Reference number : GC-2017-Q1-02582, amendment number : , agreement type : G, recipient legal name : Schwertani, Adel (McGill University)|Schwertani, Adel (Université McGill), recipient country : CA, recipient province : nan, recipient city : Quebec|Québec, recipient postal code : nan, program name english : Discovery Grants Program - Individual, program name french : Programme de subventions à la découverte - individuelles, program purpose english : Overall aims: The general aim of the current proposal is to determine mechanism of the human aortic valve interstitial cell (HAVIC) mineralization in relation to increases in lipoprotein(a)[Lp(a)] and oxidized phospholipids (OxPLs). 
Background and Rationale: Although historically HAVIC mineralization (calcification) thought to be a degenerative process, recent studies have pointed to other possible causes such as shear-stress, angiotensin system and hyperlipidemia. Lp(a) is composed of the glycoprotein apolipoprotein (a) [apo(a)] attached to apolipoprotein B-100, found in a particle highly similar to low density lipoprotein (LDL). Lp(a) also binds to OxPLs in plasma and up taken in tissue by scavenger receptors. Studies utilizing transgenic mice have supported a relationship between Lp(a) serum levels and mineralization of blood vessels. Although these studies point to a possible relationship between Lp(a) and vascular stiffness, only recent genetic population studies have pointed to an a potentially important role for Lp(a) in aortic valve calcification. How Lp(a) induces HAVICs mineralization, and the mechanisms involved remains to be determined. 
General Hypothesis: Lp(a) is uptaken by HAVICs by endocytosis or receptor binding, and subsequently decreasing cholesterol efflux and inducing mineralization and apoptosis through increased matrix vesicles (MVs) formation and reactive oxygen species (ROS). Interfering with Lp(a) and/or downstream signalling pathway would ameliorate HAVICs mineralization. 
Experimental design: We plan to determine the mechanism of: 
1) Lp(a) uptake and/or binding in HAVICs? HAVICs express ABCA1, LDL-R, SRB-1 and LRP5/6. Here, we shall assess Lp(a) catabolism and cholesterol efflux in HAVICs. 
2) Lp(a) induced HAVICs mineralization? When HAVICs undergo mineralization they produce a large number of calcium-phosphate containing matrix vesicles (MVs). Our preliminary data showed that treatment of HAVICs with Lp(a) significantly increased the number of MVs and expression of proteins and genes involved in the biogenesis of MVs accompanied by a significant reduction in Fetuin-A. Here, we plan to isolate and characterize these MVs using ultracentrifugation, NanoSight, flow cytometry and electron microscopy. We also plan to identify the functional profiles of these MVs using quantitative proteomics in order to interfere with the pathways of downstream mediators of Lp(a)-induced effects in HAVICs. 
3) Lp(a)-induced HAVICs apoptosis. Here, we plan to determine the levels of ROS and monitor mitochondrial transmembrane potential in response to Lp(a). 
4) In vivo effects of Lp(a) overexpression/inhibition and the mechanism involved. 
 
In the short term, the proposed work will determine the mechanism of HAVICs mineralization, and in the long term serves as an excellent platform for the training of graduate students and post-doctoral fellows., program purpose french : Overall aims: The general aim of the current proposal is to determine mechanism of the human aortic valve interstitial cell (HAVIC) mineralization in relation to increases in lipoprotein(a)[Lp(a)] and oxidized phospholipids (OxPLs). 
Background and Rationale: Although historically HAVIC mineralization (calcification) thought to be a degenerative process, recent studies have pointed to other possible causes such as shear-stress, angiotensin system and hyperlipidemia. Lp(a) is composed of the glycoprotein apolipoprotein (a) [apo(a)] attached to apolipoprotein B-100, found in a particle highly similar to low density lipoprotein (LDL). Lp(a) also binds to OxPLs in plasma and up taken in tissue by scavenger receptors. Studies utilizing transgenic mice have supported a relationship between Lp(a) serum levels and mineralization of blood vessels. Although these studies point to a possible relationship between Lp(a) and vascular stiffness, only recent genetic population studies have pointed to an a potentially important role for Lp(a) in aortic valve calcification. How Lp(a) induces HAVICs mineralization, and the mechanisms involved remains to be determined. 
General Hypothesis: Lp(a) is uptaken by HAVICs by endocytosis or receptor binding, and subsequently decreasing cholesterol efflux and inducing mineralization and apoptosis through increased matrix vesicles (MVs) formation and reactive oxygen species (ROS). Interfering with Lp(a) and/or downstream signalling pathway would ameliorate HAVICs mineralization. 
Experimental design: We plan to determine the mechanism of: 
1) Lp(a) uptake and/or binding in HAVICs? HAVICs express ABCA1, LDL-R, SRB-1 and LRP5/6. Here, we shall assess Lp(a) catabolism and cholesterol efflux in HAVICs. 
2) Lp(a) induced HAVICs mineralization? When HAVICs undergo mineralization they produce a large number of calcium-phosphate containing matrix vesicles (MVs). Our preliminary data showed that treatment of HAVICs with Lp(a) significantly increased the number of MVs and expression of proteins and genes involved in the biogenesis of MVs accompanied by a significant reduction in Fetuin-A. Here, we plan to isolate and characterize these MVs using ultracentrifugation, NanoSight, flow cytometry and electron microscopy. We also plan to identify the functional profiles of these MVs using quantitative proteomics in order to interfere with the pathways of downstream mediators of Lp(a)-induced effects in HAVICs. 
3) Lp(a)-induced HAVICs apoptosis. Here, we plan to determine the levels of ROS and monitor mitochondrial transmembrane potential in response to Lp(a). 
4) In vivo effects of Lp(a) overexpression/inhibition and the mechanism involved. 
 
In the short term, the proposed work will determine the mechanism of HAVICs mineralization, and in the long term serves as an excellent platform for the training of graduate students and post-doctoral fellows., agreement title english : Mechanism of human valve interstitial cell calcification, agreement title french : Mechanism of human valve interstitial cell calcification, agreement number : RGPIN, agreement value : 125000.0, agreement start date : 2017-05-10, agreement end date : nan, description english : nan, description french : nan, expected results english : nan, expected results french : nan, additional information english : Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023), additional information french : Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2022-2023), owner organization : nserc-crsng, owner organization title : Natural Sciences and Engineering Research Council of Canada | Conseil de recherches en sciences naturelles et en génie du Canada