The receptor for advanced glycation end-products in pulmonary hypertension

Farmer, David George Stephen (2012) The receptor for advanced glycation end-products in pulmonary hypertension. PhD thesis, University of Glasgow.

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The receptor for advanced glycation endproducts (RAGE) is a 35-kDa polypeptide of the immunogloblin superfamily that has been implicated as a mediator of both acute and chronic vascular inflammation. RAGE has also recently been implicated in the pathology of pulmonary hypertension (PH): a rare, progressive disease of the small pulmonary arteries characterised by pulmonary vascular remodelling, thrombosis, vasoconstriction and increased pulmonary vascular resistance. A ligand for RAGE, the calcium binding protein MTS1/S100A4, is expressed in occlusive vascular lesions of patients with advanced PH. MTS1/S100A4 is upregulated and secreted by pulmonary arterial smooth muscle cells (PASMCs) in vitro on activation of the 5HT1b receptor and 5HT transporter (5HTT). Additionally, the proliferative effect of 5HT on these cells, which is mediated by 5HT1b and 5HTT, may be inhibited by antagonism of RAGE or reduced bioavailability of MTS1/S100A4. These data suggest that MTS1/S100A4, through its action at RAGE, is a key mediator of 5HT-induced hPASMC proliferation.
Transgenic mice overexpressing MTS1/S100A4 are observed to develop obliterative pulmonary vascular disease and possess increased right ventricular pressure at baseline and after hypoxia when compared to wildtype mice (WT). These increases occur in the absence of an increase in pulmonary vascular remodelling suggesting that MTS1/S100A4 overexpression is associated with some other structural or functional change in the pulmonary circulation.
We sought to further our understanding of the role of RAGE in pulmonary hypertension through treatment with a small molecule inhibitor of monocyte chemoattractant protein 1(MCP-1), a marker of downstream of RAGE rage activation; through further characterisation of the MTS1/S100A4 mouse in a chronic hypoxic model of PAH; and through treatment with soluble RAGE (sRAGE) to reduce RAGE ligand bioavailability in vivo. In each case systolic right ventricular pressure (sRVP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling were measured in normoxic conditions or after a two week chronic hypoxia challenge to induce PH. These in vivo experiments were supplemented with functional studies in isolated intrapulmonary arteries to assess vascular reactivity and vascular elastance as well as studies of pulmonary fibroblast proliferation in vitro.
Treatment with the MCP-1 synthesis inhibitor Bindarit produced no detectable effects upon the pulmonary response of mice to chronic hypoxia, though this study may have been hampered by difficulties with the methylcellulose vehicle. MCP-1 produced no degree of proliferation in pulmonary fibroblasts and neither augmented nor inhibited proliferation induced by 5HT.
We found little evidence for the exacerbation of PH in MTS1/S100A4 mice in normoxia, hypoxia or after 4 weeks of normoxic recovery. Mean RVP was elevated above that in WT mice exposed to hypoxia. However, MTS1/S100A4 mice appeared protected against hypoxia-induced vascular remodelling and decreases in vascular elastance. No other significant differences in sRVP, RVH or remodelling were observed between strains. Vessels isolated from MTS1/S100A4 mice tended towards an enhanced contractile response to 5HT in normoxia compared with vessels in WT mice but were also more sensitive to the nitric oxide donor SNP. These differences in vasoreactivity were largely abolished by exposure to hypoxia. Treatment with soluble RAGE (sRAGE) to reduce RAGE ligand bioavailability produced a significant reduction in sRVP after hypoxia in comparison to vehicle-dosed mice -possibly associated with the prevention of a hypoxia-induced decrease in proximal vascular elastance. However, no benefit upon the development of remodelling or the extent of RVH was observed. Vessels isolated from mice treated with sRAGE and challenged with hypoxia showed a marked increase in contractility. Further work demonstrated that sRAGE produces a small, slowly developing contraction in isolated vessels and that the maximal force of contraction to 5HT was markedly augmented in the presence of sRAGE. Finally, treatment with sRAGE did not inhibit fibroblast proliferation in vitro as induced by 5HT but was observed to cause a small degree of proliferation alone and to augment hypoxia-induced proliferation.
In summary, we have reported a number of seemingly contradictory findings associated with RAGE in pulmonary hypertension. Treatment with sRAGE produced a beneficial reduction in hypoxia-induced PH associated with protection against decreased proximal vascular elastance but produced no change in hypoxia-induced RVH or remodelling as well as greatly increasing vascular contractility. MTS1/S100A4 mice show some evidence of deleterious changes to the pulmonary circulation, but these may be offset by beneficial compensatory mechanisms such as increased sensitivity to nitric oxide and protection against vascular remodelling.
MTS1/S100A4 stimulates smooth muscle cell proliferation suggesting that it may involved pulmonary vascular remodelling. However, inhibition of RAGE was observed to enhance fibroblast proliferation in response to hypoxia here. Fibroblasts are important regulators of SMC proliferation in vivo. These findings therefore suggest a more complicated relationship between RAGE, its ligands and the remodelling process. Since both MTS1/S100A4 overexpression and sRAGE treatment in vivo produced findings which are difficult to reconcile using the currently employed techniques, it is clear that furthering our understanding of RAGE will require study with greater focus upon the interaction of different cell types in the pulmonary vasculature and the manner in which the disturbance of this may lead to alterations in the physical and physiological properties of the pulmonary circulation.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: pulmonary hypertension, vascular disease, cell culture, pharmacology, pathophysiology, fibroblasts, RAGE, S100, myography, in vivo, haemodynamics, vascular tone
Subjects: Q Science > QH Natural history > QH301 Biology
R Medicine > RM Therapeutics. Pharmacology
Q Science > QP Physiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
Supervisor's Name: Kennedy, Dr. Simon and MacLean, Prof. Mandy
Date of Award: 2012
Depositing User: Mr David Farmer
Unique ID: glathesis:2012-3730
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Nov 2012
Last Modified: 10 Dec 2012 14:10

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