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A time frame permissive for Protein Kinase D2 activity to direct angiogenesis in mouse embryonic stem cells.

Müller M, Schröer J, Azoitei N, Eiseler T, Bergmann W, Köhntop R, Lin Q, Costa IG, Zenke M, Genze F, Weidgang C, Seufferlein T, Liebau S, Kleger A - Sci Rep (2015)

Bottom Line: In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer.Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs.Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine I, Ulm University, Ulm, Germany.

ABSTRACT
The protein kinase D isoenzymes PKD1/2/3 are prominent downstream targets of PKCs (Protein Kinase Cs) and phospholipase D in various biological systems. Recently, we identified PKD isoforms as novel mediators of tumour cell-endothelial cell communication, tumour cell motility and metastasis. Although PKD isoforms have been implicated in physiological/tumour angiogenesis, a role of PKDs during embryonic development, vasculogenesis and angiogenesis still remains elusive. We investigated the role of PKDs in germ layer segregation and subsequent vasculogenesis and angiogenesis using mouse embryonic stem cells (ESCs). We show that mouse ESCs predominantly express PKD2 followed by PKD3 while PKD1 displays negligible levels. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated at the time of germ layer segregation. Time-restricted PKD2-activation limits mesendoderm formation and subsequent cardiovasculogenesis during early differentiation while leading to branching angiogenesis during late differentiation. In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer. Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs. Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination.

No MeSH data available.


Related in: MedlinePlus

PKD2 driven angiogenesis in vivo.Scheme of CAM-assay preparation with EB formation, Dox treatment and EB transplantation. (B) Exemplarily display of Dox-treated and untreated CAMs in the unclosed egg. Black square represents region of transplanted EBs. (C) Immunohistochemistry for the vWF protein (violet) of Dox-treated and untreated CAMs upon transplantation of EBs. (D) Quantification of vWF positive vessels per square under respective conditions on the CAM (for details refer to method section and (A)). CAM Assay was performed n = 2. See details in Material and Methods. (D) shows the quantification of these experiments.
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f7: PKD2 driven angiogenesis in vivo.Scheme of CAM-assay preparation with EB formation, Dox treatment and EB transplantation. (B) Exemplarily display of Dox-treated and untreated CAMs in the unclosed egg. Black square represents region of transplanted EBs. (C) Immunohistochemistry for the vWF protein (violet) of Dox-treated and untreated CAMs upon transplantation of EBs. (D) Quantification of vWF positive vessels per square under respective conditions on the CAM (for details refer to method section and (A)). CAM Assay was performed n = 2. See details in Material and Methods. (D) shows the quantification of these experiments.

Mentions: Finally, we aimed to confirm the basic findings of our data in an in vivo model system. The CAM assay is a widely used method to study angiogenesis in vivo57 and previous studies have already successfully applied this tool to assess EB-derived angiogenesis58. Based on our in vitro observation that PKD2 induction at later time points of differentiation directs angiogenesis from ESCs, we transplanted untreated 4 days old EBs and subsequently triggered the PKD2 overexpression upon ectopic application of doxycycline onto EBs grafted on the CAM (Fig. 7A). After 5 days, a pronounced sprouting of chicken-derived blood vessels into the tumour-like structures of PKD2-induced EBs was observed (Fig. 7B). Moreover, we observed more vessel-like structures inside the differentiating EBs compared to untreated EBs, as demonstrated by enhanced immunoreactivity for the vWF protein (Fig. 7C,D).


A time frame permissive for Protein Kinase D2 activity to direct angiogenesis in mouse embryonic stem cells.

Müller M, Schröer J, Azoitei N, Eiseler T, Bergmann W, Köhntop R, Lin Q, Costa IG, Zenke M, Genze F, Weidgang C, Seufferlein T, Liebau S, Kleger A - Sci Rep (2015)

PKD2 driven angiogenesis in vivo.Scheme of CAM-assay preparation with EB formation, Dox treatment and EB transplantation. (B) Exemplarily display of Dox-treated and untreated CAMs in the unclosed egg. Black square represents region of transplanted EBs. (C) Immunohistochemistry for the vWF protein (violet) of Dox-treated and untreated CAMs upon transplantation of EBs. (D) Quantification of vWF positive vessels per square under respective conditions on the CAM (for details refer to method section and (A)). CAM Assay was performed n = 2. See details in Material and Methods. (D) shows the quantification of these experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493579&req=5

f7: PKD2 driven angiogenesis in vivo.Scheme of CAM-assay preparation with EB formation, Dox treatment and EB transplantation. (B) Exemplarily display of Dox-treated and untreated CAMs in the unclosed egg. Black square represents region of transplanted EBs. (C) Immunohistochemistry for the vWF protein (violet) of Dox-treated and untreated CAMs upon transplantation of EBs. (D) Quantification of vWF positive vessels per square under respective conditions on the CAM (for details refer to method section and (A)). CAM Assay was performed n = 2. See details in Material and Methods. (D) shows the quantification of these experiments.
Mentions: Finally, we aimed to confirm the basic findings of our data in an in vivo model system. The CAM assay is a widely used method to study angiogenesis in vivo57 and previous studies have already successfully applied this tool to assess EB-derived angiogenesis58. Based on our in vitro observation that PKD2 induction at later time points of differentiation directs angiogenesis from ESCs, we transplanted untreated 4 days old EBs and subsequently triggered the PKD2 overexpression upon ectopic application of doxycycline onto EBs grafted on the CAM (Fig. 7A). After 5 days, a pronounced sprouting of chicken-derived blood vessels into the tumour-like structures of PKD2-induced EBs was observed (Fig. 7B). Moreover, we observed more vessel-like structures inside the differentiating EBs compared to untreated EBs, as demonstrated by enhanced immunoreactivity for the vWF protein (Fig. 7C,D).

Bottom Line: In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer.Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs.Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine I, Ulm University, Ulm, Germany.

ABSTRACT
The protein kinase D isoenzymes PKD1/2/3 are prominent downstream targets of PKCs (Protein Kinase Cs) and phospholipase D in various biological systems. Recently, we identified PKD isoforms as novel mediators of tumour cell-endothelial cell communication, tumour cell motility and metastasis. Although PKD isoforms have been implicated in physiological/tumour angiogenesis, a role of PKDs during embryonic development, vasculogenesis and angiogenesis still remains elusive. We investigated the role of PKDs in germ layer segregation and subsequent vasculogenesis and angiogenesis using mouse embryonic stem cells (ESCs). We show that mouse ESCs predominantly express PKD2 followed by PKD3 while PKD1 displays negligible levels. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated at the time of germ layer segregation. Time-restricted PKD2-activation limits mesendoderm formation and subsequent cardiovasculogenesis during early differentiation while leading to branching angiogenesis during late differentiation. In line, PKD2 loss-of-function analyses showed induction of mesendodermal differentiation in expense of the neuroectodermal germ layer. Our in vivo findings demonstrate that embryoid bodies transplanted on chicken chorioallantoic membrane induced an angiogenic response indicating that timed overexpression of PKD2 from day 4 onwards leads to augmented angiogenesis in differentiating ESCs. Taken together, our results describe novel and time-dependent facets of PKD2 during early cell fate determination.

No MeSH data available.


Related in: MedlinePlus