Abstract
AbstractAbstract 4316We recently demonstrated that CADM1, interacting with protein C deficiency, is a novel risk factor for venous thrombosis (VT) Blood 114:3084-3091, 2009. CADM1 likely plays a role in maintaining endothelial barrier function. Prior to this study CADM1 had not been identified in endothelial cells. In this study we have determined the distribution of CADM1 in human vasculature. Methods:Human tissue samples representing all organs as well as large vessels were accessioned from archived paraffin blocks from the surgical pathology division of FAHC. Tissue sections were processed for immunoflourescence using as primary antibodies a chicken monoclonal anti-CADM1 IgY antibody (anti-SynCAM/TSLC1 clone 3E1, MBL Inc, Woburn, MA) and anti-smooth muscle actin (SMA) clone 1A4 (Sigma, St. Louis, MO), anti-von Willebrand factor (vWF) rabbit polyclonal antibody (DAKO Inc., Glostrup, DK). vWF staining was used to identify endothelial cells. The secondary antibodies for CADM1, SMA, and vWF were donkey anti chicken alexa 488, donkey anti mouse alexa 647, and donkey anti rabbit alexa 647, respectively. Appropriate negative and positive controls were run. Intensity of staining was rated as absent, trace or present by three observers (TK, DJT, and EGB). Immunoelectron microscopy (IEM) was performed on cultured human umbilical vein endothelial cells (Allcells LLC, Emeryville, CA) using a post embedding procedure with protein-A gold. Peripheral blood leukocytes were evaluated for the presence of CADM1 using the same antibody following cytospin preparation. ResultsCADM1 was found ubiquitously in the macro- and micro-vasculature of all organs as well as the aorta and saphenous vein with intensity of staining showing modest variability from organ to organ. CADM1 staining of SMA was observed in both arterial and venous vessels but was considerably stronger on the arterial side. IEM demonstrated cytoplasmic immunogold staining associated with elements of rough endoplasmic reticulum and actin filaments as well as at the membranes of filopodia. In contrast, peripheral blood monocytes, lymphocytes and granulocytes were negative for CADM1expression. Conclusions:CADM1 is expressed ubiquitously in endothelial and smooth muscle cells of the macro-and micro-vasculature with IEM evidence for its presence in filopodial cytoplasm and membranes. Peripheral blood leukocytes did not show evidence of CADM1 expression. This is the first report of CADM1 positivity in vascular smooth muscle cells. The biological role of CADM1 remains to be determined but its ubiquitous expression in the endothelium of the macro- and micro-circulation, together with an apparent role in endothelial cell motility, suggests an important role in endothelial homeostasis. Disclosures:Bovill:Haemotologic Technologies, Essex Jct., VT: Equity Ownership.
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