Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

Abstract

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PP i ), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PP i . The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PP i as substrates. While the enzymatic efficiency (k cat /K m ) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat /K m for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PP i , respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

Proteoliposomes in nanobiotechnology

“…A large number of studies have been carried out using liposomes as systems to mimic biomembranes (Daghastanli et al 2004;Camolezi et al 2002;Ciancaglini et al 2006;Benatti et al 2007;Sesana et al 2008;Simão et al 2010a, b;Ciancaglini et al 2010;Bolean et al 2010Bolean et al , 2011. Experiments confirmed that the interaction between detergentsolubilized TNAP, free of detergent excess, and the lipid bilayer of liposomes occurs via the GPI-anchor of the enzyme and our method of incorporation via direct insertion guarantees that the enzyme is associated with the external leaflet of the liposome membrane (Camolezi et al 2002).…”

Biophysical aspects of biomineralization

“…Various experiments have revealed the presence of HA crystals both inside and outside collagen fibrils in the ECM (McNally et al 2012) and also within the lumen of matrix vesicles (MVs) (Ali et al 1970;Anderson 1969;Bonucci 1970). MVs are extracellular vesicles with a diameter ranging from approximately 100 to 300 nm (Anderson 2003;Anderson et al 2010;Cui et al 2016;Golub 2009;Wuthier and Lipscomb 2011) which harbor the biochemical machinery needed to establish the appropriate inorganic pyrophosphate (PP i ) to inorganic phosphate (P i ) ratio (Ciancaglini et al 2006Harmey et al 2004;Hessle et al 2002;Johnson et al 2000;Roberts et al 2007;Yadav et al 2011;Zhou et al 2012) required to initiate the synthesis of apatitic mineral (Bechkoff et al 2008;Thouverey et al 2009;Wu et al 1997), which will subsequently be propagated onto the collagenous ECM. These vesicles are released from the hypertrophic chondrocytes and mature osteoblasts, the cells responsible for endochondral and membranous ossification.…”

“…A relevant point to be observed is that the enzyme bound to liposomes retains the ability to hydrolyze different substrates, such as ATP, ADP, AMP, PP i , and p-nitrophenylphosphate Ciancaglini et al 2006Ciancaglini et al , 2010Simao et al 2010a, b).…”

“…It is thus comparable to the median size of a natural MV Ciancaglini et al 2006) and can adequately serve as a vesicular mimetic system to examine TNAP function in the context of a lipid membrane environment that mimics the MV environment. Electron microscopy of empty DPPC liposomes and TNAP-proteoliposomes showed that enzyme reconstitution did not affect the morphology of the liposomes (Simao et al 2010a).…”

Reconstituição da Anexina V em sistemas de lipossomos: associação com a fosfatase alcalina e correlação com estudos de biomineralização

“…Um dos grandes desafios da biologia é a compreensão de como as células se diferenciam, replicam e se dividem, e um dos aspectos importantes desse problema é saber como as células formam as MVs. A nova vesícula deve ser formada com grande precisão e possuir todas as informações necessárias para criar um microambiente adequado para a formação e preservação do mineral de hidroxiapatita (Anderson, 1995;Millán, 2006;Ciancaglini et al, 2006Ciancaglini et al, , 2010.…”