Investigating the regulation of thymosin B4 by MicroRNAs in the developing human heart

Cardiovascular disease is the leading cause of death in the UK and there are over 25,000 new cases of heart failure in the UK each year. Studies in the field of regenerative medicine are seeking to find ways to repair the failing heart. Therapies which are being developed range from cell transplantation therapies to treatment with cardioprotective proteins. Thymosin β4 is a small, endogenous protein involved in wound healing and angiogenesis. Thymosin β4 is important in development of the murine heart and has also been noted to have cardioprotective effects in mouse models of myocardial infarction.Currently there is little data regarding the cellular regulation of thymosin β4. miRNAs are a class of endogenous, small RNA molecules which act as posttranscriptional regulators of protein expression. It was hypothesised that thymosin β4 is present in the developing human heart and that its expression is regulated by miRNAs.Using PCR and immunohistochemistry, thymosin β4 mRNA and protein were found to be present in human foetal hearts. Thymosin β4 protein was primarily localised to endothelial cells and was detected at higher levels in endothelial cells of the compact layer of the myocardium than the trabecular layer.Different experimental strategies were developed in order to identify miRNAs with expression inversely correlated to that of thymosin β4. Foetal heart explants were cultured under hypoxic conditions; this was hypothesised to increase thymosin β4 expression. Western blotting was used to assess thymosin β4 protein levels, however this technique was found not to be suitable and no consistent changes in thymosin β4 expression were detected. The second experimental strategy used flow cytometry to successfully detect thymosin β4-positive and –negative CD34-expressing cells in the foetal heart. The aim of this was to separate these populations by fluorescence-activated cell sorting. Unfortunately, limited access to foetal tissue hindered this approach and an alternative experimental system was therefore needed.The third experimental strategy involved culture of human umbilical vein endothelial cells on Matrigel basement membrane. This led to formation of tubules, a process during which thymosin β4 mRNA was reported to be upregulated in previous studies. In the present study no changes in thymosin β4 mRNA or protein levels were detected. Expression levels of four miRNAs predicted to target thymosin β4 (miR-148b, miR-199a-3p, miR-217 and miR495) were unchanged in tubule-forming HUVECs compared to controls