MicroRNAs Predict and Regulate Heme Oxygenase-1 Expression

The shortage of organs available for donation has resulted in the increased use of organs from deceased donors. These organs endure a relatively prolonged period without an adequate blood supply and subsequently become injured (ischaemia reperfusion injury (IRI)). IRI is associated with reduced function of the transplanted kidney immediately following transplantation and long term is associated with increased rates of transplant loss.
The drug heme arginate (HA) increases the natural protective enzyme heme oxygenase-1 (HO-1), resulting in a wide range of beneficial effects in studies examining inflammation and IRI. In particular, increases in HO-1 in the white blood immune cells 'monocytes' and their derived tissue cells 'macrophages' appear to be important for protection. There are now a number of registered clinical trials assessing the use of HA in the treatment of inflammatory conditions including pancreatitis, diabetes and renal transplantation (HOT1 study). In the upcoming multicentre 'HOT2' trial, the effect of HA on kidney transplant function in the early post-operative period will be determined.

In the HOT1 study, we noted that individuals responded variably to HA treatment, with some individuals generating higher levels of in HO-1 in blood monocytes. We have found that multiple small RNA molecules 'microRNAs' (miRs) are strongly associated with individual HO-1 response to treatment. As differences in blood miRs are evident prior to treatment, we believe that this 'miR signature' may predict treatment response and therefore could be used to personalise treatment to the individual. We also hypothesise that these miRs may regulate drug effects by interfering with the molecular pathway of HO-1 in monocytes and macrophages. By unpicking the action of these miRs, we may identify novel therapeutic targets in the regulation of HO-1.

In this study, we plan to investigate the role of miRs in a subset of kidney transplant patients recruited to the 'HOT2' clinical trial. We will determine whether our miR signature can predict HO-1 upregulation following HA treatment in the monocytes of kidney transplant patients. We will determine whether our miR signature is applicable to other populations by examining stored samples from 2 previous clinical trials where HA was given to patients undergoing liver and cardiac surgery. Determining this may allow us to personalise HA treatment to those likely to respond in future.

MiRs are known to suppress gene expression. We will use cell culture experiments using human macrophages to examine the mechanism in which these miRs regulate HO-1 expression following HA treatment. We will both increase and block the expression of key miRs before HA treatment, to see if this influences HO-1 response. We will also test factors which may regulate the expression of these miRs. This may allow us to identify novel regulators of HO-1 which could potentially be exploited as therapeutic targets in future.

This research will be conducted in the internationally renowned Queens Margaret Research Institute (QMRI) in Edinburgh which is adjacent to the Royal Infirmary of Edinburgh (RIE). The RIE is the lead centre for the HOT2 study and is supported by the Edinburgh Clinical Trials Unit. This will allow us to recruit patients and analyse samples on the same site. The QMRI is home to experts in macrophages, microRNAs, HO-1 and has supported 3 clinical trials using HA to completion previously. The local expertise available, will assist us in delivering this proposal.

This is a novel and exiting area of research with potential benefits to both renal transplant patients and the wider population. Determining if this miR signature can be used to personalise medicine is of potential immediate benefit to patients, whilst unpicking the mechanism in which miRs regulate HO-1 may allow the development new treatments that can upregulate HO-1 in all patients.

Background: We have identified a novel PBMC microRNA signature which is strongly associated with high heme oxygenase -1 (HO-1) response to heme arginate (HA) treatment.

Aims: To assess the role of miRs as both predictive biomarkers and regulators of HO-1 upregulation following HA treatment, in monocytes and macrophages.

