inducing calcium signals that overwhelm cellular homeostasis, and by causing plasma membrane rupture.
Percentage of cells skipped the calcium oscillations before cell death were 10 ± 1.9, 45 ± 4.9 and 70 ± 5 for 2.5, 12.5 and 62.5 µg/ml CaP crystals, respectively (P<0.001, 1-way ANOVA). Cells that progressed through calcium oscillation before cell death were found to be less in 2.5 µg/ml (4.6 ± 0.23) in contrast to 12.5 µg/ml 31.8 ± 4) and 62.5 µg/ml (28.2 ± 5).

Item Type: Conference or Workshop Item
Extra Information: Poster Communication
Academic Unit/School: Faculty of Science, Technology, Engineering and Mathematics (STEM) > Life, Health and Chemical Sciences
Faculty of Science, Technology, Engineering and Mathematics (STEM)
Interdisciplinary Research Centre: Biomedical Research Network (BRN)
Health and Wellbeing PRA (Priority Research Area)
Item ID: 44175
Depositing User: Katja Rietdorf
Date Deposited: 06 Oct 2015 09:37
Last Modified: 11 Sep 2017 15:32
URI: http://oro.open.ac.uk/id/eprint/44175
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