Investigation of a role for GPR35 as a novel therapeutic target in cardiovascular disease

McCallum, Jennifer Elizabeth (2014) Investigation of a role for GPR35 as a novel therapeutic target in cardiovascular disease. PhD thesis, University of Glasgow.

Abstract

G protein-coupled receptors (GPCRs) are often described as ‘gate-keepers’ of the eukaryotic cell and their roles primarily involve translating extra-cellular stimuli via G protein coupling and intracellular signalling to govern various physiological responses. These functional parameters are wide ranging and include the modulation of visual sensory organs, to the control of vasoreactivity and heart rate. Importantly, their flexible architecture can facilitate small molecule interaction within a ‘binding-pocket’ and GPCR research often focuses on this relationship to identify and design novel and effective ligands to manipulate GPCR activation and signalling.
GPR35 is a poorly characterised, Class A GPCR and despite reports of two potential endogenous activators; kynurenic acid (KYNA) and lysophosphatidic acid (LPA) in recent years, it is still widely acknowledged as an ‘orphaned’ GPCR. This is reflected by the propensity of its ligands, both endogenous and synthetic, to demonstrate extreme species orthologue-selective properties. Despite this, investigators have highlighted various roles for GPR35 relating to pain, inflammation, hypertension and heart failure, and investigations have suggested that activation of GPR35 leads to a selective coupling of Gα13, a G-protein understood to mediate Rho A and ROCK 1/2 signalling. However, a lack of functional ligand pairs has hampered further research to examine a role for GPR35 and subsequent Gα13 signalling in these disease models. In this study, we have characterised a series of highly-potent, synthetic ligands at human, rat and mouse orthologues of GPR35, revealing that GPR35 agonist, pamoic acid and antagonists, CID-2745687 and ML-145, are highly potent and selective for the human orthologue of GPR35. This has provided us with the opportunity to assess a functional role for GPR35 within a cardiovascular disease setting using functional ligand pairs in cells of a human origin, for
the first time.
Vascular smooth muscle cell (VSMC) migration and proliferation are central to neointima formation in vein graft failure. Despite a detailed understanding of VSMC migration and proliferation mechanisms, there are no pharmacological interventions which effectively
prevent vein graft failure through intimal occlusion. In this study, we demonstrated that primary vascular cells expressed robustly detectable levels of GPR35, and these were comparable to those demonstrated in the colon, which has been previously reported to highly express GPR35. Human GPR35 is potently activated by the selective agonist pamoic acid and reference ligand zaprinast and blocked by antagonists CID-2745687 and ML-145. Following exposure of VSMC to pamoic acid or zaprinast, cell migration was enhanced and these effects were blocked by co-incubation with CID-2745687 or ML-145. Pamoic acid induced HSV SMC migration was also blocked in the presence of two distinct
Rho A pathway inhibitors, Y-16 and Y-27632. Activation of this pathway was also reflected by remodelling of the cytoskeletal architecture in HSV SMCs to significantly elongate the cell and promote a contractile, migratory phenotype following pamoic acid stimulation and this effect was also blocked following co-administration with either antagonist. Additionally, we also demonstrated that following exposure to pamoic acid or zaprinast, human saphenous vein endothelial cell (HSV EC) integrity and proliferation were significantly improved and this was blocked following co-administration with either antagonist,
suggesting a protective role for GPR35 in the vascular endothelium.
Results from a previous study demonstrated that lack of GPR35 expression resulted in an elevation in systolic blood pressure (SBP) by up to 37.5mmHg in mice. The recent identification of mast-cell stabilising compounds as highly potent agonists at GPR35 provided the opportunity to pharmacologically target GPR35 within a rodent model of hypertension. Therefore, we also aimed to test if pharmacological manipulation of GPR35 via stimulation with the novel, rodent selective agonist amlexanox, modulated blood pressure and end-organ related damage in 6-12 weeks of age stroke prone spontaneously hypertensive rats (SHRSPs). Radiotelemetry acquisition of haemodynamic properties highlighted that pharmacological agonism of GPR35 exacerbated hypertension and end-organ damage in the SHRSP and this was evident following an elevation in SBP by 20mmHg throughout the trial. Moreover, quantification of heart mass and cardiomyocyte size revealed that GPR35 agonism induced cardiac hypertrophy. Collagen staining revealed enhanced renal fibrosis in both the interstitial and perivascular regions of the kidney from amlexanox treated animals, compared to vehicle controls. Additionally, large vessel myography highlighted that endothelium-dependent vasorelaxation was reduced by 20% in amlexanox treated SHRSPs. Fundamentally, these results suggest that GPR35 is involved in regulating vascular tone and we hypothesise that this may involve the Gα13-Rho A-ROCK1/2 signalling pathway demonstrated to mediate a contractile, migratory phenotype in human primary VSMCs.
Taking these data together, the results suggest that GPR35 antagonists might be of clinical use to therapeutically target and inhibit activation of GPR35 in the setting of
vascular remodelling during acute vascular injury, and hypertension and its related end organ damage.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: GPR35, G protein-coupled receptor, hypertension, vascular injury, ligand
• Subjects: R Medicine > RM Therapeutics. Pharmacology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Milligan, Professor Graeme and Nicklin, Dr. Stuart. A.
• Date of Award: 2014
• Depositing User: Jennifer Elizabeth McCallum
• Unique ID: glathesis:2014-6120
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 20 Feb 2015 08:42
• Last Modified: 20 Feb 2018 16:04
• URI: https://theses.gla.ac.uk/id/eprint/6120