Patients with chronic kidney disease, and particularly those under hemodialysis, are prone to develop cardiovascular complications, mostly due to the exacerbation of vascular calcification. Vascular calcification relies on the transdifferentiation of vascular smooth muscle cells into calcifying cells. Sphingosine 1-phosphate is a pleiotropic sphingolipid and an important regulator of osteogenesis and the cardiovascular system. Therefore, we explored the role of sphingosine 1-phosphate metabolism in chronic kidney disease-derived vascular calcification. Vascular calcification progression in chronic kidney disease and sphingosine 1-phosphate signaling were examined in calcified vascular smooth muscle cells, in aortic explants, in rats with adenine-induced chronic kidney disease, as well as in serum from hemodialysis patients. Sphingosine kinase 2 activity and sphingosine 1-phosphate secretion, under the control of phospholipase D1, were exacerbated in calcified vascular smooth muscle cells. Furthermore, phospholipase D1 knockout mice display significantly less circulating sphingosine 1-phosphate, supporting intertwined signalization cascades. Overall, sphingosine kinase expression and activity were upregulated in calcified aortic explants and in calcified aortas from rats. Sphingosine 1-phosphate was increased in the serum of rats with mild vascular calcification. The Food and Drug Administration-approved immunosuppressant drug fingolimod, a general modulator of S1P metabolism, strongly inhibited calcification in vascular smooth muscle cells and aortic explants. Additionally, fingolimod significantly reduced inflammation, attenuated metabolic syndrome and moderately inhibited aortic calcification in rats. Finally, we demonstrated for the first time that serum sphingosine 1-phosphate was significantly increased in hemodialysis patients with mild abdominal aortic calcification. Our findings open an unexplored therapeutic option, which is targeting sphingosine 1-phosphate metabolism, eventually with fingolimod, for the prevention and treatment of vascular calcification in chronic kidney disease patients.

Links between cathepsin K and the pathophysiology of osteoarthritis (OA) can be established, not least because of the overabundance of cathepsin K in the serum of OA patients and the upregulation of cathepsin K in degraded cartilage in animal models of OA. Chondrocytes, chondroclasts, or osteoclasts contribute to the accumulated cathepsin K at the diseased osteochondral junction. After a general presentation of OA and cartilage physiology, as well as its degradation processes, we describe the function of cathepsin K and its effect on cartilage degradation via type II collagen cleavage. An overview of the most promising cathepsin K inhibitors is then presented, together with their in vitro effects. Although intensive research on cathepsin K inhibitors initially focused on bone resorption, there is growing interest in the potential of these drugs to prevent cartilage degradation. In this review, we summarize the pre-clinical and clinical trials that support the use of cathepsin K inhibitors in the treatment of OA. To date, no molecules of this type are commercially available, although a few have undergone clinical trials, but we believe that the development of cathepsin K inhibitors could broaden the therapeutic arsenal for the treatment of OA.

Extracellular vesicles are released by all cell types, including vascular smooth muscle cells, endothelial cells, and macrophages. The function of extracellular vesicles depends on both the type and differentiation state of the parent cells. For instance, high phosphate levels stimulate the transdifferentiation of vascular smooth muscle cells and the release of extracellular vesicles with a protein composition different from that of the vesicles released by undifferentiated cells. Vascular smooth muscle cells secrete both small-sized and extracellular matrix-derived extracellular vesicles with different function and composition. Extracellular vesicles derived from the extracellular matrix are reminiscent of bone-associated matrix vesicles and are more efficient to induce mineral formation than extracellular vesicles. Herein, we describe the role of extracellular vesicles on cardiovascular diseases. For instance, intercellular communication pathways via extracellular vesicles represent a possible mechanism in cardiovascular diseases, although the vesicles may not represent the best cargo carrier. On the other hand, the analysis of extracellular vesicles-associated proteins may be useful to predict progression of the disease.

Hypertrophic chondrocytes and fully differentiated osteoblasts release extracellular vesicles, including apoptotic bodies, microparticles, vesicles, matrix vesicles, and exosomes. The extracellular medium contains extracellular vesicles, with a neglected amount of matrix vesicles, while most of matrix vesicles are strongly bound to collagen. Large-size vesicles (apoptotic bodies) are obtained from the extracellular medium subjected to one differential centrifugation, while small and medium-size vesicles are obtained after a second differential step from the extracellular medium. To obtain matrix vesicles, cells and the extracellular matrix, or bone and cartilage tissues, are washed to remove extracellular medium, then they are subjected to a collagenase digestion and two differential centrifugations. Determinations of tissue-nonspecific alkaline phosphatase activity, mineral profile, and the presence of apatite and protein markers (CD9, ALIX, and calnexin) allowed to characterize each class of extracellular vesicles. The extraction of organelles and of apical and basal membranes is discussed as a possible tool to determine mechanisms of the biogenesis of extracellular vesicles.

