Biochemical and epidemiological investigations of non-steroidal anti-inflammatory drug usage and related side effects in equids

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in equine veterinary practice. These drugs exert their effect by inhibiting cyclooxygenase (COX) enzymes, which control prostaglandin production, a major regulator of tissue perfusion. Two isoforms of COX enzymes exist: COX-1 is physiologically present in tissues, while COX-2 is up-regulated during inflammation and has been indicated as responsible for the negative effects of an inflammatory response. Evidence suggests that NSAIDs that inhibit only COX-2, preserving the physiological function of COX-1 might have a safer profile. Studies that evaluate the effect of NSAIDs on COX enzymes are all performed under experimental conditions and none uses actual clinical patients. The biochemical investigations in this work focus on describing the effect on COX enzymes activity of flunixin meglumine and phenylbutazone, two non-selective COX inhibitors and firocoxib, a COX-2 selective inhibitor, in clinical patients undergoing elective surgery. A separate epidemiological investigation was aimed at describing the impact that the findings of biochemical data have on a large population of equids. Electronic medical records (EMRs) from 454,153 equids were obtained from practices in the United Kingdom, United States of America and Canada. Information on prevalence and indications for NSAIDs use was extracted from the EMRs via a text mining technique, improved from the literature and described and validated within this Thesis. Further the prevalence of a clinical sign compatible with NSAID toxicity, such as diarrhoea, is reported along with analysis evaluating NSAID administration in light of concurrent administration of other drugs and comorbidities. This work confirms findings from experimental settings that NSAIDs firocoxib is COX-2 selective and that flunixin meglumine and phenylbutazone are non-selective COX inhibitors and therefore their administration carries a greater risk of toxicity. However the impact of this finding needs to be interpreted with caution as epidemiological data suggest that the prevalence of toxicity is in fact small and the use of these drugs at the labelled dose is quite safe.

Non-invasive monitoring of changes in exhaled markers of airway inflammation in Thoroughbred racehorses

Exhaled breath (EB) and exhaled breath condensate (EBC) contain numerous volatile gases and a wide-array of non-volatile compounds, several of which have been investigated as markers of lower airway inflammation in human and veterinary medicine and have been used to diagnose and monitor diseases associated with pulmonary inflammation. The identification of reliable biomarkers within EB and EBC is an active research focus with the common goal of establishing non-invasive and repeatable assessment of respiratory health and disease in mammals. The application of EB and EBC analysis holds considerable appeal in the investigation of respiratory disease in Thoroughbred racehorses, as inflammatory airway disease (IAD) is a common cause for poor performance in this population of animals. This study documented that EB and EBC samples can be safely collected from Thoroughbred racehorses in their own environment, without adverse effect or interference with the horse’s training regimen. The use of off-line collection and analysis of exhaled gases via chemiluminescence is suitable for the measurement of exhaled carbon monoxide, but is not appropriate for analyzing exhaled nitric oxide in horses. Significant changes in the concentration of exhaled CO and the pH of EBC occurred in response to strenuous exercise and when exercising in different environmental temperatures. Exhaled CO was associated with tracheal mucus score (and the number of neutrophils in the mucus) and EBC pH was significantly different in horses with evidence of neutrophilic IAD compared to horses without IAD. Numerous physiological and environmental variables were identified as confounding factors in the assessment of both exhaled CO and EBC pH, with respiratory rate prior to EB collection, and during EBC collection, consistently identified as an explanatory variable influencing the concentration of exhaled biomarkers. Further studies in EB and EBC analysis in horses need to focus on objectively accounting for key respiratory dynamics during sample collection.

