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Beta-Adrenoceptor Activation Reduces Both Dermal Microvascular Endothelial Cell Migration via a cAMP-Dependent Mechanism and Wound Angiogenesis.

O'Leary AP, Fox JM, Pullar CE - J. Cell. Physiol. (2015)

Bottom Line: They play a role in wound repair but their specific role in angiogenesis is unknown.In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic growth factors and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning β-AR-mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. β-AR activation reduced HDMEC migration via cyclic adenosine monophosphate (cAMP)-dependent and protein kinase A (PKA)-independent mechanisms as demonstrated through use of an EPAC agonist that auto-inhibited the cAMP-mediated β-AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective.In more complex environments, β-AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. β-AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, β-AR agonists could be promising anti-angiogenic modulators in skin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.

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Related in: MedlinePlus

cAMP and EPAC-dependent pathways transduce the β-AR-mediated decrease in HDMEC single cell migration. The rate of HDMEC cell migration was determined as described in methods; the rate of migration of an average of N untreated cells or those treated with 10 μM Iso are shown for comparison on each panel and alongside cells treated with 50 μM sp cAMP (sp) (control N = 2219; Iso N = 382; sp cAMP N = 132) (A). HDMECs pre-incubated with the PKA inhibitor rp cAMP (rp; 50 µM) for 1 h before imaging and the addition of either 50 μM rp alone or in combination with 10 μM Iso (control N = 2219; Iso N = 382; rp cAMP N = 70; rp cAMP + Iso N = 81) (B). HDMECs pre-incubated with the EPAC agonist 8-pCPT (10 μM) for 1 h before the addition of 10 μM 8-pCPT alone or in combination with 10 μM Iso (control N = 2219; Iso N = 382; 8-pCPT N = 105; 8-pCPT + Iso N = 103) (C). Data were combined from 3–88 independent experiments using three separate cell strains. The data were averaged, statistically analysed and graphically represented with the bars representing the means ± SEM. (**P < 0.01).
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fig04: cAMP and EPAC-dependent pathways transduce the β-AR-mediated decrease in HDMEC single cell migration. The rate of HDMEC cell migration was determined as described in methods; the rate of migration of an average of N untreated cells or those treated with 10 μM Iso are shown for comparison on each panel and alongside cells treated with 50 μM sp cAMP (sp) (control N = 2219; Iso N = 382; sp cAMP N = 132) (A). HDMECs pre-incubated with the PKA inhibitor rp cAMP (rp; 50 µM) for 1 h before imaging and the addition of either 50 μM rp alone or in combination with 10 μM Iso (control N = 2219; Iso N = 382; rp cAMP N = 70; rp cAMP + Iso N = 81) (B). HDMECs pre-incubated with the EPAC agonist 8-pCPT (10 μM) for 1 h before the addition of 10 μM 8-pCPT alone or in combination with 10 μM Iso (control N = 2219; Iso N = 382; 8-pCPT N = 105; 8-pCPT + Iso N = 103) (C). Data were combined from 3–88 independent experiments using three separate cell strains. The data were averaged, statistically analysed and graphically represented with the bars representing the means ± SEM. (**P < 0.01).

Mentions: HDMECs from three different donors were plated at 3.5 × 103 cells/cm2 on collagen I-coated (30 μg/ml; Cohesion Technologies, Invitrogen) 35 mm plastic cell culture dishes for 2 h at 37 °C. In some experiments, HDMECs were serum-starved for 24 h in ECGM without MGS. HDMECs were incubated with ECGM alone or ECGM containing Iso at time 0 or following a 60 min preincubation. The 35 mm dishes were placed in a heating chamber at 37 °C on a Nikon Eclipse phase contrast microscope. Time-lapse images were taken every 10 min over a 60 min period using a Hamamatsu digital camera under automation via Volocity or Openlab software (v5.0 or v5.5.2, Perkin Elmer, Coventry, UK) and cell tracking was performed with the same software (Pullar et al., 2007). A control experiment was always performed to ensure that the cells were healthy. Cells (15–40) were imaged and tracked from control or treatment experiments with the cell data for each group being pooled from a minimum of four individual experiments prior to analysis. The cell data from all control experiments (88) were pooled to give a large total cell number (2219). The cell data from all experiments for a particular treatment were pooled to give a minimum pooled cell number above 60 (a minimum of 15 cells from each of four experiments). The experiments performed in Figure 2 and Figure 4 were all performed under the same control conditions (no pre-treatments; ECGM alone or treatments added at time 0) and therefore the same pooled control data set was used.


Beta-Adrenoceptor Activation Reduces Both Dermal Microvascular Endothelial Cell Migration via a cAMP-Dependent Mechanism and Wound Angiogenesis.

O'Leary AP, Fox JM, Pullar CE - J. Cell. Physiol. (2015)

cAMP and EPAC-dependent pathways transduce the β-AR-mediated decrease in HDMEC single cell migration. The rate of HDMEC cell migration was determined as described in methods; the rate of migration of an average of N untreated cells or those treated with 10 μM Iso are shown for comparison on each panel and alongside cells treated with 50 μM sp cAMP (sp) (control N = 2219; Iso N = 382; sp cAMP N = 132) (A). HDMECs pre-incubated with the PKA inhibitor rp cAMP (rp; 50 µM) for 1 h before imaging and the addition of either 50 μM rp alone or in combination with 10 μM Iso (control N = 2219; Iso N = 382; rp cAMP N = 70; rp cAMP + Iso N = 81) (B). HDMECs pre-incubated with the EPAC agonist 8-pCPT (10 μM) for 1 h before the addition of 10 μM 8-pCPT alone or in combination with 10 μM Iso (control N = 2219; Iso N = 382; 8-pCPT N = 105; 8-pCPT + Iso N = 103) (C). Data were combined from 3–88 independent experiments using three separate cell strains. The data were averaged, statistically analysed and graphically represented with the bars representing the means ± SEM. (**P < 0.01).
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fig04: cAMP and EPAC-dependent pathways transduce the β-AR-mediated decrease in HDMEC single cell migration. The rate of HDMEC cell migration was determined as described in methods; the rate of migration of an average of N untreated cells or those treated with 10 μM Iso are shown for comparison on each panel and alongside cells treated with 50 μM sp cAMP (sp) (control N = 2219; Iso N = 382; sp cAMP N = 132) (A). HDMECs pre-incubated with the PKA inhibitor rp cAMP (rp; 50 µM) for 1 h before imaging and the addition of either 50 μM rp alone or in combination with 10 μM Iso (control N = 2219; Iso N = 382; rp cAMP N = 70; rp cAMP + Iso N = 81) (B). HDMECs pre-incubated with the EPAC agonist 8-pCPT (10 μM) for 1 h before the addition of 10 μM 8-pCPT alone or in combination with 10 μM Iso (control N = 2219; Iso N = 382; 8-pCPT N = 105; 8-pCPT + Iso N = 103) (C). Data were combined from 3–88 independent experiments using three separate cell strains. The data were averaged, statistically analysed and graphically represented with the bars representing the means ± SEM. (**P < 0.01).
Mentions: HDMECs from three different donors were plated at 3.5 × 103 cells/cm2 on collagen I-coated (30 μg/ml; Cohesion Technologies, Invitrogen) 35 mm plastic cell culture dishes for 2 h at 37 °C. In some experiments, HDMECs were serum-starved for 24 h in ECGM without MGS. HDMECs were incubated with ECGM alone or ECGM containing Iso at time 0 or following a 60 min preincubation. The 35 mm dishes were placed in a heating chamber at 37 °C on a Nikon Eclipse phase contrast microscope. Time-lapse images were taken every 10 min over a 60 min period using a Hamamatsu digital camera under automation via Volocity or Openlab software (v5.0 or v5.5.2, Perkin Elmer, Coventry, UK) and cell tracking was performed with the same software (Pullar et al., 2007). A control experiment was always performed to ensure that the cells were healthy. Cells (15–40) were imaged and tracked from control or treatment experiments with the cell data for each group being pooled from a minimum of four individual experiments prior to analysis. The cell data from all control experiments (88) were pooled to give a large total cell number (2219). The cell data from all experiments for a particular treatment were pooled to give a minimum pooled cell number above 60 (a minimum of 15 cells from each of four experiments). The experiments performed in Figure 2 and Figure 4 were all performed under the same control conditions (no pre-treatments; ECGM alone or treatments added at time 0) and therefore the same pooled control data set was used.

Bottom Line: They play a role in wound repair but their specific role in angiogenesis is unknown.In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic growth factors and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning β-AR-mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. β-AR activation reduced HDMEC migration via cyclic adenosine monophosphate (cAMP)-dependent and protein kinase A (PKA)-independent mechanisms as demonstrated through use of an EPAC agonist that auto-inhibited the cAMP-mediated β-AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective.In more complex environments, β-AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. β-AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, β-AR agonists could be promising anti-angiogenic modulators in skin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.

Show MeSH
Related in: MedlinePlus