Collagen promotes matrix vesicle-mediated mineralization by vascular smooth muscle cells.

Vascular calcification (VC) is a hallmark of atherosclerotic plaques. Calcification of advanced plaques shares common features with endochondral ossification of long bones and appears to be protective. On the other hand, microcalcification of early plaques, which is poorly understood, is thought to be harmful. Tissue-nonspecific alkaline phosphatase (TNAP) and collagen are the two proteins necessary for physiological mineralization. Here, we demonstrate the presence of membrane-bound TNAP, detected by immunofluorescence, that seems to form clusters on the plasma membrane of vascular smooth muscle cells (VSMCs) cultured in mineralizing conditions. We observed that TNAP activity and mineralization were increased when VSMCs were cultured in the presence of ascorbic acid (AA) and ß-glycerophosphate (ß-GP). Increased TNAP activity was observed in whole cell lysates, total membrane fractions and, more particularly, in matrix vesicles (MVs). We have shown that TNAP-enriched MVs released from VSMCs subjected to collagenase contained more apatite-like mineral than the less TNAP-rich/TNAP-enriched vesicles isolated without collagenase treatment. These results suggest a role for collagen in promoting calcification induced by TNAP in atherosclerotic plaques.

Functions of Rho family of small GTPases and Rho-associated coiled-coil kinases in bone cells during differentiation and mineralization.

BACKGROUND: Members of Rho-associated coiled-coil kinases (ROCKs) are effectors of Rho family of small GTPases. ROCKs have multiple functions that include regulation of cellular contraction and polarity, adhesion, motility, proliferation, apoptosis, differentiation, maturation and remodeling of the extracellular matrix (ECM). SCOPE OF THE REVIEW: Here, we focus on the action of RhoA and RhoA effectors, ROCK1 and ROCK2, in cells related to tissue mineralization: mesenchymal stem cells, chondrocytes, preosteoblasts, osteoblasts, osteocytes, lining cells and osteoclasts. MAJOR CONCLUSIONS: The activation of the RhoA/ROCK pathway promotes stress fiber formation and reduces chondrocyte and osteogenic differentiations, in contrast to that in mesenchymal stem cells which stimulated the osteogenic and the chondrogenic differentiation. The effects of Rac1 and Cdc42 in promoting chondrocyte hypertrophy and of Rac1, Rac2 and Cdc42 in osteoclast are discussed. In addition, members of the Rho family of GTPases such Rac1, Rac2, Rac3 and Cdc42, acting upstream of ROCK and/or other protein effectors, may compensate the actions of RhoA, affecting directly or indirectly the actions of ROCKs as well as other protein effectors. GENERAL SIGNIFICANCE: ROCK activity can trigger cartilage degradation and affect bone formation, therefore these kinases may represent a possible therapeutic target to treat osteoarthritis and osseous diseases. Inhibition of Rho/ROCK activity in chondrocytes prevents cartilage degradation, stimulate mineralization of osteoblasts and facilitate bone formation around implanted metals. Treatment with osteoprotegerin results in a significant decrease in the expression of Rho GTPases, ROCK1 and ROCK2, reducing bone resorption. Inhibition of ROCK signaling increases osteoblast differentiation in a topography-dependent manner.

TNAP stimulates vascular smooth muscle cell trans-differentiation into chondrocytes through calcium deposition and BMP-2 activation: Possible implication in atherosclerotic plaque stability.

Atherosclerotic plaque calcification varies from early, diffuse microcalcifications to a bone-like tissue formed by endochondral ossification. Recently, a paradigm has emerged suggesting that if the bone metaplasia stabilizes the plaques, microcalcifications are harmful. Tissue-nonspecific alkaline phosphatase (TNAP), an ectoenzyme necessary for mineralization by its ability to hydrolyze inorganic pyrophosphate (PPi), is stimulated by inflammation in vascular smooth muscle cells (VSMCs). Our objective was to determine the role of TNAP in trans-differentiation of VSMCs and calcification. In rodent MOVAS and A7R5 VSMCs, addition of exogenous alkaline phosphatase (AP) or TNAP overexpression was sufficient to stimulate the expression of several chondrocyte markers and induce mineralization. Addition of exogenous AP to human mesenchymal stem cells cultured in pellets also stimulated chondrogenesis. Moreover, TNAP inhibition with levamisole in mouse primary chondrocytes dropped mineralization as well as the expression of chondrocyte markers. VSMCs trans-differentiated into chondrocyte-like cells, as well as primary chondrocytes, used TNAP to hydrolyze PPi, and PPi provoked the same effects as TNAP inhibition in primary chondrocytes. Interestingly, apatite crystals, associated or not to collagen, mimicked the effects of TNAP on VSMC trans-differentiation. AP and apatite crystals increased the expression of BMP-2 in VSMCs, and TNAP inhibition reduced BMP-2 levels in chondrocytes. Finally, the BMP-2 inhibitor noggin blocked the rise in aggrecan induced by AP in VSMCs, suggesting that TNAP induction in VSMCs triggers calcification, which stimulates chondrogenesis through BMP-2. Endochondral ossification in atherosclerotic plaques may therefore be induced by crystals, probably to confer stability to plaques with microcalcifications.

Membranes and pathophysiological mineralization.

Vascular calcification accompanies the pathological process of atherosclerotic plaque formation. Artery calcification results from trans-differentiation of vascular smooth muscle cells (VSMCs) into cells resembling mineralization-competent cells such as osteoblasts and chondrocytes. The activity of tissue-nonspecific alkaline phosphatase (TNAP), a GPI-anchored enzyme necessary for physiological mineralization, is induced in VSMCs in response to inflammation. TNAP achieves its mineralizing function being anchored to plasma membrane of mineralizing cells and to the surface of their derived matrix vesicles (MVs), and numerous important reports indicate that membranes play a crucial role in initiating the crystal formation. In this review, we would like to highlight various functions of lipids and proteins associated to membranes at different stages of both physiological mineralization and vascular calcification, with an emphasis on the pathological process of atherosclerotic plaque formation.