Annexins are Ca2+- and lipid-binding proteins involved in Ca2+ homeostasis in bone cells and in extracellular vesicles. Annexins have been studied at the Nencki Institute since early 1990s with emphasis on the mineralization processes under physiological and pathological conditions. This chapter describes the family of annexins, the annexin knockout mice models for mineralization, and the expression and functions of annexins in chondrocytes, osteoblasts, mesenchymal stromal cells, and vascular smooth muscle cells. The functions, including the ability to form a nucleation core, bind to collagen fibrils, transport Ca2+, and bind to nucleotides, of annexin A2 (AnxA2), AnxA5, and AnxA6 in media vesicles (exosomes), and matrix vesicles are discussed. The overall findings support the model that annexins are not essential for ossification; however, due to their ability to induce mineralization, they contribute to prevent bone defects and facilitate biomineralization.

The observation, over 40 years ago, that matrix vesicles accumulate calcium and inorganic phosphate ions to grow apatite minerals in their lumen led to the discovery that matrix vesicles are equipped with a nucleational core that regulates the formation of apatite minerals. The discovery of the nucleational core can be divided into three steps. First, the matrix vesicles were disassembled to extract the matrix vesicles' core and characterize its physical, chemical, and biochemical properties. Then, a nucleational core mimicking the matrix vesicle kinetics of ion uptake and possessing the same biophysical properties of the native matrix vesicles' core was reconstituted from pure components. Finally, the biochemical machinery that enables the conversion of the nucleational core to a more crystalline structure was identified. In this chapter, we will describe the current knowledge on the structure and biological functions of the nucleational core.

The proteomes of matrix vesicles, cartilage articular extracellular vesicles, and extracellular vesicles are compared. Matrix vesicles and cartilage articular extracellular vesicles bound to the extracellular matrix are extracted after enzymatic digestion from the extracellular matrix. Extracellular vesicles, not bound to the extracellular matrix, are extracted from the extracellular medium without any enzymatic digestion. Matrix vesicles from the growth plate cartilage and articular extracellular vesicles from the articular cartilage bear different proteomes. Matrix vesicles lack inflammatory complements, while cartilage articular extracellular vesicles lack several chaperones. Matrix vesicles from the growth plate cartilage, Saos-2, and MC3T3-E1 osteoblast cell lines are distinct than extracellular vesicles from SV-HFO and from calvaria primary osteoblasts. β-actin, copine III, heat shock 70 kDa protein 1A, IQ motif-containing GTPase activating protein 1, Na+/K+ ATPase α2, and tubulin β are present in all matrix vesicles from osteoblasts and chondrocytes, while they were not detected in extracellular vesicles. Tissue-nonspecific alkaline phosphatase and several annexins are highly enriched in matrix vesicles but were also present in extracellular vesicles. Extracellular vesicles have several lysosomal and exosomal markers, which were undetected in matrix vesicles. Around 93% of the matrix vesicles' proteins originate from microvilli indicating that matrix vesicles originate from microvilli.

Matrix vesicles are a special class of extracellular vesicles contributing to both physiologic and pathologic biomineralization. Biomimetic models, including proteoliposomes, Langmuir monolayers, and Langmuir–Blodgett films, have been used to assess the biochemical and functional properties of matrix vesicles. Proteoliposomes can reproduce the spherical geometry and the lipid and protein content of matrix vesicles. Mimicking the function and membrane organization of natural vesicles, proteoliposomes can provide insights into the mechanisms of matrix vesicle-mediated calcification. Moreover, although Langmuir monolayers and Langmuir–Blodgett films cannot reproduce the spherical geometry of the vesicles, they are useful for determining the thermodynamic parameters of lipid–lipid and lipid–protein interactions. In this chapter, we will describe the use of biomimetic models to shed light on the role of proteins already described in the proteome of matrix vesicles, including tissue-nonspecific alkaline phosphatase (TNAP), ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1), and members of the annexin family, in the biomineralization processes.

