Non-coding RNA-based therapeutics and biomarkers for treatment and detection of vascular disease

Cardiovascular disease (CVD), with atherosclerosis as its main underlying pathology, is the most prominent cause of death worldwide. Progression and rupture of atherosclerotic plaques lead to potential adverse pathological events such as myocardial infarction and stroke. Although largely successful, primary and secondary prevention strategies have thus far been insufficient in minimizing the vast consequences of atherosclerotic disease progression on global health. Abdominal aortic aneurysm (AAA) disease shares a similar risk profile with atherosclerosis. A consequence of undiagnosed AAAs can be their subsequent rupture, which up to 90% of patients will not survive. In both atherosclerosis and AAA, treatment and prevention are complicated by the fact that they progress silently and rarely lead to significant health impacts in their early stages. In addition, different pathological processes are known to be of importance as the diseases progress. These are also affected by patient-specific genetic and environmental risk factors. It would therefore be of benefit to find better ways of stratifying patient-specific disease risk and develop novel treatments. In the past decades, non-coding RNAs have emerged as powerful disease regulators in CVD and have been implicated as disease biomarkers in several research fields. In this thesis, we have sought to: (1) identify novel long non-coding RNAs (lncRNAs) involved in late-stage atherosclerotic disease and AAA, (2) establish techniques of their targeted delivery to affected vasculature, and (3) identify novel microRNA biomarkers of AAA with direct roles in disease development and progression. In study I, we have identified lncRNA MIAT as a novel regulator of vascular smooth muscle cell (VSMC) dynamics in carotid atherosclerotic disease, with positive effects on their survival – a beneficial trait in late-stage disease. Its effects on earlier disease stages were however detrimental through regulation of VSMC phenotypic switching into macrophage-like phenotypes and through regulation of macrophage-specific processes. In study II, we identified the lncRNA NUDT6, the natural antisense transcript of FGF2, to be up-regulated in fibrous caps of vulnerable vs stable plaques. NUDT6 was also up-regulated in AAA vs control aortic tissues. In experimental animal models of atherosclerosis and AAA, FGF2 de-repression by the way of NUDT6 inhibition had a beneficial effect on disease phenotypes and was successful in limiting the progression of these diseases. In studies II and III, we successfully used drug-eluting balloons to deliver therapeutics to the abdominal aorta of the translational mini-pig model of AAA. In addition, in study III, we observed beneficial effects of lenvatinib (VEGF-signaling inhibitor) on experimental AAA disease phenotype through positive effects on VSMC contractility and decreased diameter growth. Finally, in study IV, we identified miR-15a-5p as a novel disease biomarker of AAA. We showed miR-15a-5p to be relevant in AAA pathogenesis through its ability to modulate VSMCs into more inflammatory phenotypes, and its inhibition was able to limit experimental murine AAA diameter growth. In conclusion, our studies not only confirm that non-coding RNAs are promising targets for treatment of CVD, but also underline the translational feasibility of their use

Targeting long non-coding RNA NUDT6 enhances smooth muscle cell survival and limits vascular disease progression

Long non-coding RNAs (lncRNAs) orchestrate various biological processes and regulate the development of cardiovascular diseases. Their potential therapeutic benefit to tackle disease progression has recently been extensively explored. Our study investigates the role of lncRNA Nudix Hydrolase 6 (NUDT6) and its antisense target fibroblast growth factor 2 (FGF2) in two vascular pathologies: abdominal aortic aneurysms (AAA) and carotid artery disease. Using tissue samples from both diseases, we detected a substantial increase of NUDT6, whereas FGF2 was downregulated. Targeting Nudt6 in vivo with antisense oligonucleotides in three murine and one porcine animal model of carotid artery disease and AAA limited disease progression. Restoration of FGF2 upon Nudt6 knockdown improved vessel wall morphology and fibrous cap stability. Overexpression of NUDT6 in vitro impaired smooth muscle cell (SMC) migration, while limiting their proliferation and augmenting apoptosis. By employing RNA pulldown followed by mass spectrometry as well as RNA immunoprecipitation, we identified Cysteine and Glycine Rich Protein 1 (CSRP1) as another direct NUDT6 interaction partner, regulating cell motility and SMC differentiation. Overall, the present study identifies NUDT6 as a well-conserved antisense transcript of FGF2. NUDT6 silencing triggers SMC survival and migration and could serve as a novel RNA-based therapeutic strategy in vascular diseases

The circular RNA Ataxia Telangiectasia Mutated regulates oxidative stress in smooth muscle cells in expanding abdominal aortic aneurysms

An abdominal aortic aneurysm (AAA) is a pathological widening of the aortic wall characterized by loss of smooth muscle cells (SMCs), extracellular matrix degradation, and local inflammation. This condition is often asymptomatic until rupture occurs, leading to high morbidity and mortality rates. Diagnosis is mostly accidental and the only currently available treatment option remains surgical intervention. Circular RNAs (circRNAs) represent a novel class of regulatory non-coding RNAs that originate from backsplicing. Their highly stable loop structure, combined with a remarkable enrichment in body fluids, make circRNAs promising disease biomarkers. We investigated the contribution of circRNAs to AAA pathogenesis and their potential application to improve AAA diagnostics. Gene expression analysis revealed the presence of deregulated circular transcripts stemming from AAA-relevant gene loci. Among these, the circRNA to the Ataxia Telangiectasia Mutated gene (cATM) was upregulated in human AAA specimens, in AAA-derived SMCs, and serum samples collected from aneurysm patients. In primary aortic SMCs, cATM increased upon angiotensin II and doxorubicin stimulation, while its silencing triggered apoptosis. Higher cATM levels made AAA-derived SMCs less vulnerable to oxidative stress, compared with control SMCs. These data suggest that cATM contributes to elicit an adaptive oxidative-stress response in SMCs and provides a reliable AAA disease signature

