BONE MORPHOGENETIC PROTEINS: NOVEL MEDIATORS OF ATHEROTHROMBOSIS

Following the specific aims and plan of the project for 2013 we found an interesting correlation between preoperative and postoperative TF levels as well as an inverse correlation between TF and heart failure severity. The PhD presented these data as an oral poster presentation on the XXIV Congress of The International Society on Thrombosis and Haemostasis (ISTH) in Amsterdam in June 2013. During 2013 we also assessed our data on the levels of soluble BMPs in the blood of individuals with asymptomatic carotid atherosclerosis. The resulting data was also presented on the XXIV ISTH Congress in June 2013. Further, we obtained findings pointing to the novel role of BMP7 and its antagonists Noggin in monocyte motility. Pilot experiments were performed at University of Tromsø and later confirmed by stipendiate and collaborators at the laboratory of Dr. V. Bogdanov, University of Cincinnati. The data were accepted for publication in 2016 in Thrombosis Research journal. During 2014 we further extended our work on BMPs and their role in monocyte migration/adhesion. Following the initial monocyte motility experiments, the PhD stipendiate obtained data that strongly indicates that BMP-6 and -7 significantly increase the rate of monocyte transmigration through endothelial monolayers. The PhD learned techniques vital to study cell adhesion, cell migration, and techniques for scrutinizing protein interactions in the cell. Data, obtained by the PhD during his visit to Dr. Bogdanovs' lab at the University of Cincinnati for 8 months in 2014, provided a strong basis for two resulting publications in 2015 and 2016. Following our progress plan, we continued to further study histological characteristics of the advanced atherosclerotic lesions in 2013/14. This part of the study was based on the collection of calcified and lipid-rich advanced atherosclerotic plaques from human patients collected by our collaborator Dr. J.T. Fallon of Mount Sinai School of Medicine / New York medical College. This work was done at University of Tromsø in collaboration with prof. B. Sveinbjørnsson?s research group. We found that macrophages are located in the same areas of atherosclerotic plaques rich in BMP6 and BMP7. BMP7 also present in high quantities in the vascular endothelium, supporting our findings on its role in monocyte extravasation into plaques. In 2015 the PhD stipendiate has re-established relevant experimental routines such as Boyden chamber assay, orbital shear assay and static adherence assay at the University of Tromsø laboratory, as well as performed additional studies concerning the signaling mechanisms, regulating BMP7-induced monocyte migration. The signaling data was proved by the application of selective protein kinase inhibitors. Data on monocyte migration was presented as oral communication at the ISTH Congress in Toronto in June 2015, published as an abstract at the meeting. Together with the data on the use BMP signaling inhibitors Noggin and Dorsomorphin, these data were summarized as an article accepted for publication in Thrombosis Research journal. Proceeding with our data on the role of BMPs in macrophage polarization, a model for monocyte macrophage differentiation was developed by the PhD student and Next Generation RNA sequencing was performed at UoC Childrens Hospital. The results identify BMP6 and BMP7 as potential players in regulation of the macrophage phenotype. However, we were not able to verify RNA-seq data by the conventional RT-PCR. Taken together, the data of the current project financed by The Research Council of Norway, resulted in two articles in peer-reviewed journal, three abstracts, two posters, as well as two oral presentations on international scientific meetings.

Bone morphogenetic proteins (BMPs) are newly discovered potent regulators of vascular calcification. Our own data support the role of BMPs in regulation of plaque thrombogenicity. This translational project is focused on characterization and pharmacologic al attenuation of BMPs, novel mediators of plaque thrombogenicity in a population of patients with asymptomatic and advanced atherosclerosis. During step I of this project, in a cross-sectional population-based study, we will measure plasma levels of seve ral BMPs and markers of blood thrombogenicity (soluble and monocyte tissue factor [TF], and tissue factor pathway inhibitor [TFPI]) in the groups of asymptomatic patients with early atherosclerosis. The patients with lipid-rich or calcified carotid plaque s were already enrolled in Tromsø IV health study. The association between plaque burden, BMPs, and markers of blood thrombogenicity will be found. During step II, in a cross-sectional study, we will measure BMP, TF, and TFPI protein levels in human surgi cal carotid endarterectomy material from patients with advanced atherosclerosis. The levels of BMPs and thrombotic markers within the plaque will be correlated with the degree of plaque calcification and plasma concentrations of BMPs, TF, and TFPI. During step III, the effects of BMP family members on TF/TFPI expression will be tested in freshly isolated or cryopreserved monocytes collected from healthy individuals and from patients enrolled to a step I of this study. BMP-dependent TF/TFPI regulation will be studied by activation of transcriptional regulators Smads, Jun, and NF-kB. The role of noggin and gremlin, endogenous BMP antagonists, in targeting TF/TFPI system will be tested. In the present application, we are seeking for the support of a doctoral stipendiate who will work on finding the relationship between increased atherosclerotic plaque thrombogenicity and BMPs in a population of patients with asymptomatic or advanced atherosclerosis.