Specific Visualization of Nitric Oxide in the Vasculature with Two-Photon Microscopy Using a Copper Based Fluorescent Probe

Mitrajit Ghosh,Marc A M J Van Zandvoort,Michael D Pluth,Mark J Post,Karolina A P Wijnands,Daniel G M Molin,Christian Weber,Stephen J Lippard,Lindsey E Mcquade,Martijn Poeze,Nynke M S Van Den Akker,David D Roberts

doi:10.1371/journal.pone.0075331

Abstract

To study the role and (sub) cellular nitric oxide (NO) constitution in various disease processes, its direct and specific detection in living cells and tissues is a major requirement. Several methods are available to measure the oxidation products of NO, but the detection of NO itself has proved challenging. We visualized NO production using a NO-sensitive copper-based fluorescent probe (Cu 2FL2E) and two-photon laser scanning microscopy (TPLSM). Cu 2FL2E demonstrated high sensitivity and specificity for NO synthesis, combined with low cytotoxicity. Furthermore, Cu 2FL2E showed superior sensitivity over the conventionally used Griess assay. NO specificity of Cu 2FL2E was confirmed in vitro in human coronary arterial endothelial cells and porcine aortic endothelial cells using various triggers for NO production. Using TPLSM on ex vivo mounted murine carotid artery and aorta, the applicability of the probe to image NO production in both endothelial cells and smooth muscle cells was shown. NO-production and time course was detected for multiple stimuli such as flow, acetylcholine and hydrogen peroxide and its correlation with vasodilation was demonstrated. NO-specific fluorescence and vasodilation was abrogated in the presence of NO-synthesis blocker L-NAME. Finally, the influence of carotid precontraction and vasorelaxation validated the functional properties of vessels. Specific visualization of NO production in vessels with Cu 2FL2E-TPLSM provides a valid method for studying spatial-temporal synthesis of NO in vascular biology at an unprecedented level. This approach enables investigation of the pathways involved in the complex interplay between NO and vascular (dys) function.

Highlights

Produced vascular nitric oxide (NO) affects important biological processes such as platelet and leukocyte adhesion, smooth muscle cell (SMC) migration, and endothelial regeneration in blood vessels [1,2,3,4]
To underscore the sensitivity of Cu 2FL2E to NO, we started with its spectroscopic behaviour in response to various concentrations of SNAP as NO donor [17] in phosphate buffer solution (PBS)
The present study demonstrates the feasibility of using Cu 2FL2E as a direct, sensitive, specific, non-toxic, and rapid NO probe in vascular biology

Summary

Introduction

Produced vascular nitric oxide (NO) affects important biological processes such as platelet and leukocyte adhesion, smooth muscle cell (SMC) migration, and endothelial regeneration in blood vessels [1,2,3,4]. The regulation of blood flow through induction of vasodilation is a major function of endothelial-derived NO. Cellular NO is produced by three different enzymes (i.e. iNOS, eNOS, nNOS) [3], of which endothelial nitric oxide synthase (eNOS), expressed in endothelial cells (ECs), is essential for physiological NO (order of nanomolar range) [5,6] production in healthy blood vessels. NO diffuses to the neighboring SMCs, where it induces vasodilation through SMC relaxation and subsequently increases vessel lumen diameter [4,5] and blood flow. The direct and specific detection of NO in living cells and tissues is a major, hitherto unmet, requirement for investigating the role and (sub) cellular NO constitution in various disease processes

Methods

Results

Conclusion