Abstract Body (Do not enter title and authors here): Background: Tissue-nonspecific alkaline phosphatase (TNAP) has emerged as a key contributor to vascular calcification. Prior studies have linked elevated plasma TNAP levels to increased coronary artery calcium scores. Our group demonstrated that TNAP overexpression in endothelial cells promotes calcification and accelerates coronary atherosclerosis. In the current study, we hypothesize that plasma TNAP can directly contribute to vascular calcification. We generated a transgenic mouse model with hepatocyte-targeted TNAP overexpression (hTNAPOE) on a low-density lipoprotein receptor mutant background.

Methods: hTNAPOE male and female mice and littermate controls (n=10-12 per group) were fed a western diet to induce atherosclerosis. Cardiac function parameters, along with ascending aortic velocity, were assessed using electrocardiograms. At the terminal time point (42 weeks), the mice were fasted for 5 hours, and plasma was collected by cardiac puncture. Livers and hearts were dissected for histological evaluation. ALP activity was assessed in liver sections to confirm hTNAP overexpression. Plasma alkaline phosphatase (ALP), cholesterol, and triglycerides were measured. Micro-computed tomography (μCT) was employed to detect vascular calcification. Data were analyzed using a two-way ANOVA, accounting for sex and genotype.

Results: Our results demonstrated a 10-fold increase in circulating ALP in the plasma of hTNAPOE female mice and a 100-fold increase in male mice compared to control mice (p<0.0001). There was no significant difference in triglycerides; cholesterol levels were mildly elevated in hTNAPOE mice (36% in males and 14% in females, p<0.05). hTNAPOE mice exhibited significantly greater calcification in whole-heart preparations, confirming our hypothesis (p<0.01). Surprisingly, we didn’t see increased aortic root calcification in hTNAPOE mice; however, coronary artery calcification was significantly increased (p<0.05). hTNAPOE mice presented with increased left ventricular mass normalized to body weight, implying the pathophysiologic significance of calcification. While hTNAPOE mice showed higher calcification, ascending aortic velocity, an indicator of stenosis, did not differ between the groups.

Conclusion: Overexpression of hTNAP in mice resulted in a significant increase in circulating ALP in plasma and increased coronary calcification.