for a more clinically relevant setting was conducted using the porcine stenting model. Human BMS were initially coated with PLA, followed by siPORT/miRNA and allowed to dry before deploying using the methodology developed in Chapter 4. After 48 h the pigs were sacrificed and the arteries harvested for RNA extraction, and quantification of the locally delivered miRNA was carried out using qRT-PCR.
It was important to identify a suitable housekeeper to allow meaningful comparisons to assess whether delivery of the miRNA had been successful. U6 and miR-103 were tested as potential candidates for the endogenous control on the pig samples, the standard deviation when miR-103 was used across the groups was 0.988735 whereas the standard deviation when U6 was used was 1.800209, therefore, miR-103 was used as a housekeeper for the study (Figure 5-2).
Figure 5-2 Comparison of U6 and miR 103 as endogenous controls for in vivo porcine stenting study.
Relative expression of miR-103 and U6 following RNA extraction and quantification by Taqman®qRTPCR from porcine coronary arteries from in vivo stenting study. Data is expressed as Ct value, U6 n=31/group, miR-103 n=30/group.
Using the same methodology for coating the stents for the murine in vivo study, stents were deployed and harvested and after 48 h. Delivery of miR-39 was observed at this time point, so evaluation as to whether the delivery system worked was straightforward as the expression levels for miR-39 were ~7500 fold above the background levels from the control groups (Figure 5-3).
Figure 5-3 Local delivery of miR-39-3p from PLA coated stents to porcine coronary arteries, 48 h after deployment using porcine in vivo stenting model.
2.5% wt/wt PLA dissolved in chloroform was coated onto porcine stents and dried overnight. miR- 39-3p (20 μM, 4.5 μL) was added to siPORT (9 μL) was then applied to the PLA coted stents. Stents were washed in PBS before deployment into the porcine coronary arteries.Arteries were harvested at 48h post deployment, and immediately stored at -80oC. RNA was extracted from the
arteries and miR-39-3p expression was quantified by Taqman®qRT-PCR and normalised to miR- 103 expression. Data is expressed as RQ values ± RQ max. Data was analysed by performing a One way ANOVA and post-hoc Tukey test relative to the BMS control, ***p<0.001, ns=non- significant, n=6/group.
Delivery of miRNAs that are relevant to the process of neointima hyperplasia, miR-145-5p and antimiR-21, were attempted. However, expression of miR-145- 5p was not upregulated enough to signify significance within the porcine in vivo setting, although the RQ value for PLA BMS miR-145-5p stent was doubled. This could have been due to the fact that miR-145-5p was highly expressed within the artery walls. Ct values for the control groups were, BMS 15.1 and PLA BMS 16.7, therefore saturation of the signal from the miRNA which was delivered from the stent could have occurred (Figure 5-4).
Figure 5-4 Local delivery of miR-145-5p from PLA coated stents to porcine coronary arteries, 48 h after deployment using porcine in vivo stenting model.
2.5% wt/wt PLA dissolved in chloroform was coated onto porcine stents and dried overnight.miR- 145-5p (20 μM, 4.5 μL) was added to siPORT (9 μL) was then applied to the PLA coted stents. Stents were washed in PBS before deployment into the porcine coronary arteries.Arteries were harvested at 48h post deployment, and immediately stored at -80oC. RNA was extracted from the
arteries and miR-145-5p expression was quantified by Taqman®qRT-PCR and normalised to miR- 103 expression. Data is expressed as RQ values ± RQ max. Data was analysed by performing a One way ANOVA and post-hoc Tukey test relative to the BMS control, ns=non-significant, n=4 for PLA BMS + miR-145-5p and n=6 for control BMS and PLA BMS groups.
AntimiR-21 was also delivered from the porcine stent surface and the levels of miR-21 were recorded at 48 h post-deployment. Firstly, we noted a change in the miRNA-21 expression profile between the non-stented arteries (NTC) and the control groups (PLA BMS and PLA BMS + S/C), which agrees with other reports which have documented miR-21 release after response to stent deployment and corresponding vessel injury (McDonald et al., 2015). miR-21 expression was decreased by 50% after local delivery of antimiR-21 from the stent surface; however the change was not statistically significant. This study was not highly powered however, with low numbers in the treatment group (n=3) and the scramble control group (n=2), so further testing to elicit whether the trend continued. Increasing the concentration of antimiR-21 could also help to ensure a decrease in miR-21 expression was observed at the 48 h time-point which we could then use to investigate the therapeutic potential and efficacy of this miR- based treatment (Figure 5-5).
