Pulmonary arterial hypertension: role of miRNAs in animal models and pathological samples.
PhD thesis, University of Glasgow.
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Pulmonary arterial hypertension (PAH) is a disease of the small pulmonary arteries (PAs), characterized by an increase in pulmonary arterial pressure and vascular remodelling leading to a progressive increase in pulmonary vascular resistance. The consequence of vascular obliteration is right heart failure and high mortality. Germline mutations in the gene coding for the bone morphogenetic protein (BMP) type-2 receptor (BMPR2), a receptor for the transforming growth factor (TGF)-beta super-family, have been identified in approximately 70% of patients with the heritable form of PAH (HPAH). Moreover, BMPR2 expression is markedly reduced in PAH cases in the absence of mutations in this gene (idiopathic PAH, IPAH). In pulmonary artery smooth muscle cells (PASMCs) mutations in BMPR2 are associated with an abnormal growth response to BMPs and TGF-beta. In endothelial cells (PAECs), these mutations increase the susceptibility of cells to apoptosis. The absence of BMPR2 mutations in some families and in the majority of IPAH cases suggests that further pathological mechanisms still need to be identified. The serotonin system has also been implicated in both experimental and human PAH. In fact, an additional genetic risk factor for the development of this pathology has been identified in the serotonin transporter (SERT), dysregulated in IPAH patients. Mice over-expressing SERT (SERT+ mice) exhibit PAH and exaggerated hypoxia-induced PAH.
Although different advanced PAH therapies are currently available, they can only provide a symptomatic relief, and mortality rates remain high. Therefore, the identification of novel therapeutic approaches for the treatment of this pathology is urgently required.
MicroRNAs (miRNAs) are a class of small, endogenous and non-coding RNAs able to negatively regulate gene expression by targeting specific messenger RNAs (mRNAs) and inducing their degradation or translational repression. These non-coding sequences are transcribed from endogenous loci as long precursors, converted in single-stranded molecules of approximately 20 nucleotides after a series of enzymatic maturation steps. miRNAs carry out their activity in association with the RNA-induced silencing complex (RISC), interacting with the 3’ untranslated region (3’UTR) of specific target mRNAs which they bind with imperfect complementarity. Several recent studies have assessed the direct role of miRNAs in vascular inflammation and in the development of cardiovascular pathologies. The aim of this project was to investigate the role of miRNAs in the development of PAH.
In Chapter 3, two distinct and well established rat models (hypoxic and monocrotaline) of PAH were used to determine the regulation of miRNAs during disease initiation and progression. We demonstrate time and insult-dependent changes in a specific group if miRNAs and this dysregulation was also confirmed in vitro in rat and human PA cells exposed to chronic hypoxia. Moreover, the stimulation of rat cells with TGF-beta and BMP4 mimicked the alteration of miRNA expression observed in vivo. An analysis of the expression level of the main enzymes involved in miRNA maturation (i.e. Dicer, Drosha, DGCR8 and Exp5) revealed the significant down-regulation of Dicer in response to chronic hypoxia both in vivo and in vitro, suggesting that the manipulation of this enzyme could re-establish a normal miRNA expression level in pathological samples. We also identified selective targets altered in response to miRNA dysregulation, suggesting the possibility of future interventional studies.
In Chapter 4 the specific role of miR-143 and miR-145 in the development of PAH was evaluated. We report the significant up-regulation of these miRNAs in WT mice exposed to chronic hypoxia and that genetic ablation of miR-145 is protective against the development of PAH (with no effects on miR-143 expression), assessed via measurement of systolic right ventricular pressure (sRVP), pulmonary vascular remodelling and right ventricular hypertrophy (RVH). miR-145 KO has also an effect on the expression of specific targets, including kruppel-like factor 4 and 5 (KLF4 and 5), which are regulators of smooth muscle proliferation and differentiation. Further, both miR-143 and miR-145 are up-regulated in mice heterozygous for a BMPR2 mutation. In human tissues we confirm the elevated expression of the miR-143/145 cluster observed in hypoxic mice in pathological samples compared with unaffected controls, suggesting a conserved regulation of these miRNAs in the two species. The study described in this chapter is the first to report a critical role for miR-145 in the development of PAH in vivo.
Finally, in Chapter 5 a preliminary study focused on miR-21 regulation and function on PAH development is shown. An analysis of the expression of this miRNA in WT mice revealed its up-regulation in response to chronic hypoxia, whereas the genetic ablation of miR-21 induced an exaggerated hypoxia-induced PAH phenotype. However, the analysis of human pathological samples showed a reduced expression of this miRNA in comparison with unaffected controls, suggesting its differential regulation in hypoxic mice and patients, although the differences observed between the animal and the human pathology could be the cause of this different phenothipe. The identification of dysregulated targets in both the species will give more informations about the effect of miR-21 alteration in the development of PAH.
In summary, the results presented in this thesis support a role for defined miRNAs in the development of PAH, both in animal models and patients. Whether this specific alteration of selective miRNAs can be used as a novel therapeutical approach still need to be evaluated, but represent an attractive possibility to assess in the longer term.
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