uu.seUppsala University Publications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Exocyst Complex Component 3-like 2 (EXOC3L2) Associates with the Exocyst Complex and Mediates Directional Migration of Endothelial Cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Show others and affiliations
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.

Place, publisher, year, edition, pages
2011. Vol. 286, no 27, 24189-24199 p.
Keyword [en]
Angiogenes, VEGF, Cell migration, Chemotaxis, Exocyst, Microarray
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-145508DOI: 10.1074/jbc.M110.212209ISI: 000292294900061OAI: oai:DiVA.org:uu-145508DiVA: diva2:396299
Available from: 2011-02-09 Created: 2011-02-09 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Directing Angiogenesis: Cellular Responses to Gradients in vitro
Open this publication in new window or tab >>Directing Angiogenesis: Cellular Responses to Gradients in vitro
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Blood vessels are essential for the delivery of nutrients and oxygen to tissues, as well as for the removal of waste products. Patients with tumors, wounds or diabetes all have active angiogenesis, formation and remodeling of blood vessels, a process that is initiated and manipulated by gradients of secreted signaling proteins.

This thesis describes the development of new microfluidic in vitro assays where directed migration of single endothelial cells and three dimensional vascular structures can be monitored in real time. Combining these assays with live imaging microscopy we have studied the behavior of endothelial cells in gradients of proangiogenic factors as well as directed sprouting in embryonic kidneys and stem cell cultures.

With the 2D assay we have quantified endothelial cell chemotaxis towards FGF2, VEGFA165 and VEGFA121 and we also demonstrate that constant levels of VEGFA165, but not of FGF2, are able to reduce chemokinesis of endothelial cells.

In the 3D migration chamber we have studied directed endothelial cell sprouting in mouse embryonic kidneys and embryoid bodies in response to VEGFA gradients. In both models directed angiogenesis is detected towards increasing levels of growth factor.

Using the microarray technique on differentiating embryonic stem cells we have been able to identify the gene exoc3l2 as potentially involved in angiogenesis and endothelial cell migration and we present evidence that ExoC3l2 is associated with the exocyst complex; an important regulator of cell polarity. We have also shown that siRNA mediated gene silencing of exoc3l2 results in impaired VEGFR2 phosphorylation as well as loss of directionality in response to a VEGFA gradient.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 52 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 643
Keyword
Angiogenesis, Endothelial cell, Cell migration, Chemotaxis, Gradients, Microfluidics, VEGFA, VEGFR2, Exocyst, Exocytosis
National Category
Biochemistry and Molecular Biology
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:uu:diva-145525 (URN)978-91-554-8006-6 (ISBN)
Public defence
2011-04-15, B22, BMC, Husargatan 3, 74123, Uppsala, 09:15 (English)
Opponent
Supervisors
Note
(Faculty of Medicine)Available from: 2011-03-08 Created: 2011-02-09 Last updated: 2011-05-04
2. Myofibroblasts and the Vascular Endothelium: Impact of Fibrin Degradation Products and miRNA on Vascular Motility and Function
Open this publication in new window or tab >>Myofibroblasts and the Vascular Endothelium: Impact of Fibrin Degradation Products and miRNA on Vascular Motility and Function
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
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:nbn:se:uu:diva-196884 (URN)978-91-554-8622-8 (ISBN)
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
3. Identification and Characterization of Proteins and MicroRNAs that Modulate Receptor Signaling, Vesicular Trafficking and Cell Migration in Vascular Cells
Open this publication in new window or tab >>Identification and Characterization of Proteins and MicroRNAs that Modulate Receptor Signaling, Vesicular Trafficking and Cell Migration in Vascular Cells
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Blood vessels deliver nutrients and oxygen to tissues. Importantly, the functions and growth of blood vessels are commonly altered in disease. The inside of all blood vessels are lined with endothelium, a thin specialized layer of endothelial cells that separate the blood from other tissues. This thesis deals with the identification and functional characterization of proteins and microRNAs that have key roles as modulators of growth factor signaling and directed cell migration of endothelial cells and other vascular cells.

A previously uncharacterized protein of the exocyst complex, Exocyst complex component 3-like 2 (ExoC3L2) was identified and shown to be highly expressed in endothelial cells of sprouting vessels. Suppression of ExoC3L2 resulted in reduced VEGF-A signaling together with reduced chemotaxis in response to VEGF-A gradients. VEGF-A-signaling via its receptor VEGFR-2 is thus modulated by the exocyst complex and ExoC3L2.

Expression profiling of highly vascularized tissues were used to identify several microRNAs selectively expressed in blood vessels. miR-145, targeting the transcription factor Fli1, was shown to be expressed in pericytes and mural cells. Elevated levels of miR-145 reduced chemotaxis of both endothelial cells and fibroblasts in response to growth factor gradients. miR-145 depletion in fibroblasts was shown to inhibit chemotaxis in response to PDGF-BB.

The guanine nucleotide exchange factor FGD5 was shown to be selectively expressed in endothelial cells and to regulate Cdc42 activity. FGD5 was shown to regulate the turnover of activated VEGF-receptors. Suppression of FGD5 impaired endothelial cell chemotaxis, suggesting that FGD5 is required for efficient and sustained VEGF-A signaling.

Inactivation of RhoD, a regulator of endosomal trafficking, resulted in an increased pool of acetylated and stable microtubules. Knockdown of RhoD in human fibroblasts resulted in a loss of cell polarity. A link between PDGFR-β and RhoD was implicated by the finding that PDGF-BB was shown to trigger formation of GTP-bound RhoD. Chemotaxis towards PDGF-BB was severely inhibited in cells with reduced RhoD expression, suggesting a role for RhoD in chemotaxis via its regulation of microtubule dynamics.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 961
Keyword
Angiogenesis, cell migration, signaling, VEGF, FGD5, ExoC3L2, microRNA, RhoD
National Category
Cell and Molecular Biology Other Basic Medicine
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-212949 (URN)978-91-554-8833-8 (ISBN)
Public defence
2014-02-14, A1:111a, Husargatan 3, BMC, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2014-01-23 Created: 2013-12-16 Last updated: 2014-02-10

Open Access in DiVA

No full text

Other links

Publisher's full text

Authority records BETA

Barkefors, IrmeliFredlund Fuchs, PederHeldin, JohanBergström, TobiasForsberg-Nilsson, KarinKreuger, Johan

Search in DiVA

By author/editor
Barkefors, IrmeliFredlund Fuchs, PederHeldin, JohanBergström, TobiasForsberg-Nilsson, KarinKreuger, Johan
By organisation
Department of Medical Biochemistry and MicrobiologyScience for Life Laboratory, SciLifeLabDepartment of Genetics and Pathology
In the same journal
Journal of Biological Chemistry
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 1314 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf