Beta-Adrenoceptor Activation Reduces Both Dermal Microvascular Endothelial Cell Migration via a cAMP-Dependent Mechanism and Wound Angiogenesis.
Bottom Line: Beta-adrenoceptors (β-AR) are G protein-coupled receptors (GPCRs) expressed on all skin cell-types.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.
Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.Show MeSH
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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.
Affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK.