Interactions between mts 1 and serotonin in vascular remodelling

Thickening of blood vessels (known as remodelling) occurs in many diseases associated with the cardiovascular system and is related to high blood pressure (hypertension). This can occur in the blood vessels that supply the lungs (pulmonary arteries), those that supply the heart and the brain as well as those that supply blood to the rest of the body (systemic arteries). Many factors contribute to the remodelling of arteries. Recent evidence suggests that a chemical in the body known as serotonin can interact with another chemical known as mts1 to cause pulmonary arterial remodelling. This has been shown in isolated cells but whether or not this occurs in the whole animal requires investigation. We have established techniques designed to investigate pulmonary and systemic arteries in transgenic mice, both in the whole animal, at the level of the very small arteries and at the cellular level. Application of these techniques to mice that have an artificial increase in the expression of the pore that allows serotonin to enter the cell (the serotonin transporter) and mts1 will enable us to investigate this potentially important mechanism for vascular remodelling. In addition, lack of oxygen (hypoxia) is an important mediator of pulmonary arterial remodelling and we have developed techniques for exposing mice and cells to a hypoxic environment. This will be applied to study the interaction between hypoxia, serotonin and mts1. The major aim of the work is to establish, in the whole animal, if there is an important interaction between mts1 and serotonin that causes a remodelling of the pulmonary arteries. This will be done by examining these interactions in mice over-expressing mts1 and mice over-expressing the serotonin transporter. These mice will also be cross-bred to develop mice that over-express both the serotonin transporter and mts1. Further experiments will be carried out on blood vessels derived from these 'models' and from cells grown up in culture from these blood vessels. This will give a clear picture of how intracellular interactions relate to whole body function. There are many benefits and applications of this work, including knowledge of how mts1, serotonin and hypoxia affect vascular function, how this changes in disease and how such changes could be prevented. It will suggest novel therapies for remodelling diseases such as hypertension.

Pulmonary vascular remodelling is associated with vascular smooth muscle cell and fibroblast proliferation. This in turn is associated with increased expression of the serotonin transporter (5HTT). Recently, we have demonstrated that this vascular proliferation may be associated with an increase in the expression of the calcium binding protein, mts1. The objectives of this study are to translate these observations from a cellular to an in vivo model and integrate further in vitro/in vivo studies. We have established techniques designed to investigate pulmonary and systemic arteries in transgenic mice, both in the whole animal, at the level of the microvasculature and at the cellular level. Hypoxia is an important mediator of pulmonary vascular remodelling but its interaction with the mts1/serotonin system is unknown. We have also developed techniques for exposing mice and cells to a hypoxic environment which will be utilised to study this interaction in vivo and in vitro. Specifically we will: 1. Test the hypothesis that serotonin-induced Mts1 production is directly related to both 5-HT1B receptor and 5HTT activity in vivo under normoxic conditions. To this end, we will study mice that over-express the gene for human 5HTT (5HTT+ mice) and 5HTT knockout mice (5HTT- mice), as well as mice that over-express mts1 (mts1+ mice). In these mice, we will examine pulmonary and systemic haemodynamics and how this relates to the vascular localisation of mts1 and 5HTT and how this, in turn, is related to pulmonary vascular remodelling. We will also examine the expression of the receptor for mts1 (RAGE) and the expression of fibulin 5 which is a target protein for mts1. Isolated pulmonary and systemic arteries will be studied using myography techniques to relate vascular reactivity with mts1/5HTT expression. Pulmonary and systemic arterial fibroblasts will be derived from these arteries and further detailed cellular studies carried out to examine the intra-cellular interactions between 5HTT and mts1. The ability of these cells to proliferate, and the roles that these mediators play in proliferation will be clarified. 2. Test the hypothesis that over-expression of Mts1 plus increased 5HTT expression will synergise in terms of their effects on proliferation and vascular reactivity. This will be studied in vitro and in vivo (as described above) by studying the progeny of a 5-HTT+ x S100A4/Mts1+ cross at a whole animal and cellular level. 3. Test the hypothesis that serotonin-induced mts 1 expression may depend on the 5-HT receptor in hypoxic conditions. i.e. there is altered regulation of cellular proliferation under hypoxic conditions. We will expose mice to two weeks of hypoxia to test this hypothesis and then carry out studies as described above. In addition, we will expose arterial fibroblasts to acute hypoxic conditions and examine changes in the intracellular interactions between mts1 and 5HTT. Hence, we will test our hypothesis in the whole animal using novel transgenic models and translate these observations into studies on isolated arteries and through to cultured cellular preparations. This study will undoubtedly suggest novel therapeutic approaches to prevent proliferation and such knowledge can be taken back into the whole animal (i.e. via chronic dosing studies) to continue this translational approach.