Cortisol dysregulation in equine endocrinopathic laminitis

Abstract

Laminitis is a common and severe disease of the horse's hoof occurring most frequently in association with the endocrine diseases Pituitary Pars Intermedia Dysfunction (PPID) or Equine Metabolic Syndrome (EMS) and referred to as "endocrinopathic laminitis" (EL).

Inadequate blood supply to the foot results in irreparable damage to the highly specialised structure of the hoof rendering the horse unable to walk and often necessitating euthanasia.

There is a significant paucity of knowledge regarding how and why dysfunction of the endocrine system in the horse results in failure ofthe hoofvasculature. This thesis addresses the hypothesis that dysregulation of Cortisol metabolism is a central feature of PPID and EMS and contributes to the development of laminitis.

Two approaches were used to address this hypothesis; analysis of the mechanisms of (1) vascular and (2) metabolic dysfunction in EL. The role of impaired vascular function in EL was addressed using ex vivo analysis of isolated laminar and systemic vessels. This demonstrated marked blunting of endothelial-dependent vasorelaxation in both laminar and systemic vessels of horses with EL. In addition, vessels from the hooves of these horses demonstrated increased contractile responses.

EL is commonly a chronic disease and once the blood supply to the hoof has been compromised is does not return to normal. In the light of our data demonstrating endothelial cell dysfunction and Cortisol dysregulation in horses with EL, we hypothesised that inadequate angiogenesis was a potential mechanism for this failure of the blood supply to return to normal. The angiogenic response of equine vessels to Cortisol was determined in horses with and without EL. Unlike the vessels of rodents, in which Cortisol inhibits angiogenesis, equine vessels had a significant pro-angiogenic response to Cortisol. This effect appears to be mediated through the glucocorticoid receptor. The angiogenic responses of horses with EL were not different from healthy horses.

The role of metabolic dysregulation was investigated with a detailed study of glucocorticoid metabolism in healthy horses, horses with PPID and EMS; addressing the hypothesis that these conditions are syndromes of glucocorticoid excess. Equine Cortisol metabolism and the relative contribution of different clearance pathways were found to differ significantly from that of humans and rodents. 20p-Dihydrocortisol (20(TDHF) was shown to be the predominant glucocorticoid metabolite in the horse; present in the urine, plasma, adipose and liver. PPID is a disease characterised by very high plasma ACTE1 but paradoxically normal Cortisol; this study showed marked elevation in Cortisol clearance with HPA activation. 11 p-HSDl mRNA transcript levels were increased in peri-renal adipose tissue. The role of peripheral glucocorticoid metabolism dysregulation and its interaction with the HPA axis is not clear in PPID but these findings suggest clearance may play a vital role in the pathogenesis of the disease. In EMS Cortisol clearance was also found to be significantly increased compared to healthy horses while plasma Cortisol concentrations remained within normal limits and plasma ACTH was mildly elevated. This adds weight to the argument that enhanced Cortisol clearance might drive ACTH secretion. Plasma 20p-DHF was increased in horses with EMS. This was accompanied by increases in adipose lip-HSDl mRNA transcript levels and suggests that, as in human metabolic syndrome, Cortisol dysregulation contributes to the pathogenesis of EMS.

The physiological role of 20P-DHF was explored in cell experiments designed to test its ability to activate the glucocorticoid receptor (GR). 20P-DHF was found to bind, dimerize and translocate GR. It was able to induce transcription of glucocorticoid responsive genes and inhibit transcription of inflammatory cytokines. Carbonyl reductase 1 was investigated as a potential candidate enzyme responsible for converting Cortisol into 20(B-DHF and was found to catalyse this reaction in the presence of NADPH. CBR1 mRNA transcript levels were found to be increased in the adipose of horses with EMS.

In conclusion, this thesis presents the first detailed study in horses investigating the role of Cortisol in the pathogenesis of EL. Significant and likely clinically relevant vascular dysfunction was identified in these horses. Species-specific responses to Cortisol were demonstrated and pose significant challenges to our understanding of Cortisol as an "angiostatic" agent. This thesis was the first to present a detailed description of glucocorticoid metabolism in the horse and to identify significant abnormalities in horses with EL. It also presents novel data on, endogenous glucocorticoid metabolite, 20|3-DHF.