Background
: Myosin VI is an actin-based molecular motor that is involved in endocytic trafficking of plasma membrane receptors. Myosin VI has multiple binding partners including the cytoplasmic PDZ protein synectin that has recently been shown to be involved in VEGF signaling and VEGF receptor endocytosis.
Hypothesis
: We assessed the hypothesis that disruption of the myosin VI gene results in altered VEGF signaling and receptor trafficking in endothelial cells (EC), leading to altered arterial morphogenesis.
Methods
: Myosin VI
−/−
mice were used for in vivo and in vitro studies. Primary myosin VI
−/−
(KO) and
+/+
(WT) EC were isolated from mouse heart, lung, inferior vena cava and abdominal aorta by dissection followed by PECAM-1 cell sorting.
Results:
In response to VEGF-A, primary EC treated with an siRNA targeted against myosin VI showed decreased activation of Erk1/2 as determined by Western blotting. Further experiments using myosin VI
−/−
and
+/+
EC confirmed a reduction in the activation of Erk1/2 in response to VEGF in cells lacking myosin VI. Activation of VEGFR-2 in myosin VI
−/−
endothelial cells is also decreased as assessed by diminished phosphorylation of tyrosine 1175 within the cytoplasmic domain of the receptor. To determine whether myosin VI is involved in vascular development and angiogenesis, micro CT analysis of myosin VI
−/−
and
+/+
murine kidney vasculature was performed. Results suggest that myosin VI
−/−
animals have fewer large arterial vessels compared with the WT controls.
Conclusions:
VEGFR-2 activation and downstream signaling is decreased in myosin VI
−/−
endothelial cells in response to VEGF-A. Myosin VI
−/−
mice show altered vasculature structure as assessed by micro CT analysis. Similar perturbations in VEGF signaling and vasculature structure were seen in synectin
−/−
mice and endothelial cells. The decrease in VEGF signaling associated with reduced myosin VI and synectin levels points to a potential role of VEGF receptor trafficking resulting in these defects. Together these results suggest that the interaction of myosin VI and synectin is important in vascular development and angiogenesis.
Delayed Contrast Enhancement Imaging of a Murine Model for Ischemia Reperfusion with Carbon Nanotube Micro-CT