- Title
- Extracellular matrix protein 1 as a mediator of inflammation-to-fibrosis progression after myocardial infarction
- Creator
- Hardy, Sean
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2023
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Background: Fibrosis is a hallmark of heart disease, particularly following myocardial infarction (MI) and in heart failure. We previously identified a key role for ECM1 in wound healing post-MI but the precise cellular origin and mechanism of ECM1 remained elusive. Here, we aimed to investigate the spatiotemporal cellular origin of ECM1 in healthy and diseased human and mouse hearts, and ECM1 dependent human cardiac fibroblast (HuCFb) signalling mechanisms. Methods: Immunoblotting, immunohistochemistry and mRNA in-situ hybridisation were conducted on non-failing, ischemic (ICM) and dilated (DCM) failing human heart tissue. In-situ hybridisation, flow cytometry and immunoblotting were conducted on mouse heart and bone marrow samples. ECM1 specific analysis of existing single-cell/-nuclei RNA sequencing (sc/snRNAseq) data was conducted. HuCFbs were treated with recombinant ECM1 and phosphoproteomics mass spectrometry (MS), MS of ECM1-HuCFb cell binding partners, wound healing assay, MTT assay, qPCR, and immunoblotting was conducted. Results: ECM1 expression was upregulated in human ICM and DCM, localised interstitially to fibrotic, inflammatory, and peri-vascular areas. ECM1 originates from fibroblasts, macrophages, and pericytes/vascular cells in uninjured human and mouse hearts. In mouse hearts post-MI, ECM1 predominantly originates from M1 macrophages/monocytes (MΦ/Mo) at day-3, and myofibroblasts and M2MΦ’s at day-7. Further, ECM1 expression was correlated with cell-cell communication, collagen organisation, inflammation, cell adhesion and migration. In vitro, ECM1 inhibited HuCFb migration and CCL2 mRNA expression, and stimulated mRNA expression of inflammatory (IL-6, IL-1β), fibrotic (TGF-β1, Col1a2), and non-canonical Wnt gene Wnt5a. MS experiments showed ECM1 stimulates HuCFb Rho protein, cell-cell adhesion, and chemotactic signalling pathways, potentially initiated by ECM1-CTNND1 protein binding. Finally, we created an ECM1 genetic knockout animal (ECM1Δ2). Homozygous ECM1Δ2 knockout resulted in lower body weight, smaller body size, and a significantly lower rate of survival over the first 16 weeks of life, relative to WT. Conclusions: ECM1 represents a novel mechanism in facilitating inflammation-fibrosis crosstalk post-MI and thus a potential therapeutic target.
- Subject
- cardiology; myocardial infarction; cardiac fibrosis; inflammation; molecular biology; biochemistry
- Identifier
- http://hdl.handle.net/1959.13/1471397
- Identifier
- uon:48668
- Rights
- Copyright 2023 Sean Hardy
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 3 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 731 KB | Adobe Acrobat PDF | View Details Download |