Figure 5-5 Local delivery of antimiR-21 from PLA coated stents to porcine coronary arteries, 48 h after deployment using porcine in vivo stenting model.
2.5 % wt/wt PLA dissolved in chloroform was coated onto porcine stents and dried overnight.antimiR-21 (20 μM, 4.5 μL) was added to siPORT (9 μL) was then applied to the PLA coted stents Stents were washed in PBS before deployment into the porcine coronary arteries.Arteries were harvested at 48h post deployment, and immediately stored at -80oC. RNA
was extracted from the arteries and miR-21 expression was quantified by Taqman®qRT-PCR and normalised to miR-103 expression. Data is expressed as RQ values ± RQ max. Data was analysed by performing a One way ANOVA and post-hoc Tukey test relative to the PLA BMS + antimiR scramble control, *p<0.05, ns=non-significant, n=3 for antimiR 21 PLA BMS group, n=2 for antimiR 21 S/C PLA BMS, n=6 for PLA BMS and n=8 for NTC group.
5.3 Discussion
In summary, it has been demonstrated that localised delivery of miRNA from stent surfaces can be achieved in both murine and porcine in vivo stenting models, using the technique of coating the BMS stent initially with PLA and then with the miRNA/siPORT. This was clearly demonstrated when using miR-39-3p as a reporter miRNA, as it is non-endogenous to mammals and therefore detection was easier to recognise.
When preliminary studies were conducted delivering antimiR-21 and miR-145-5p from the stent, we did not observe significant differences in miRNA expression between the control groups and the treatment groups. This could be due to a number of reasons. Firstly, since miR-145 is one of the most abundantly expressed miRNAs in arteries, saturation of the miR-145 expression signal could occur as the high background levels of miR-145 could prevent the detection of the miR-145 delivered from the stent being observed. In the case of antimiR-21, the experiment was not highly powered enough to ascertain conclusively whether antimiR-21 could be delivered from the stent and correspond to a decrease in arterial miR-21 expression, so a more highly powered study would aid this. In addition higher concentrations of therapeutic miRNA could be administered to ensure the change in miRNA expression was observed. Finally a method for spray coating the stent with miRNA/siPORT could ensure uniform distribution of miRNA coating (as done with DES) and this could decrease the variability within the treatment groups, which does seem to be an issue to enable consistent results to be generated.
The ability to deliver the miRNA in two different animal models has a lot of future potential for further scientific investigations, as genetically-modified mice could be used to further probe miRNA action on development of ISR. The porcine model however, has the advantage of being much more relevant to the human stent setting. Therefore further investigation using therapeutic miRNAs attached to the surface using this model to see whether alteration of the miRNAs usual expression profile could be altered and most importantly whether a therapeutic response can be monitored would provide a novel therapy for the prevention of ISR.
Recently, the first example of a miRNA eluting stent was published in the rat model, whereby antimiR-21 was delivered from the stent surface which exhibited a decrease in neointima formation (Wang et al., 2015) and also circumvented issues which were present with systemic delivery of the miRNA when delivered intravenously. This has highlighted the advantages of localised delivery and the powerful effects which therapeutic miRNA could have when administered in this way.
Further to the in vitro testing detailed in Chapter 4 of miR 99b/let 7e/125a on the effect of VSMC proliferation, a porcine in vivo 28 day efficacy study was carried out delivering this cluster from stent surfaces. Although completed, at the time of writing the RNA extractions and analysis of the vessels by optical coherence tomography have not yet been completed. These results will indicate whether or not the cluster miRNA eluting stent would be effective at preventing ISR.
Finally, using a PLA platform for miRNA delivery would enable BMSs to be used as demonstrated in these studies; it also however could be used with the new technology of biodegradable stents which are composed of PLA, to create a transient PLA miRNA eluting stent. This could aid prevention of ISR through miRNA action and then dissolve overtime which would avoid chronic inflammatory responses caused by the stent struts.
To date the work presented here represents the second study detailing methodology for miRNA-eluting stents, with the first study being published a few months before the completion of the writing of this text (Wang et.al., 2015). The methods which have been used here are novel in that they can be universally applied to any BMS, and represent a simple approach for miRNA delivery within this setting. In addition this is the first time that PLA has been used to absorb miRNA onto for use as a miRNA delivery device in the context of coronary stents. Since PLA is a common material for medical implants this technique for miRNA delivery could be applied in different medical contexts.