Chitinase 3 like 1 is a regulator of smooth muscle cell physiology and atherosclerotic lesion stability

AIMS  Atherosclerotic cerebrovascular disease underlies the majority of ischaemic strokes and is a major cause of death and disability. While plaque burden is a predictor of adverse outcomes, plaque vulnerability is increasingly recognized as a driver of lesion rupture and risk for clinical events. Defining the molecular regulators of carotid instability could inform the development of new biomarkers and/or translational targets for at-risk individuals. METHODS AND RESULTS  Using two independent human endarterectomy biobanks, we found that the understudied glycoprotein, chitinase 3 like 1 (CHI3L1), is up-regulated in patients with carotid disease compared to healthy controls. Further, CHI3L1 levels were found to stratify individuals based on symptomatology and histopathological evidence of an unstable fibrous cap. Gain- and loss-of-function studies in cultured human carotid artery smooth muscle cells (SMCs) showed that CHI3L1 prevents a number of maladaptive changes in that cell type, including phenotype switching towards a synthetic and hyperproliferative state. Using two murine models of carotid remodelling and lesion vulnerability, we found that knockdown of Chil1 resulted in larger neointimal lesions comprised by de-differentiated SMCs that failed to invest within and stabilize the fibrous cap. Exploratory mechanistic studies identified alterations in potential downstream regulatory genes, including large tumour suppressor kinase 2 (LATS2), which mediates macrophage marker and inflammatory cytokine expression on SMCs, and may explain how CHI3L1 modulates cellular plasticity. CONCLUSION  CHI3L1 is up-regulated in humans with carotid artery disease and appears to be a strong mediator of plaque vulnerability. Mechanistic studies suggest this change may be a context-dependent adaptive response meant to maintain vascular SMCs in a differentiated state and to prevent rupture of the fibrous cap. Part of this effect may be mediated through downstream suppression of LATS2. Future studies should determine how these changes occur at the molecular level, and whether this gene can be targeted as a novel translational therapy for subjects at risk of stroke

Long Noncoding RNA MIAT Controls Advanced Atherosclerotic Lesion Formation and Plaque Destabilization

BACKGROUND Long noncoding RNAs (lncRNAs) are important regulators of biological processes involved in vascular tissue homeostasis and disease development. The present study assessed the functional contribution of the lncRNA myocardial infarction-associated transcript (MIAT) to atherosclerosis and carotid artery disease. METHODS We profiled differences in RNA transcript expression in patients with advanced carotid artery atherosclerotic lesions from the Biobank of Karolinska Endarterectomies. The lncRNA MIAT was identified as the most upregulated noncoding RNA transcript in carotid plaques compared with nonatherosclerotic control arteries, which was confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. RESULTS Experimental knockdown of MIAT, using site-specific antisense oligonucleotides (LNA-GapmeRs) not only markedly decreased proliferation and migration rates of cultured human carotid artery smooth muscle cells (SMCs) but also increased their apoptosis. MIAT mechanistically regulated SMC proliferation through the EGR1 (Early Growth Response 1)-ELK1 (ETS Transcription Factor ELK1)-ERK (Extracellular Signal-Regulated Kinase) pathway. MIAT is further involved in SMC phenotypic transition to proinflammatory macrophage-like cells through binding to the promoter region of KLF4 and enhancing its transcription. Studies using Miat$^{-/-}$ and Miat$^{-/-}$ApoE$^{-/-}$ mice, and Yucatan LDLR$^{-/-}$ mini-pigs, as well, confirmed the regulatory role of this lncRNA in SMC de- and transdifferentiation and advanced atherosclerotic lesion formation. CONCLUSIONS The lncRNA MIAT is a novel regulator of cellular processes in advanced atherosclerosis that controls proliferation, apoptosis, and phenotypic transition of SMCs, and the proinflammatory properties of macrophages, as well

miR-29b Mediates the Chronic Inflammatory Response in Radiotherapy-Induced Vascular Disease

Summary: As a consequence of the success of present-day cancer treatment, radiotherapy-induced vascular disease is emerging. This disease is caused by chronic inflammatory activation and is likely orchestrated in part by microRNAs. In irradiated versus nonirradiated conduit arteries from patients receiving microvascular free tissue transfer reconstructions, irradiation resulted in down-regulation of miR-29b and up-regulation of miR-146b. miR-29b affected inflammation and adverse wound healing through its targets pentraxin-3 and dipeptidyl-peptidase 4. In vitro and in vivo, we showed that miR-29b overexpression therapy, through inhibition of pentraxin-3 and dipeptidyl-peptidase 4, could dampen the vascular inflammatory response. Key Words: arteriosclerosis, inflammation, microRNA, radiotherap