Biomineralization is a complex process that requires a biological synchronization of cells. These cells orchestrate the production of an extracellular matrix composed of collagen fibrils, noncollagenous proteins, and proteoglycans that undergoes mineralization. Mineral competent odontoblasts, chondrocytes, and osteoblasts are able to produce extracellular matrix and to release matrix vesicles, ranging from 100 to 300 nm diameter. Several pieces of experimental evidence suggest that matrix vesicles are the initial site of calcification in dentin, cartilage, bone, as well in turkey tendon. Matrix vesicles are bound to collagen fibrils, in contrast to other types of extracellular vesicles and function as mineral nanoreactors that facilitate the initial formation of apatite, the main mineral component in teeth and bones. Matrix vesicles harbor all the machinery necessary to induce apatite, especially tissue-nonspecific alkaline phosphatase, a mineralization marker which is highly enriched in these vesicles. During the initial mineralization phase, apatite is formed in the lumen of matrix vesicles, then it is released in the extracellular medium, where mineralization continues with the supplementation of extracellular Pi and Ca2+. Here, we report the accumulated experimental evidence on apatite formation in the lumen of matrix vesicles, on the binding of matrix vesicles to collagen fibers, and on the biogenesis of matrix vesicles from mineral competent cells.

Mineralization-competent cells, including hypertrophic chondrocytes, mature osteoblasts, and osteogenic-differentiated smooth muscle cells secrete media extracellular vesicles (media vesicles) and extracellular vesicles bound to the extracellular matrix (matrix vesicles). Media vesicles are purified directly from the extracellular medium. On the other hand, matrix vesicles are purified after discarding the extracellular medium and subjecting the cells embedded in the extracellular matrix or bone or cartilage tissues to an enzymatic treatment. Several pieces of experimental evidence indicated that matrix vesicles and media vesicles isolated from the same types of mineralizing cells have distinct lipid and protein composition as well as functions. These findings support the view that matrix vesicles and media vesicles released by mineralizing cells have different functions in mineralized tissues due to their location, which is anchored to the extracellular matrix versus free-floating.

Calcium accumulation in atherosclerotic plaques predicts cardiovascular mortality, but the mechanisms responsible for plaque calcification and how calcification impacts plaque stability remain debated. Tissue-nonspecific alkaline phosphatase (TNAP) recently emerged as a promising therapeutic target to block cardiovascular calcification. In this study, we sought to investigate the effect of the recently developed TNAP inhibitor SBI-425 on atherosclerosis plaque calcification and progression. TNAP levels were investigated in ApoE-deficient mice fed a high-fat diet from 10 weeks of age and in plaques from the human ECLAGEN biocollection (101 calcified and 14 non-calcified carotid plaques). TNAP was inhibited in mice using SBI-425 administered from 10 to 25 weeks of age, and in human vascular smooth muscle cells (VSMCs) with MLS-0038949. Plaque calcification was imaged in vivo with 18F-NaF-PET/CT, ex vivo with osteosense, and in vitro with alizarin red. Bone architecture was determined with µCT. TNAP activation preceded and predicted calcification in human and mouse plaques, and TNAP inhibition prevented calcification in human VSMCs and in ApoE-deficient mice. More unexpectedly, TNAP inhibition reduced the blood levels of cholesterol and triglycerides, and protected mice from atherosclerosis, without impacting the skeletal architecture. Metabolomics analysis of liver extracts identified phosphocholine as a substrate of liver TNAP, who's decreased dephosphorylation upon TNAP inhibition likely reduced the release of cholesterol and triglycerides into the blood. Systemic inhibition of TNAP protects from atherosclerosis, by ameliorating dyslipidemia, and preventing plaque calcification.

The diagnosis of breast cancer in the early stage is essential for a favorable prognosis. Extracellular vesicles isolated from body fluids have a central role in breast cancer development due to their biochemical components. Among the biochemical components, surface proteins mediate vesicle interactions with elements of the extracellular milieu, the extracellular matrix, and neighboring cells. The identification of specific surface proteomic profile has been regarded as an easy and reproducible means to define cancer parameters, identify markers for a diagnosis, and determine targets for therapeutical treatments. In this review, we will focus on annexins, tetraspanins, integrins, immune checkpoint proteins, and growth factor receptors that have been identified on the surface of extracellular vesicles isolated from the serum of patients with breast cancer and that have been found to be relevant diagnostic and prognostic biomarkers.

