Abstract 13953: Specialized Matrix Vesicles Induce Microcalcification by Aggregation Within a 3-Dimensional Collagen Network_Novel Implications for Plaque Vulnerability
Background: Recent studies have identified an important role for microcalcifications in thin fibrous cap biomechanical instability. We hypothesized that fibrous cap thinning and microcalcification formation are interrelated, whereby reduced collagen in the cap allows aggregation and subsequent calcification of matrix vesicles (MVs).
Methods and Results: Growth media collected from human arterial smooth muscle cells (SMCs) cultured up to 21 days in normal (NM) or osteogenic media (OM) was added to 3-dimensional collagen I gels simulating fibrous caps of various densities. MV presence within the media was confirmed using Nanoparticle Tracking Analysis, showing MVs between 30 and 300 nm. Confocal reflection imaging reveals MV aggregates adjacent to collagen fibers (Fig. A). CellLight fluorescent labeling confirms that MV aggregates are plasma membrane derived. MV aggregate size decreases (11-fold) with increasing collagen content (P<0.001, Fig. B). A near-infrared fluorescent hydroxyapatite-specific probe reveals microcalcification nucleation within MV aggregates from calcific SMCs (day 14, Fig. C) in a pattern similar to calcified plaques from Apoe-/- mice (Fig. D). Mass spectrometry reveals 52 proteins with ≥ 2-fold expression in MVs from calcific SMCs (day 14) compared to controls, including alkaline phosphatase (ALP), which exhibits a 7-fold higher activity in calcific MVs. Addition of an ALP inhibitor to SMC culture blocks MV-induced calcification in collagen gels and gross calcification of matrix in the culture dish as shown by Alizarin red staining.
Conclusion: Our data demonstrate that SMCs cultured in OM produce specialized ALP-enriched MVs required for microcalcification nucleation. Further, MV aggregation is inversely correlated to the density of the collagen network (Fig. B) suggesting that a thinning fibrous cap may be the impetus for formation of MV-derived microcalcification, thus unifying two prominent theories of plaque instability.
- © 2013 by American Heart Association, Inc.