Abstract 309: Osteogenic Induction of p-ERK 1/2 Signaling Associated with NOTCH1 Inactivation in Human Aortic Valvar Interstitial Cells
Introduction Heart valve disease affects over 2% of the population, leading to at least 20,000 deaths and $1 billion in direct costs in the US each year. Calcific aortic valve disease (CAVD) is linked to loss of function mutations in NOTCH1 that result in osteogenic gene induction. In vitro animal studies have established that multiple osteogenic targets are mediated through induction of p-ERK 1/2 signaling; however, the relationship between NOTCH1 and p-ERK has not been examined in primary human aortic valvar interstitial cells (AVICs).
Hypothesis We tested the hypothesis that chemically-induced NOTCH1 suppression results in mineralization due to p-ERK 1/2 activation in human AVICs.
Methods Human AVICs were isolated from CAVD specimens at the time of valve replacement. AVICs were cultured for two weeks in complete media containing DMEM and 10% FBS, and treated with calcific media (10mM b-glycerol phosphate; 60μM ascorbic acid-2-phosphate; 1μM dexamethasone), which has been shown to suppress NOTCH1 signaling. Standard real-time RT-PCR, western blot, and immunocytochemistry were used to compare groups.
Results Treatment with calcific media resulted in a 2.3 fold decrease in the level of NOTCH1 expression and a 3 fold increase in p-ERK 1/2 expression, assessed with both western blots and immunocytochemistry. Interestingly, calcific media also induced a 3.5 fold decrease in VEGF, consistent with early osteogenic differentiation. Additionally, the AVICs treated with calcific media demonstrated overt calcification by Alizarin Red staining.
Conclusions Human AVICs demonstrated calcification associated with p-ERK 1/2 activation and reduced NOTCH1 levels, suggesting p-ERK inhibition may mitigate the effects of NOTCH loss of function, thereby representing a potential pharmacologic therapy for CAVD. Further investigation of the molecular mechanisms linking NOTCH1 and p-ERK are warranted, including studies that specifically inhibit NOTCH1 signaling and define the impact of mechanical forces on molecular pathways, thereby identifying mechanisms underlying CAVD progression.
- © 2013 by American Heart Association, Inc.