Methods
HOT2 is a multicentre trial powered to determine if HA improves primary function following renal transplant. A local subset of patients will be recruited to HOT2R (HOT2-Research) to investigate: 1) the stability and validity of miRs in predicting HO-1 response (protein and mRNA) to HA 2) the impact of HA treatment on monocyte phenotype 3) the effect of HO-1 response on clinical outcomes. MiR expression in two completed RCTs employing HA in surgery (HACS, Cardiac and HOLS; Liver) will be performed to determine the generalisability. Concordance between PBMC and serum miR expression will be determined and further correlated with end organ (renal and liver) HO-1 expression in HOT2R and HOLS samples respectively.
To mechanistically interrogate factors associated with HO-1 response, we will first use flow cytometry/cell sorting, to examine the cell surface characteristics and miR profile of isolated monocytes in HA treated patients. Using an in vitro model of human monocyte derived macrophages (MDM), the regulation of HA response by miRs will be probed. Anti-miRs and lenti-viruses will be used block and overexpress miRs prior to HA treatment and the effect on MDM HO-1 measured. The impact of miR and HO-1 expression on monocyte and macrophage phenotype will be determined. The transcriptional regulation of these miRs will be explored in vitro and results correlated with trial samples.

Outcomes: The potential benefits are twofold: as biomarkers to predict HO-1 response, thereby allowing a stratified treatment approach; and enhancing the understanding of the molecular regulation of HO-1, potentially identifying novel therapeutic targets.

Clinical and Academic Impact
HO-1 upregulation has demonstrated protective effects in a wide range of experimental models, resulting in intense interest in translating this into clinical benefit. There are 11 registered clinical trials utilising hemin or heme arginate (HA) on clinicaltrials.gov and a further 30 studies where HO-1 levels are stated as an outcome measure. Within transplantation, HA treatment to induce HO-1 demonstrates promise as a therapeutic agent to reduce ischaemia reperfusion injury (IRI). The shortfall of organs available for donation necessitates the use of organs from extended criteria deceased (ECD) donors. This is associated with increased severity of IRI, resulting in delayed graft function (DGF) in up to 50% of renal allografts and ultimately, increased rates of graft loss. Currently there are no specific therapies available to treat IRI. In renal transplantation, the efficacy of HA in reducing DGF is currently under evaluation in the multi-centre trial 'HOT2.'

This proposal examines microRNAs as biomarkers of and potential barriers to, the effective induction of HO-1 in all patients. Understanding this relationship may allow stratification of patients to treatment or optimisation of HO-1 upregulating agents through the identification of novel therapeutic targets. This would be of significant cross-disciplinary clinical relevance.

In addition to the potential benefit to clinical academics studying HO-1 related conditions, the mechanistic insights generated by the study of microRNAs in this context may generate impact of relevance to the scientific community. For example, by validating the gene targets of examined miRs or by understanding the common transcriptional regulation of these miRs.

Socioeconomic Impact
Positive socioeconomic gains are anticipated to be generated by i) development of strategies which effectively induce HO-1 and improve health ii) stratification of patients to HO-1 modulating treatments and iii) direct economic outputs of the knowledge generated.

In the context of end stage renal failure, renal transplantation is effective as compared to dialysis; reducing direct health care costs and improving individual productivity, resulting in benefit to society. Therefore therapeutic strategies which effectively induce HO-1 resulting reduced graft loss, increased utilisation of ECD organs and reducing the number of patients waiting for transplant would be beneficial. Further gains may be made in the application of this knowledge to other conditions.

Heme arginate (HA) costs £400 per infusion. It has a well established safety profile in the context of porphyria and this makes it an appealing clinically applicable HO-1 inducing agent. Development of a miR panel which can accurately stratify patients to those most likely to respond would result in economic benefits, particularly in the case of studies where multiple infusions are planned.

The intellectual assets created may generate economic benefit either through the patenting of a miR panel that can be used to predict individual HO-1 responsiveness or, through the identification of novel therapeutic targets. The latter may stimulate industry partnerships to develop pharmaceutical agents (e.g. antisense oligonucleotides) or widen the application of agents currently in use (e.g. heme arginate). This will generate economic benefit for the University of Edinburgh and would enhance collaborations between industry, researchers and funding bodies ultimately increasing the impact of UK based research.