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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - NORVAS (Therapeutic and Biomarker Potential of long non-coding RNAs in Vascular Disease)

Teaser

Summary

Main aim of NORVAS is to identify non-coding RNA based therapies and biomarkers that enable us to combat the burden of vascular diseases in general, and abdominal aortic aneurysms and carotid artery stenosis and subsequent stroke in particular. Abdominal aortic aneurysms (AAAs) are defined as a permanent dilation of the abdominal aorta that predisposes to the fatal consequence of rupture. The diagnosis of a AAA is commonly an accidental finding, although there is an increasing number of screening programs targeting high-risk populations. A number of screens demonstrate that the disease prevalence is approximately 5% in men and 1 % in women over 60 years of age. The most feared clinical consequence of AAA progression is acute rupture, which carries a mortality of 80%. Sixty percent of patients with AAAs die of other cardiovascular causes, such as stroke or myocardial infarction, suggesting a relationship between AAAs and atherosclerosis. Currently, the only available treatment option remains surgical repair, with the classic surgical approach being the insertion of an intraluminal graft via open access to the aneurysmal aorta. However, this approach has largely been replaced by endovascular stenting. Besides being not feasible to treat the early stages of the disease, both interventional procedures do carry a potential operative risk, and thus appear only effective in preventing aortic rupture. The characteristic pathology of AAAs is characterized by progressive aortic dilation, promoted by dying vascular smooth muscle cells (SMCs) and limited proliferation, as well as impaired synthesis and degradation of extracellular matrix (ECM) components, which at least partially is the result of transmural inflammation and its disruptive effect on vessel wall homeostasis. With a mortality rate of 30%, stroke is the fourth leading cause of death and also the leading cause of adult disability in Western countries, with ischemic stroke accounting for approximately 85% of all cases. Vulnerable atherosclerotic plaques in the common carotid arteries are considered the most dominant initiator for ischemic forms of stroke (approx. 85% of all strokes). Some of the mechanisms proposed to contribute to unstable carotid atheroma development are identical to the ones being involved in AAA development and rupture, including apoptosis and insufficient proliferation of SMCs within the fibrous cap of advanced atherosclerotic carotid lesions. Again, the most common form of treatment in patients with an ischemic stroke is surgical removal of the affected carotid artery plaque. Thus, here all novel forms of treatments that can stabilise late stage and rupture prone carotid artery plaques are highly desirable.
Multiple research groups have started to uncover the complex genetic and epigenetic machinery necessary to maintain cardiovascular homeostasis. In particular, the key contribution of non-coding RNAs in regulating gene expression has recently received great attention. Using transcriptomic profiling technology on unique diseased human biobank material, we have identified several microRNAs and long non-coding RNAs as novel key regulators of smooth muscle cell survival in the vascular system. We have initiated studies in disease-relevant experimental in vivo models (rodents and LDLR-/- Yucatan mini-pigs) to functionally assess how inhibition of these non-coding RNAs influence aortic aneurysm progression and atherosclerotic plaque vulnerability. To enhance the translational feasibility of our findings, we are utilizing clinically established delivery tools, such as drug eluting stents and balloons, to locally administer our ncRNA-modulators of interest to the arterial wall. Furthermore, we have access to longitudinal cohort studies that have screened and followed-up on individuals at different stages of aortic aneurysm disease, enabling us to investigate the prospective biomarker potential of ncRNAs in recognizing aneurysm growth patterns and patientâ€™s a

Work performed

The biggest translational setback of all current administration forms for antisense oligonucleotides (ASOs) in cardiovascular diseases are reported off-target effects on organ systems in which these modulators assimilate to a much higher extent than the targeted vasculature (e.g., in liver, kidney, spleen). The main focus of our investigations in fighting the pathologies of aneurysm and carotid artery disease have thus shifted towards the development of local delivery tools and techniques for ASOs, taking us away from systemic injections that carry the disadvantage of substantial side effects. To test the feasibility of drug eluting stent/balloon delivery of ASOs we completed a study that proves the efficiency of inhibiting miR-21 in a humanized model of in-stent restenosis (Wang et al, Arterioscler Thromb Vasc Biol 2016). For our current study, have we decided to utilize a novel atherosclerosis and cardiovascular disease-relevant LDLR-/- Yucatan mini-pig model, allowing us to test our therapeutic approach of local, DEB-assisted delivery of ASOs to the pig aorta.
Recently (Li DY et al, Circulation 2018) were we able to show that blocking the long non-coding RNA H19 halts abdominal aortic aneurysm progression through suppression of the transcription factor HIF1a. This was the first ever published study that identifies a functional role for a lncRNA in aneurysm disease development and progression. Other NORVAS-supported studies in our lab revealed that local miR-21 inhibition is feasible by using drug eluting stents to limit the occurrence of in-stent restenosis (Wang H et al, Arterioscler Thromb Vasc Biol 2016; planned Phase I clinical trial in 2019/2020), and inhibition of miR-210 enhances smooth muscle cell survival and plaque stabilization via regulation of the canonical Wnt-pathway (Eken SM et al, Circ Res 2017). Also, we were able to show that triggering local uptake of miR-21 mimics through the support of ultrasound-mediated nanoparticle burst can also stabilize advanced atherosclerotic lesions through inhibition of the transcription factor REST in smooth muscle cells of fibrous caps (Jin H et al, Mol Ther 2018). Further have we sufficiently tested our large animal (mini-pig) model in regards of feasibility to mimic human aneurysm and carotid artery disease.

Final results

By the end of the project we believe that we have identified the role and therapeutic benefit of non-coding RNAs, mainly long non-coding RNAs, in aortic aneurysm and carotid artery disease. We will have tested their potential as novel molecular treatments to limit the burden of these vascular diseases, for which currently very limited forms of treatment exist (apart from surgical intervention). Our novel disease models (preclinical large animal and in vitro aorta-on-a-chip) will be extremely useful tools for drug development in aneurysm disease and late stage atherosclerosis. Utilizing these advanced disease models enables us to test our novel ncRNA-based strategies in a feasible translational setting before entering Phase 1 clinical trials in humans and affected patients. Furthermore are we currently working on the identification of a first suitable biomarker in aortic aneurysm disease to predict future expansion rates and the risk of rupture. This biomarker will also be derived from non-coding RNA transcripts, and will most likely be tissue specific (being exclusively expressed in aortic smooth muscle cells). Confirmation of this biomarker in related longitudinal aortic aneurysm biobanks is currently being assessed.