Ratiometric fluorophores to ONOO- in living cells and monitoring endothelial dysfunction

초록

Peroxynitrite (ONOO-), generated from nitric oxide and superoxide, is implicated in cellular dysfunction and death. To detect ONOO- in live cells, we developed two hemicyanine-based ratiometric fluorophores, P-mito and P-boc. These ratiometric fluorophores were successfully used to monitor ONOO- fluctuations in cancer cells and endothelial cells. P-mito predominantly accumulated in the mitochondria of live cells and, upon selective reaction with ONOO-, increased the ratio of the fluorescence intensity from blue channel to that from red channel. Treatment with an ONOO- scavenger, N-acetyl cysteine, suppressed this ratio increase. Notably, in a model of endothelial dysfunction induced by treating pulmonary arterial endothelial cells with TGF beta 2 and IL-1 beta, our system successful demonstrated increased ONOO- levels, effectively highlighting the utility of the hemicyaninebased fluorescence response to ONOO-. Therefore, our hemicyanine-based ONOO- detection system provides an effective platform for spatiotemporal monitoring and quantitative analysis of ONOO- within live cells, enabling molecular-level research into various ONOO--related pathological conditions.

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