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Title: Characterisation of a novel nested encoding Laf4ir gene in the cardiovascular system
Author: Ehteramyan, Mazdak
ISNI:       0000 0004 8500 4480
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2019
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Rationale: Recently, a novel nested intronic gene was discovered from the microarray profiling of the laminar flow-upregulated genes in mouse embryonic stem cells (ESCs). This gene is located in the intron 6 of the lymphoid transcription factor gene Laf4/Aff3. Therefore, this novel gene is referred as Laf4 intron resident (Laf4ir). Laf4ir exhibits 7 exons and two transcript variants. Laf4 and Laf4ir utilise opposite DNA strands for transcription. Laf4ir mRNA sequence contains a few potential open reading frames (ORFs) for the translation of polypeptides. Objective: In the present study, I aimed to verify whether Laf4ir is an encoding gene, explore the expression profile of Laf4ir, potential functions and its underlying mechanisms. I also aimed to investigate the role of Laf4ir in cardiovascular remodelling and utilise the global knockout mouse model to further understand the functions of Laf4ir. Methods and results: Mass spectrometry detected multiple peptides associated with two ORF polypeptides from the Laf4ir gene, demonstrating that Laf4ir is an encoding gene. Laf4ir ORF1 and Laf4ir ORF2 polypeptide expression was found in various adult organs, different stages of embryonic development, different cell types and different subcellular localisations. Immunoprecipitation and immunofluorescence staining revealed the physical association of Laf4ir ORF1 polypeptide with LAF4 protein suggesting the potential co-ordinated regulation of the parent and nested genes. Overexpression of Laf4ir ORF2 via adenoviral gene transfer could enhance laminar flow- and VEGF-induced endothelial cell (EC) differentiation and reduce cell proliferation. This could be due to cell cycle arrest via the retention of mini- chromosome maintenance protein 3 (MCM3) in the cytosol and as a consequence assist differentiation towards EC lineage. Overexpression of Laf4ir ORF2 also promoted endothelial survival under oxidative stress induced by hydrogen peroxide. To investigate the potential contribution of Laf4ir in cardiovascular repair and pathology, several different cardiovascular remodelling in-vivo models were conducted. Results indicated the upregulation of Laf4ir ORF2 polypeptide in transverse aortic constriction-mediated pressure overload in the heart, and femoral artery wall following vascular injury and ischaemia. An alteration in distribution of Laf4ir ORF2 expression was also observed in the aorta from ApoE-/- mice. To explore the functionality of Laf4ir further, a Cre-loxP in-vivo global knockout model was developed and utilised. A significant reduction in blood perfusion recovery was detected in Laf4ir heterozygous mice when compared to wild type mice at day 14-post surgery of hindlimb ischaemia. This suggests the potential contribution of Laf4ir in vascular repair. Phenotypic examination of Laf4ir knockout mice displayed a reduction in body size, an enlargement in heart size, malocclusion, abnormal arch posture and abnormal hindlimb vessel growth. Sca1+ adventitia cells isolation from transgenic mice exhibited an increase in proliferation, further supporting the inhibitory role of Laf4ir in proliferation. Conclusion: Overall, the novel nested Laf4ir gene may contribute to cardiovascular remodelling via spatiotemporal translation of different ORFs. Further detailed investigation on Laf4ir will undoubtedly shed new insights into cardiovascular biology and pathology. This study could also potentially encourage new discoveries for other nested mammalian genes with diverse functions.
Supervisor: Zeng, Lingfang ; Hu, Yanhua Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available