University of Vermont

College of Medicine

Department of Pathology and Laboratory Medicine

bovill_lab

Bovill Lab Research

Blood Magazine Cover

Dr Bovill's research is focused on genotypic and phenotypic investigation of the pathogenesis of venous thrombosis.

Gene Discovery in Venous Thrombophilia
The Bovill laboratory is focused on improving understanding of the pathogenesis of Venous Thromboembolic disease. A major project in the laboratory has involved applying modern genomic tools and genetic epidemiology 52to a large, protein C deficient, thrombophilic family of French Canadian Origin who reside in Vermont and Quebec (n=800). The protein C mutation (3363InsC) increases the risk of thrombosis but did not fully account for the pattern of inheritance. Using successive STR and SNP genotyping technologies we were able to identify a probable candidate gene in the 11q23 region as well as two other loci. Subsequent exonic resequencing of 109 genes in these regions identified the 300kb, Cell Adhesion Molecule 1 gene (CADM1) gene at 11q23 as the putative causative variant. We then resequenced the entire CADM1 gene as well as 1.5 mega-bases up and downstream, which narrowed the area of interest to 25kb within the gene. No exonic variants were identified; however the intronic regions within the 25 kb segment appeared rich in methylated histone binding sites suggesting there may be a regulatory variant. We are currently using a combination of Sanger and Ilumina HiSeq1000 resequencing to identify indels and/or copy number variants in this region.

The CADM1 protein is a member of the Immunoglobulin Super Family and in epidermally derived cells appears to play a role in cell-cell adhesion and has been described as a tumor suppressor gene in various epithelial cancers. CADM1 had not been identified in endothelial cells prior our 2009 publication (Blood 114:3084-3090, 2009). We were able show that endothelial cells cultured from whole blood in affected compared with unaffected family members demonstrated decreased expression in affected members. In addition, whereas confluent epidermally derived cells exhibit CADM1 at cell-cell junctions, in confluent endothelial cells CADM1 is located in the cytoplasm. By contrast, migrating endothelial cells exhibited localization of CADM1 in the cytoplasm and membranes of filopodia and lammelopodia suggesting a role in cell motility. Both Activated Protein C and CADM1 affect the actin cytoskeleton through RAC1. See Blood cover art from this work.

These observations support an hypothesis that in the presence of protein C deficiency, abnormal or decreased CADM1, could lead to decreased maintenance of endothelial barrier function with an associated increased risk of venous thrombosis. We are presently focused on the phenotypic characterization of the role of CADM1 in the endothelium. We have demonstrated the presence of CADM1 in human umbilical vein lysates by immunoprecipitation at a lower molecular weight than that observed in epithelium suggesting a unique CADM1 variant in endothelium. We are currently characterizing the endothelial protein and its associated intracellular pathways and its effect on endothelial barrier function. In addition we are the levels of CADM1 in the plasma of affected and unaffected family members. The vascular wall plays a major role in thrombogenesis but little is known about specific mechanisms. CADM1 would appears likely to be an important and novel risk factor for venous thrombosis that may shed considerable light on venous thrombogenesis.

The Venous Endothelial Microenvironment
A related line of investigation pursued by the laboratory is the characterization of the venous valvular sinus endothelium. The venous valvular sinus experiences periodic blood flow stasis which on occasion can last for hours (eg during sleep). The pO2 at the base of the sinus has been shown to drop to near 0 after 2 hours in animal models. We hypothesized that the venous valvular sinus endothelium would have a phenotype characterized by a coagulant/anticoagulant balance tilted in favor of anticoagulant activity when compared to the non valvular endothelium. In a study of human venous valvular sinus versus non-valvular sinus endothelium from saphenous veins collected from coronary bypass surgery we demonstrated that the endothelium lining the valvular sinus had increased expression of the anticoagulants, thrombomodulin and endothelial protein C receptor, compared to the procoagulant von Willebrand factor (Blood 114:12761279. 2009). A subsequent study demonstrated a marked inter-individual variability in the relative degree of expression of these proteins suggesting that this variability may play a role in venous thrombosis risk (Histochemistry and Cell Biology 135:141-152, 2011). The laboratory is presently further characterizing the venous vascular wall with respect to intimal changes that occur in association with aging, as aging is a poorly understood but major risk factor for venous thrombosis.

Bovill Lab Graphic Fig. 4A: Split and merge confocal images of valve. DAPI (blue) is a fluorescent dye that binds to DNA and thus serves as a general nuclear marker. Von Willebrand Factor protein expression (white) is upregulated in lumenal epithelium, Enodthelial Protein C Receptor (red) amd Thrombomodulin (green) protein expression appear upregulated in valve sinus as opposed to non-sinus epithelium. Note that in confocal miscroscopy, the overlap of red and green fluorphores is perceived as yellow.

Last modified June 11 2012 01:17 PM