The role of endothelial tryptophan hydroxylase in pulmonary vascular remodelling: an integrated approach

Serotonin is a chemical that can act on the blood vessels of the lungs to cause them to thicken. This can lead to high blood pressure in the lungs. The enzyme responsible for the synthesis of serotonin that affects the lungs is tryptophan hydroxlase 1. Blood vessels have three layers, an outer layer of adventitial cells, a muscular layer and an inner layer of cells called endothelial cells. We will determine if tryptophan hydroxlase 1 causes synthesis of serotonin by the endothelial cells and if this serotonin can then act on the adjacent muscle and adventitial cells to make them grow and cause thickening of the artery. We will also determine if hypoxia (a reduction in oxygen) can stimulate an increase in endothelial cell serotonin synthesis and release and that this contributes to hypoxia-induced thickening of the blood vessels. We will use genetically modified mice which lack tryptophan hydroxlase 1 in our studies as well as isolated smooth muscle, adventitial and endothelial cells. We will also examine the feasilbility of knocking out tryptophan hydroxlase 1 in the endothelial cells in the whole animal using novel gene transfer techniques.

Serotonin has increasingly been associated with hypoxia-induced pulmonary vascular remodelling and thought to act via activation of serotonin receptors as well as through entering the pulmonary arterial smooth muscle cells (PASMCs) via the serotonin transporter with subsequent activation of proliferative signalling pathways. Tryptophan hydroxylase 1 (TPH1) is the rate-limiting enzyme in the synthesis of peripheral serotonin. We have recently shown that TPH1 knockout (Tph1-/-) mice are protected against hypoxia-induced pulmonary vascular remodelling. Mice that express a Tph1-specific miniribozyme (tRz186 mice) demonstrate reduced 5-HT in plasma and whole blood. Hence, these studies have identified TPH1 as a potential key player in the control of thromboembolism, thrombosis, hypoxia-induced pulmonary vascular remodelling and PASMC proliferation. Novel hypothesis: We will test the following hypothesese: 1: Endothelium-derived TPH1 plays a pivotal role in the in vivo response of PASMCs (and adventitial cells) to hypoxia. 2: Selective ablation of endothelial TPH1 in vivo will protect against hypoxia-induced pulmonary vascular remodelling. 3: Activities of the serotonin transporter, the 5-HT1B receptor and TPH1 are co-regulated in the proliferative response of PASMCs to hypoxia. Adopting an integrative approach, applying novel genetic information and models to molecular, cellular and novel gene transfer technologies, we will determine how TPH1 and the serotonin system contributes at the whole animal level as well as at a tissue and cellular level. Specifically we will study Tph1 knockout mice and mice with reduced plasma serotonin, lacking Tph1 through selective knockout with novel miniribozymes. We will selectively knockout pulmonary endothelial Tph1 in vivo using novel gene transfer techniques and examine the effects of this on hypoxia-induced PASMC proliferation in vivo and ex vivo.