Investigation of MiR-140-5p in Humans and Experimental Models of Pulmonary Arterial Hypertension.

Pulmonary Arterial Hypertension:
Pulmonary arterial hypertension (PAH) is a devastating and debilitating disease affecting 5 people per million. It is caused by narrowing of the arteries of the lungs and leads to high blood pressure in the lungs and failure of the heart. Severe shortness of breath, heart failure and death are common among sufferers despite recent advances in scientific understanding and clinical treatment of the disease.

What Causes Pulmonary Arterial Hypertension?
The causes of PAH are incompletely understood. Specific gene mutations are common within families with PAH. This finding had lead to the identification of cellular pathways important in causing PAH. Mutations of BMPR2, a cell surface receptor protein, reduce signalling in the BMPR2 pathway and cause PAH; however, not all patients with mutations of BMPR2 develop the disease. Mutations in other genes leading to reduced BMPR2 signalling can cause PAH and reduced BMPR2 signalling is seen in several experimental models of PAH. These observations have lead to the 'multiple hit' model of pathology in which several genetic 'hits' each reduce BMPR2 signalling and together cause PAH.

What are microRNA?
MicroRNA are short RNAs that alter the expression of genes within the cell by specifically targeting and preventing the production of proteins. Blood microRNA expression levels have been used as markers of disease in cancer and heart disease. Manipulation of microRNA levels is also used experimentally to treat several diseases. The first human trial of a microRNA based drugs are being undertaken currently.

New Findings and Opportunities:
The Pulmonary Vascular Research Group in Sheffield has extensive experience in PAH research with expertise in cell culture and experimental models of PAH. The Sheffield Pulmonary Vascular Unit is one of five national centers for clinical investigation of PAH. Prospective collection from patients with newly diagnosed pulmonary hypertension referred to the Sheffield Pulmonary Vascular Unit has created an extensive biobank of blood samples with associated clinical data from over 300 patients.

New results from our lab show that patients with PAH have altered microRNA expression patterns in their blood. Further experiments show that reproducing these microRNA changes in human lung artery cells alters the levels of proteins known to regulate BMPR2 signalling. These changes act in a way that would reduce BMPR2 signalling in the manner seen in PAH.

Hypotheses:
1 Reduced levels of specific microRNA are an important change leading to PAH
2 Blood microRNA expression levels may be used as a marker of PAH

Aims:
The aim is to complete our study of microRNA expression in blood samples taken from PAH, examine the effects of altered microRNA expression in human lung cells and test these microRNA as a potential treatment in experimental models of PAH.

Experiments:
We aim to test our hypothesis by manipulating microRNA levels in human lung artery cells. To determine the effect of the changes in microRNA levels we will asses levels of mRNA and protein of targets within the cell and examine cell growth and movement of cells as indicators of cell function. We will examine expression levels of microRNA expression in experimental models of PAH. Finally we will test a microRNA drug to determine if the changes prevent and treat PAH in experimental models.

Conclusion:
New findings suggest an exciting link between blood microRNA changes and the mechanisms causing PAH. The Sheffield Pulmonary Vascular Unit blood sample collection provides a unique opportunity to investigate blood microRNAs a biomarker for PAH and the expertise of the Pulmonary Vascular Research Group provide an opportunity to investigate how the observed microRNA changes affect experimental models of PAH.

Pulmonary arterial hypertension (PAH) is a devastating and life threatening condition for which there is no pharmacological cure. Dissecting the molecular mechanisms that drive pathogenesis is crucial for the development of effective treatments. Several studies have highlighted the importance the Bone Morphogenetic Protein Receptor Type II (BMPR2) and related signalling pathways in PAH, but how these cause the disease remains unclear. MicroRNA, a recently described class of RNA, are proposed to play a key role in regulating health and disease by modulating the translation of target mRNA. In-house data that I have performed demonstrate that whole blood microRNA-140-5p (miR-140-5p) is decreased in patients with idiopathic and scleroderma associated PAH. In Silico analysis predicts that miR-140-5p targets molecules downstream of BMPR2. I therefore hypothesise that miR-140-5p expression is decreased in PAH, resulting in reduced BMPR2 signalling and contributing to disease pathogenesis; even in the absence of a BMPR2 mutation. To test this hypothesis I will use a combination of clinical samples, in vitro cell culture and rodent models of PAH to validate the initial microRNA (miR) array results, and subsequently quantify changes in the key predicted targets of miR-140-5p in pulmonary artery endothelial cells (PAEC) and pulmonary artery smooth muscle cells (PASMC). We will measure the effect of miR-140-5p on PASMC and PAEC phenotype, before determining the effect of exogenous miR-140-5p on disease phenotype in rodent models. This data will provide clear and novel information on the function of miR-140-5p in pulmonary vascular cells and the temporal expression in two animal models of PAH. This will allow direct translation to our clinical data showing reduced miR-140-5p in PAH, and establish whether reduced miR-140-5p levels are causative, or a bystander in PAH pathogenesis. Finally this will provide insight into the utility of miR-140-5p levels as a biomarker in PAH.

Pulmonary arterial hypertension (PAH) is a rare but debilitating disease. Despite resent advances in the understanding and treatment of PAH clinical, scientific and public awareness of the disease is low. To maximise the potential impact of high quality research it is essential that appropriate steps are undertaken to increase awareness. I aim to communicate the findings of my research to engage a range of audiences; scientists, clinicians, industrial partners, patients, potential new researchers and the public.

Scientific, Clinical and Industry Communication: My work has the potential to influence the work of other groups in PAH research. I plan to communicate the findings of my research to the scientific community via publication in high impact peer reviewed journals. This will be furthered by presentation at International and National meetings with science, clinical practice and industry audiences such as: American Thoracic Society, American Heart Association, British Thoracic Society. I have presented preliminary findings at the UK Pulmonary Vascular Science Meeting and the PH Physicians Forum. Research from Dr Lawrie's group has led directly to patented IP and an ongoing collaboration with MRC Technology demonstrating the groups attitude and success in producing high impact translational research.

Patient Public Involvement: Human sample collection has been undertaken through the Cardiovascular Biomedical Research Unit (CVBRU) in Sheffield. The CVBRU has an integrated patient advisory panel who advise on research and sample collection. Prior to sample analysis the patient panel discussed and approved use of whole blood samples use for the investigation of microRNA changes in PAH. Regular science updates are held by the CVBRU to inform patients and the public of research progress. I have presented to both the expert patient panel and the general public at the resent CVBRU fellows research event. Patient involvement is regarded as a high priority within the CVBRU and as such I intend to maximise patient and public involvement in my research through University events and the Pulmonary Hypertension Association UK.

Engaging the Public and the Next Generation: Over a number of years I have been actively involved in engaging students in medicine and science. I have spoken at events at my old school and local dyslexic groups, and teach medical students and junior doctors at a range of levels. I aim to continue my current teaching and broaden potential public engagement by involvement in Science Brainwaves, an outreach programme run by a Sheffield based charity with the aim of 'advancing the public understanding, accessibility and accountability of the sciences and engineering', and by giving a talk on my research at the National Science Week. These activities will provide a good platform engage the public and potential new scientists in my research. This work will be complemented by promoting the results of my research on the University of Sheffield and Cardiovascular Science Department websites.