The role of non-coding RNA in the development of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease of the small pulmonary arteries, characterised by pulmonary vascular remodelling due to excessive proliferation and resistance to apoptosis of pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs). The increased pulmonary vascular resistance and elevated pulmonary artery pressures result in right heart failure and premature death. Germline mutations of the bone morphogenetic protein receptor-2 (bmpr2) gene, a receptor of the transforming growth factor beta (TGF-β) superfamily, account for approximately 75%-80% of the cases of heritable form of PAH (HPAH) and 20% of sporadic cases or idiopathic PAH (IPAH). IPAH patients without known bmpr2 mutations show reduced expression of BMPR2. However only ~ 20% of bmpr2-mutation carriers will develop the disease, due to an incomplete penetrance, thus the need for a ‘second hit’ including other genetic and/or environmental factors is accepted. Diagnosis of PAH occurs most frequently when patients have reached an advanced stage of disease. Although modern PAH therapies can markedly improve a patient’s symptoms and slow the rate of clinical deterioration, the mortality rate from PAH remains unacceptably high. Therefore, the development of novel therapeutic approaches is required for the treatment of this multifaceted disease. Noncoding RNAs (ncRNAs) include microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). MiRNAs are ~ 22 nucleotide long and act as negative regulators of gene ex-pression via degradation or translational inhibition of their target mRNAs. Previous studies showed extensive evidence for the role of miRNAs in the development of PAH. LncRNAs are transcribed RNA molecules greater than 200 nucleotides in length. Similar to classical mRNA, lncRNAs are translated by RNA polymerase II and are generally alternatively spliced and polyadenylated. LncRNAs are highly versatile and function to regulate gene expression by diverse mechanisms. Unlike miRNAs, which exhibit well-defined actions in negatively regulating gene expression via the 3’-UTR of mRNAs, lncRNAs play more diverse and unpredictable regulatory roles. Although a number of lncRNAs have been intensively investigated in the cancer field, studies of the role of lncRNAs in vascular diseases such as PAH are still at a very early stage. The aim of this study was to investigate the involvement of specific ncRNAs in the development of PAH using experimental animal models and cell culture. The first ncRNA we focused on was miR-143, which is up-regulated in the lung and right ventricle tissues of various animal models of PH, as well as in the lungs and PASMCs of PAH patients. We show that genetic ablation of miR-143 is protective against the development of chronic hypoxia induced PH in mice, assessed via measurement of right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling. We further report that knockdown of miR-143-3p in WT mice via anti-miR-143-3p administration prior to exposure of mice to chronic hypoxia significantly decreases certain indices of PH (RVSP) although no significant changes in RVH and pulmo-nary vascular remodelling were observed. However, a reversal study using antimiR-143-3p treatment to modulate miR-143-3p demonstrated a protective effect on RVSP, RVH, and muscularisation of pulmonary arteries in the mouse chronic hypoxia induced PH model. In vitro experiments showed that miR-143-3p overexpression promotes PASMC migration and inhibits PASMC apoptosis, while knockdown miR-143-3p elicits the opposite effect, with no effects observed on cellular proliferation. Interestingly, miR-143-3p-enriched exosomes derived from PASMCs mediated cell-to-cell communication between PASMCs and PAECs, contributing to the pro-migratory and pro-angiogenic phenotype of PAECs that underlies the pathogenesis of PAH. Previous work has shown that miR-145-5p expression is upregulated in the chronic hypoxia induced mouse model of PH, as well as in PAH patients. Genetic ablation and pharmacological inhibition (subcutaneous injection) of miR-145-5p exert a protective against the de-velopment of PAH. In order to explore the potential for alternative, more lung targeted delivery strategies, miR-145-5p expression was inhibited in WT mice using intranasal-delivered antimiR-145-5p both prior to and post exposure to chronic hypoxia. The decreased expression of miR-145-5p in lung showed no beneficial effect on the development of PH compared with control antimiRNA treated mice exposed to chronic hypoxia. Thus, miR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while the inhibition of miR-143-3p prevented the development of experimental pulmonary hypertension. We focused on two lncRNAs in this project: Myocardin-induced Smooth Muscle Long noncoding RNA, Inducer of Differentiation (MYOSLID) and non-annotated Myolnc16, which were identified from RNA sequencing studies in human coronary artery smooth muscle cells (HCASMCs) that overexpress myocardin. MYOSLID was significantly in-creased in PASMCs from patients with IPAH compared to healthy controls and increased in circulating endothelial progenitor cells (EPCs) from bmpr2 mutant PAH patients. Exposure of PASMCs to hypoxia in vitro led to a significant upregulation in MYOSLID expres-sion. MYOSLID expression was also induced by treatment of PASMC with BMP4, TGF-β and PDGF, which are known to be triggers of PAH in vitro. Small interfering RNA (siR-NA)-mediated knockdown MYOSLID inhibited migration and induced cell apoptosis without affecting cell proliferation and upregulated several genes in the BMP pathway in-cluding bmpr1α, bmpr2, id1, and id3. Modulation of MYOSLID also affected expression of BMPR2 at the protein level. In addition, MYOSLID knockdown affected the BMP-Smad and BMP-non-Smad signalling pathways in PASMCs assessed by phosphorylation of Smad1/5/9 and ERK1/2, respectively. In PAECs, MYOSLID expression was also induced by hypoxia exposure, VEGF and FGF2 treatment. In addition, MYOSLID knockdown sig-nificantly decreased the proliferation of PAECs. Thus, MYOSLID may be a novel modulator in pulmonary vascular cell functions, likely through the BMP-Smad and –non-Smad pathways. Treatment of PASMCs with inflammatory cytokines (IL-1 and TNF-α) significantly in-duced the expression of Myolnc16 at a very early time point. Knockdown of Myolnc16 in vitro decreased the expression of il-6, and upregulated the expression of il-1 and il-8 in PASMCs. Moreover, the expression levels of chemokines (cxcl1, cxcl6 and cxcl8) were sig-nificantly decreased with Myolnc16 knockdown. In addition, Myolnc16 knockdown decreased the MAP kinase signalling pathway assessed by phosphorylation of ERK1/2 and p38 MAPK and inhibited cell migration and proliferation in PASMCs. Thus, Myolnc16 may a novel modulator of PASMCs functions through anti-inflammatory signalling pathways. In summary, in this thesis we have demonstrated how miR-143-3p plays a protective role in the development of PH both in vivo animal models and patients, as well as in vitro cell cul-ture. Moreover, we have showed the role of two novel lncRNAs in pulmonary vascular cells. These ncRNAs represent potential novel therapeutic targets for the treatment of PAH with further work addressing to investigate the target genes, and the pathways modulated by these ncRNAs during the development of PAH.