Matrix vesicles (MVs) are 100-300 nm spherical structures released by mineralization competent cells to initiate formation of apatite, the mineral component in bones. Among proteins present in MVs, annexin A6 (AnxA6) is thought to be ubiquitously distributed in the MVs' lumen, on the surface of the internal and external leaflets of the membrane and also inserted in the lipid bilayer. To determine the molecular mechanism(s) that lead to the different locations of AnxA6, we hypothesized the occurrence of a pH drop during the mineralization. Such a change would induce the AnxA6 protonation, which in turn, and because of its isoelectric point of 5.41, would change the protein hydrophobicity facilitating its insertion into the MVs' bilayer. The various distributions of AnxA6 are likely to disturb membrane phospholipid organization. To examine this possibility, we used fluorescein as pH reporter, and established that pH decreased inside MVs during apatite formation. Then, 4-(14-phenyldibenzo[a,c]phenazin-9(14H)-yl)-phenol, a vibration-induced emission fluorescent probe, was used as a reporter of changes in membrane organization occurring with the varying mode of AnxA6 binding. Proteoliposomes containing AnxA6 and 1,2-Dimyristoyl-sn-glycero-3phosphocholine (DMPC) or 1,2-Dimyristoyl-sn-glycero-3phosphocholine: 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (DMPC:DPPS 9:1), to mimic the external and internal MV membrane leaflet, respectively, served as biomimetic models to investigate the nature of AnxA6 binding. Addition of Anx6 to DMPC at pH 7.4 and 5.4, or DMPC:DPPS (9:1) at pH 7.4 induced a decrease in membrane fluidity, consistent with AnxA6 interactions with the bilayer surface. In contrast, AnxA6 addition to DMPC:DPPS (9:1) at pH 5.4 increased the fluidity of the membrane. This latest result was interpreted as reflecting the insertion of AnxA6 into the bilayer. Taken together, these findings point to a possible mechanism of AnxA6 translocation in MVs from the surface of the internal leaflet into the phospholipid bilayer stimulated upon acidification of the MVs' lumen during formation of apatite.

Phospholipids are essential components of biological membranes and are involved in cell signalization, in several enzymatic reactions, and in energy metabolism. In addition, phospholipids represent an evolutionary and non-negligible step in life emergence. Progress in the past decades has led to a deeper understanding of these unique hydrophobic molecules and their most pertinent functions in cell biology. Today, a growing interest in “prebiotic lipidomics” calls for a new assessment of these relevant biomolecules.

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanosized particles released by all cell types during physiological as well as pathophysiological processes to carry out diverse biological functions, including acting as sources of cellular dumping, signalosomes and mineralisation nanoreactors. The ability of EVs to perform specific biological functions is due to their biochemical machinery. Among the components of the EVs' biochemical machinery, surface proteins are of critical functional significance as they mediate the interactions of EVs with components of the extracellular milieu, the extracellular matrix and neighbouring cells. Surface proteins are thought to be native, that is, pre-assembled on the EVs' surface by the parent cells before the vesicles are released. However, numerous pieces of evidence have suggested that soluble proteins are acquired by the EVs' surface from the extracellular milieu and further modulate the biological functions of EVs during innate and adaptive immune responses, autoimmune disorders, complement activation, coagulation, viral infection and biomineralisation. Herein, we will describe the methods currently used to identify the EVs' surface proteins and discuss recent knowledge on the functional relevance of the soluble proteins acquired by EVs.

The biochemical machinery involved in matrix vesicles-mediated bone mineralization involves a specific set of lipids, enzymes, and proteins. Annexins, among their many functions, have been described as responsible for the formation and stabilization of the matrix vesicles′ nucleational core. However, the specific role of each member of the annexin family, especially in the presence of type-I collagen, remains to be clarified. To address this issue, in vitro mineralization was carried out using AnxA6 (in solution or associated to the proteoliposomes) in the presence or in the absence of type-I collagen, incubated with either amorphous calcium phosphate (ACP) or a phosphatidylserine-calcium phosphate complex (PS–CPLX) as nucleators. Proteoliposomes were composed of 1,2-dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoylphosphatidylcholine: 1,2-dipalmitoylphosphatidylserine (DPPC:DPPS), and DPPC:Cholesterol:DPPS to mimic the outer and the inner leaflet of the matrix vesicles membrane as well as to investigate the effect of the membrane fluidity. Kinetic parameters of mineralization were calculated from time-dependent turbidity curves of free Annexin A6 (AnxA6) and AnxA6-containing proteoliposomes dispersed in synthetic cartilage lymph. The chemical composition of the minerals formed was investigated by Fourier transform infrared spectroscopy (FTIR). Free AnxA6 and AnxA6-proteoliposomes in the presence of ACP were not able to propagate mineralization; however, poorly crystalline calcium phosphates were formed in the presence of PS–CPLX, supporting the role of annexin-calcium-phosphatidylserine complex in the formation and stabilization of the matrix vesicles’ nucleational core. We found that AnxA6 lacks nucleation propagation capacity when incorporated into liposomes in the presence of PS–CPLX and type-I collagen. This suggests that AnxA6 may interact either with phospholipids, forming a nucleational core, or with type-I collagen, albeit less efficiently, to induce the nucleation process.

