Lyso-phosphatidylcholine induces osteogenic gene expression and phenotype in vascular smooth muscle cells

Abstract

Objective: Calcifying vascular cells in human atherosclerotic plaques actively contribute to ectopic vascular mineralization. Lyso-phosphatidylcholine (LPC), a product of oxidized phosphatidylcholine hydrolysis, is found at concentrations of 1-12 microg/g tissue throughout the atheroma. The objective of this study was to determine if LPC induces an osteogenic phenotype in vascular smooth muscle cells.

Methods and results: Proliferating human aortic smooth muscle cells were treated with a wide-range of LPC concentrations (0.1 nM to 100 microM) over 14 days. Von Kossa, Alizarin Red S, and alkaline phosphatase staining were used to identify mineralizations. RT-PCR, ELISA, alkaline phosphatase activity, and 45Ca incorporation assays were used to evaluate the osteo-inductive effect of LPC on smooth muscle phenotype. Histology and morphometry revealed that cells treated with as little as 10 nM LPC produced calcium phosphate deposits in culture. LPC-treated vascular smooth muscle cells showed a significant increase in 45Ca incorporation and alkaline phosphatase activity. Furthermore, LPC treatment induced a significant loss of Schnurri 3 protein, a key repressor of Runt-related transcription factor 2 stability. Genomic studies revealed that osteogenic gene expression was significantly up-regulated in LPC-treated cells, which is attributed to increased Runt-related transcription factor 2 expression and transcriptional activity.

Conclusion: LPC induces osteogenic morphology, physiology, gene expression, and phenotype in vascular smooth muscle cells. The present study suggests that localized concentrations of LPC in human atherosclerotic plaques may be a contributing factor to the generation of calcifying vascular cells.

Figure 1. LPC correlates to CEA mineralization

(A) LPC abundance in human CEA segments; I, internal; B, bifurcation; C, common; E, external. LPC (μg) used as standard curve. Arrow = LPC (B) LPC (μg) correlates to CEA total calcium (soluble calcium + pelleted calcification) (μg). Linear regression with 95% confidence lines. n=14 (C) LPC induces VSMC mineralizations in culture. Morphometric analysis of calcific ridges, Alizarin Red S stain. Green boxes outline the edges of the uniform grid used to score positive mineralizations.

Figure 2. LPC-treated VSMCs produce calcium phosphate deposits in culture

(A) von Kossa stain, 14d, 4X. Arrows indicate calcium phosphate ridges. (B) Alizarin Red S calcium stain, 14d, 4X. Arrows indicate calcium structures. Scale bar = 200μm (C) Total phosphate levels; n=3, p<0.05, phosphate (μg) normalized to total protein (μg) (D) Calcium incorporation assay; ⁴⁵CaCl₂, n=6,*p<0.01, #p<0.05. CPM was normalized to total protein (μg/μL) concentrations (CPM/μg total protein). Error bars represent ±SD.

Figure 3. LPC induces osteogenic gene expression in VSMCs

A–C, Real-time PCR.14d, n=3-7. (A) Alkaline phosphatase. (B) Collagen 1α. (C) Osteopontin. (D) Osteopontin (protein) ELISA Assay, 14d, n=6. *p<0.01, # p<0.05. Error bars represent ±SD.

Figure 4. LPC induces alkaline phosphatase activity in VSMCs

(A) Alkaline phosphatase activity assay. 1X upper panels; 4X lower panels. Scale bar = 200μm. (B) Alkaline phosphatase mass. *p<0.01 (C) Alkaline phosphatase specific activity (RFU/AP mass (μg/μL)) at 1 and 21 min (D) Alkaline phosphatase specific activity over time.

Figure 5. RUNX2 expression and transcriptional activity are increased in LPC-treated VSMCs

(A) Real-time PCR, RUNX2, 14d, n=3-7, # p<0.05. (B) RUNX2-Luciferase Reporter Assay; 14d, n=3, *p<0.001. RLU, relative luciferase units. (C) Temporal Luciferase Assay, 10d-14d, n=3, *p<0.01, # p<0.05. Error bars represent ±SD. (D) Schnurri 3 (protein) ELISA; n=3 *p<0.05. Schnurri 3 protein (μg) normalized to total protein (μg).