Abstract: TH-PO545
Extracellular Matrix Stiffness Modulates Calcification of Vascular Smooth Muscle Cells via Phosphate Uptake
Session Information
- Bone and Mineral Metabolism: Basic
November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Bone and Mineral Metabolism
- 401 Bone and Mineral Metabolism: Basic
Authors
- Liu, I-Chia, University of Florida, Gainesville, Florida, United States
- To, Brandon, University of Florida, Gainesville, Florida, United States
- Li, Shiyu, University of Florida, Gainesville, Florida, United States
- Mohandas, Rajesh, University of Florida, Gainesville, Florida, United States
Background
In patients with chronic kidney disease (CKD), stiffening and calcification of blood vessels are common and predict mortality and adverse cardiovascular events. Our preliminary data suggests that stiffening occurs early in CKD and could be independent of calcification. Stiffness of the extracellular matrix (ECM) has also been shown to influence differentiation of pluripotent stem cells. Thus, we hypothesize that stiffness of the ECM will increase phosphate mediated osteoblastic transformation and calcification of vascular smooth muscle cells.
Methods
Human aortic smooth muscle cells (HASMCs) were plated on polyacrylamide gels of varying stiffness: 8kPa, 50kPa, or plastic (~10,000kPa). Cells were cultured in either control or calcification medium (3.0mM phosphate and 2.7mM calcium). After 7 days in culture, alkaline phosphatase activity (ALP), intracellular phosphate and calcium content in cells were measured and normalized to total protein content.
Results
There was a 4-fold increase in calcium in HASMCs plated on 8kPa (243 ± 124 μg/mg of protein) compared to those on 50kPa (954 ± 59 μg/mg of protein, P<0.05) and a 9-fold increase from 50kPa to plastic (8246 ± 1324 μg/mg of protein, P<0.05) (Fig 1). HASMCs on plastic had a near 2-fold increase in ALP activity when cultured in calcification medium (0.58 ± 0.12 vs 0.33 ± 0.05 mU/µg of protein, P<0.05), while cells on 8kPa (0.36 ± 0.02 vs 0.35 ± 0.04 mU/µg of protein, P>0.05) and 50kPa (0.33 ± 0.03 vs 0.39 ± 0.05 mU/µg of protein, P>0.05) showed no appreciable increase in ALP activity. Similar to ALP data, phosphate uptake was increased in cells plated on plastic (2.21-fold increase, p<0.05) but not for the 8kPa and 50kPa gels in calcification media (Fig 2).
Conclusion
Our study showed that ECM stiffness increases calcification of HASMC. Osteoblastic transformation was abolished on soft gels. The decrease in osteoblastic transformation appears secondary to downregulation of phosphate uptake. Targeting molecular pathways that mediate stiffness induced upregulation of phosphate uptake might decrease calcification and improve cardiovascular outcomes in CKD.
Funding
- Other NIH Support