The bones can be viewed as both an organ and a material. As an organ, the bones give structure to the body, facilitate skeletal movement, and provide protection to internal organs. As a material, the bones consist of a hybrid organic/inorganic three-dimensional (3D) matrix, composed mainly of collagen, noncollagenous proteins, and a calcium phosphate mineral phase, which is formed and regulated by the orchestrated action of a complex array of cells including chondrocytes, osteoblasts, osteocytes, and osteoclasts. The interactions between cells, proteins, and minerals are essential for the bone functions under physiological loading conditions, trauma, and fractures. The organization of the bone's organic and inorganic phases stands out for its mechanical and biological properties and has inspired materials research. The objective of this review is to fill the gaps between the physical and biological characteristics that must be achieved to fabricate scaffolds for bone tissue engineering with enhanced performance. We describe the organization of bone tissue highlighting the characteristics that have inspired the development of 3D cell-laden collagenous scaffolds aimed at replicating the mechanical and biological properties of bone after implantation. The role of noncollagenous macromolecules in the organization of the collagenous matrix and mineralization ability of entrapped cells has also been reviewed. Understanding the modulation of cell activity by the extracellular matrix will ultimately help to improve the biological performance of 3D cell-laden collagenous scaffolds used for bone regeneration and repair as well as for in vitro studies aimed at unravelling physiological and pathological processes occurring in the bone.

The mineralization process is initiated by osteoblasts and chondrocytes during intramembranous and endochondral ossifications, respectively. Both types of cells release matrix vesicles (MVs), which accumulate Pi and Ca2+ and form apatites in their lumen. Tissue non-specific alkaline phosphatase (TNAP), a mineralization marker, is highly enriched in MVs, in which it removes inorganic pyrophosphate (PPi), an inhibitor of apatite formation. MVs then bud from the microvilli of mature osteoblasts or hypertrophic chondrocytes and, thanks to the action of the acto-myosin cortex, become released to the extracellular matrix (ECM), where they bind to collagen fibers and propagate mineral growth. In this report, we compared the mineralization ability of human fetal osteoblastic cell line (hFOB 1.19 cells) with that of osteosarcoma cell line (Saos-2 cells). Both types of cells were able to mineralize in an osteogenic medium containing ascorbic acid and beta glycerophosphate. The composition of calcium and phosphate compounds in cytoplasmic vesicles was distinct from that in extracellular vesicles (mostly MVs) released after collagenase-digestion. Apatites were identified only in MVs derived from Saos-2 cells, while MVs from hFOB 1.19 cells contained amorphous calcium phosphate complexes. In addition, AnxA6 and AnxA2 (nucleators of mineralization) increased mineralization in the sub-membrane region in strongly mineralizing Saos-2 osteosarcoma, where they co-localized with TNAP, whereas in less mineralizing hFOB 1.19 osteoblasts, AnxA6, and AnxA2 co-localizations with TNAP were less visible in the membrane. We also observed a reduction in the level of fetuin-A (FetuA), an inhibitor of mineralization in ECM, following treatment with TNAP and Ca channels inhibitors, especially in osteosarcoma cells. Moreover, a fraction of FetuA was translocated from the cytoplasm towards the plasma membrane during the stimulation of Saos-2 cells, while this displacement was less pronounced in stimulated hFOB 19 cells. In summary, osteosarcoma Saos-2 cells had a better ability to mineralize than osteoblastic hFOB 1.19 cells. The formation of apatites was observed in Saos-2 cells, while only complexes of calcium and phosphate were identified in hFOB 1.19 cells. This was also evidenced by a more pronounced accumulation of AnxA2, AnxA6, FetuA in the plasma membrane, where they were partly co-localized with TNAP in Saos-2 cells, in comparison to hFOB 1.19 cells. This suggests that both activators (AnxA2, AnxA6) and inhibitors (FetuA) of mineralization were recruited to the membrane and co-localized with TNAP to take part in the process of mineralization.