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Myofibroblasts and the Vascular Endothelium: Impact of Fibrin Degradation Products and miRNA on Vascular Motility and Function
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Gerwins)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Angiogenesis is the formation of new blood vessels from pre-existing vasculature and is important during development as well as wound healing and tissue remodeling. Angiogenesis also occurs during pathological conditions such as diabetic retinopathy and cancer. This thesis is centered on the biology of endothelial cells, lining the blood vessels, and myofibroblasts, important for wound healing.

We investigated an endothelial cell specific gene, ExoC3l2, and its role in VEGFR2 signaling and migration. EXOC3L2 co-localize with members of the exocyst complex, involved in vesicular transport, as well as VEGFR2. Reducing the level of EXOC3L2 in microvascular endothelial cells results in reduced VEGFR2 signaling and subsequently reduced chemotactic response to VEGF-A.

MicroRNA (miRNA) have been shown to be regulators of gene transcription and cell type specific miRNAs have been identified. We investigated two miRNAs, miR-145 and miR-24. miR-145 is expressed in pericytes and fibroblasts but was shown to regulate fli1, an endothelial transcription factor. miR-145 overexpression reduced chemotaxis in both fibroblasts and endothelial cells, as did suppression of the endogenous miR-145 level in fibroblasts.

miR-24 in contrast is expressed by endothelial cells and are able to target Ndst1, important for heparan sulfate (HS) sulfation. Sulfation of HS is important for many processes, amongst them growth factor signaling. Overexpression of miR-24 resulted in lower sulfation of HS chains, decreasing the ability of HS to interact with VEGF-A. Overexpressing miR-24 resulted in disturbed chemotaxis, similar to suppressing Ndst1 using siRNA.

Myofibroblast recruitment is an important step in wound healing. The myofibroblasts contract the wound, synthesize new extracellular matrix and contribute to revascularization by looping angiogenesis. Maturation from resting fibroblast to myofibroblast is dependent on TGF-β. We found that fibrin fragment E (FnE), a degradation product of fibrin, potentiated the response of fibroblasts to TGF-β thus enhancing TGF-β-induced myofibroblast differentiation. FnE was also found to influence the migration of fibroblasts. These responses are dependent on integrins and toll-like receptors.

These findings may serve to further increase the understanding of angiogenesis and wound healing to develop new therapies against pathological conditions.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 54 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 878
Keyword [en]
Angiogenesis, Vascular biology, Chemotaxis, Endothelial cell, Myofibroblast, Wound healing, miRNA, Heparan sulfate
National Category
Cell and Molecular Biology Biochemistry and Molecular Biology
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-196884ISBN: 978-91-554-8622-8 (print)OAI: oai:DiVA.org:uu-196884DiVA: diva2:611164
Public defence
2013-05-03, A1:111a, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-04-11 Created: 2013-03-14 Last updated: 2013-08-30Bibliographically approved
List of papers
1. Exocyst Complex Component 3-like 2 (EXOC3L2) Associates with the Exocyst Complex and Mediates Directional Migration of Endothelial Cells
Open this publication in new window or tab >>Exocyst Complex Component 3-like 2 (EXOC3L2) Associates with the Exocyst Complex and Mediates Directional Migration of Endothelial Cells
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2011 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 27, 24189-24199 p.Article in journal (Refereed) Published
Abstract [en]

The exocyst is a protein complex that ensures spatial targeting of exocytotic vesicles to the plasma membrane. We present microarray data obtained from differentiating mouse embryonic stem cell cultures that identify an up-regulation of exocyst complex component 3-like 2 (exoc3l2) mRNA in sprouting blood vessels. Vascular expression of exoc3l2 is confirmed by qPCR analysis of different mouse tissues and immunofluorescence analyses of mouse brain sections. We detect an up-regulation of exoc3l2 mRNA synthesis in primary human endothelial cells in response to VEGFA, and this response is enhanced when the cells are grown on a three-dimensional collagen I matrix. Myc-tagged EXOC3L2 co-precipitates with the exocyst protein EXOC4, and immunofluorescence detection of EXOC3L2 shows partial subcellular colocalization with EXOC4 and EXOC7. Finally, we show that exoc3l2 silencing inhibits VEGF receptor 2 phosphorylation and VEGFA-directed migration of cultured endothelial cells.

Keyword
Angiogenes, VEGF, Cell migration, Chemotaxis, Exocyst, Microarray
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Cell Biology
Identifiers
urn:nbn:se:uu:diva-145508 (URN)10.1074/jbc.M110.212209 (DOI)000292294900061 ()
Available from: 2011-02-09 Created: 2011-02-09 Last updated: 2017-12-11Bibliographically approved
2. Discovery of microvascular miRNAs using public gene expression data: miR-145 is expressed in pericytes and is a regulator of Fli1
Open this publication in new window or tab >>Discovery of microvascular miRNAs using public gene expression data: miR-145 is expressed in pericytes and is a regulator of Fli1
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2009 (English)In: Genome Medicine, ISSN 1756-994X, E-ISSN 1756-994X, Vol. 1, no 11, 108- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND

A function for the microRNA (miRNA) pathway in vascular development and angiogenesis has been firmly established. miRNAs with selective expression in the vasculature are attractive as possible targets in miRNA-based therapies. However, little is known about the expression of miRNAs in microvessels in vivo. Here, we identified candidate microvascular-selective miRNAs by screening public miRNA expression datasets.

METHODS

Bioinformatics predictions of microvascular-selective expression were validated with real-time quantitative reverse transcription PCR on purified microvascular fragments from mouse. Pericyte expression was shown with in situ hybridization on tissue sections. Target sites were identified with 3' UTR luciferase assays, and migration was tested in a microfluid chemotaxis chamber.

RESULTS

miR-145, miR-126, miR-24, and miR-23a were selectively expressed in microvascular fragments isolated from a range of tissues. In situ hybridization and analysis of Pdgfb retention motif mutant mice demonstrated predominant expression of miR-145 in pericytes. We identified the Ets transcription factor Friend leukemia virus integration 1 (Fli1) as a miR-145 target, and showed that elevated levels of miR-145 reduced migration of microvascular cells in response to growth factor gradients in vitro.

CONCLUSIONS

miR-126, miR-24 and miR-23a are selectively expressed in microvascular endothelial cells in vivo, whereas miR-145 is expressed in pericytes. miR-145 targets the hematopoietic transcription factor Fli1 and blocks migration in response to growth factor gradients. Our findings have implications for vascular disease and provide necessary information for future drug design against miRNAs with selective expression in the microvasculature.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-119570 (URN)10.1186/gm108 (DOI)19917099 (PubMedID)
Note

Article 108

Available from: 2010-02-26 Created: 2010-02-26 Last updated: 2017-12-12Bibliographically approved
3. miR-24 suppression of NDST1 reduces endothelial cellresponsiveness to VEGFA
Open this publication in new window or tab >>miR-24 suppression of NDST1 reduces endothelial cellresponsiveness to VEGFA
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-196883 (URN)
Available from: 2013-03-14 Created: 2013-03-14 Last updated: 2013-08-30
4. Fibrin fragment E stimulates fibroblast migration and enhances TGF-β induced myofibroblast differentiation through an integrin β3-dependent activation of Toll-like receptor 4
Open this publication in new window or tab >>Fibrin fragment E stimulates fibroblast migration and enhances TGF-β induced myofibroblast differentiation through an integrin β3-dependent activation of Toll-like receptor 4
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-196882 (URN)
Available from: 2013-03-14 Created: 2013-03-14 Last updated: 2013-08-30

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