Two-photon microscopic imaging in the vasculature : a sub-cellular window for imaging nitric oxide and thrombus

Abstract

The domain of the vascular biology is a complex system consisting of different types of blood vessels, cells, and signaling molecules. It is now recognized that these vessels and molecules are part of a subtle regulatory system with differential properties along the vascular tree. Altered production of these molecules is a molecular clue for dysfunctionality of cells or alterations of vessel properties that may lead to various acute and chronic diseases. Current knowledge of such vascular alterations is mostly based on histological studies of isolated samples that have lost their viability. Functional properties of various compounds are still largely unknown. Better understanding of the functionality of these molecules in the context of cardiovascular functioning can increase insight in early stage of disease condition. Thus studying these properties in vivo or in viable arteries ex vivo is indispensable. This thesis focuses on the vessel wall of large murine arteries and cultured cell systems using TPLSM as an imaging tool for studying alterations in vessel wall properties as well as important molecules and their functional consequences. In this thesis, I first addressed the concept of NO imaging in vasculature with a novel method. Nitric oxide analysis with Cu2FL2E in combination with TPLSM allowed specific detection and semi-quantification of endogenous NO production both in vitro and ex vivo. With the use of Cu2FL2 and TPLSM we were able to unravel the structural-functional relationship of NO in the vessel wall. Presence of NO in various vascular cells could be monitored ex vivo for several physiological NO-stimuli. The Cu2FL2E-TPLSM approach provides a valid method to study the role of NO in vascular biology at an unprecedented level and can enable investigation of the regulatory pathways in the complex interplay between NO and vascular (dys)function. In the second part, NO production was studied with TPLSM in murine endotoxemia. Impact of arginine supplementation in wild type and ARG-1 -/- mouse, in NO metabolism was investigated. A disturbed arginine-nitric oxide metabolism is associated with endotoxemia. Therefore, the effect of L-arginine supplementation on eNOS-induced intracellular NO production was studied in wild type and a non-lethal prolonged endotoxemia model in mice. TPLSM revealed that the L-arginine supplementation restored intracellular NO production during endotoxemia. However, further investigation is needed to find out if this NO improves the microcirculation during endotoxemia. Arginase-I also contributes to endothelial dysfunction during endotoxemia as it competes with NOS3 for arginine availability. Therefore, I investigated the effects of cell-specific arginase-1 deficiency on the arginine availability, the NOS3-derived NO production during murine endotoxemia. Arginase-I plays a crucial role in controlling NOS2 during inflammation in endothelial cells. Modulating the arginase activity resulted in an inflammatory response an increased NO production by NOS2. Therefore, modulating arginase activity during endotoxemia needs to aim at restoring the balance between arginase and NOS2 during inflammatory conditions, as this may be the key to an improved endothelial function. In the final part, visualization of fresh thrombus formation has been described using fibrin-targeted peptide conjugated with C-Dot. Thrombosis plays a major role in several vascular diseases and early detection of thrombus formation is a hitherto unmet requirement in clinical practice. Further validation of this method might help in translation of early detection strategy of thrombus in clinical scenario. In general discussion, I contemplate various ideas regarding the topics described and open new avenues and possible future outcomes for application of these novel studies. I conclude that the described techniques (in the forefront of TPLSM imaging) offer a new and different view on healthy and diseased arteries and provide new insight in various structural and functional properties of vessels. Further development of these techniques holds potential for future applications in both a scientific and clinical environment