Alpha-1 antitrypsin inhibits RANKL-induced osteoclast formation and functions.

Osteoporosis is a global public health problem affecting more than 200 million people worldwide. We previously showed that treatment with alpha-1 antitrypsin (AAT), a multifunctional protein with anti-inflammatory properties, mitigated bone loss in an ovariectomized mouse model. However, the underlying mechanisms of the protective effect of AAT on bone tissue are largely unknown. In this study, we investigated the effect of AAT on osteoclast formation and function in vitro. Our results showed that AAT dose-dependently inhibited the formation of RANKL (receptor activator of nuclear factor κB ligand) induced osteoclasts derived from mouse bone marrow macrophages/monocyte (BMM) lineage cells and the murine macrophage cell line, RAW 264.7 cells. In order to elucidate the possible mechanisms underlying this inhibition, we tested the effect of AAT on the gene expression of cell surface molecules, transcription factors, and cytokines associated with osteoclast formation. We showed that AAT inhibited M-CSF (macrophage colony-stimulating factor) induced cell surface RANK expression in osteoclast precursor cells. In addition, AAT inhibited RANKL-induced TNF-α production, cell surface CD9 expression, and dendritic cell-specific transmembrane protein (DC-STAMP) gene expression. Importantly, AAT treatment significantly inhibited osteoclast-associated mineral resorption. Together, these results uncovered new mechanisms for the protective effects of AAT and strongly support the notion that AAT has therapeutic potential for the treatment of osteoporosis.

Carbonic anhydrase XII in valve interstitial cells promotes the regression of calcific aortic valve stenosis.

AIMS: Calcific aortic valve stenosis (CAVS) is the most common heart valve disease. In the present work we sought to determine the reversibility of mineralization in the aortic valve. METHODS AND RESULTS: By using in vitro analyses we found that valve interstitial cells (VICs) have the ability to resorb minerals. We documented that agonist of P2Y2 receptor (P2Y2R) promoted the expression of carbonic anhydrase XII (CAXII) at the cell membrane of VICs, whereby minerals are resorbed. P2Y2R-mediated mineral resorption was corroborated by using mouse VICs isolated from wild type and P2Y2R(-/-) mice. Measurements of extracellular pH (pHe) by using core-shell nanosensors revealed that P2Y2R-mediated CAXII export to the cell membrane led to an acidification of extracellular space, whereby minerals are resorbed. In vivo, we next treated LDLR(-/-)/ApoB(100/100)/IGF2 mice, which had developed CAVS under a high-fat/high-sucrose diet for 8 months, with 2-thioUTP (a P2Y2R agonist) or saline for the next 2 months. The administration of 2-thioUTP (2mg/kg/day i.p.) reduced the mineral volume in the aortic valve measured with serial microCT analyses, which improved hemodynamics and reduced left ventricular hypertrophy (LVH). Examination of leaflets at necropsy confirmed a lower level of mineralization and fibrosis along with higher levels of CAXII in mice under 2-thioUTP. In another series of experiment, the administration of acetazolamide (a CA inhibitor) prevented the acidification of leaflets and the regression of CAVS induced by 2-thioUTP in LDLR(-/-)/ApoB(100/100)/IGF2 mice. CONCLUSION: P2Y2R-mediated expression of CAXII by VICs acidifies the extracellular space and promotes the regression of CAVS.

Multinucleated giant cells in atherosclerotic plaques of human carotid arteries: Identification of osteoclast-like cells and their specific proteins in artery wall.

UNLABELLED: The mechanism(s) mediating atherosclerotic calcification may be similar to those governing bone remodeling, and osteoblast-like cells have been observed in plaque. We tested the hypothesis that osteoclast-like cells (OLCs) also exist in atherosclerotic arteries. In 205 tissue blocks obtained from 21 patients undergoing carotid endarterectomy, we performed histopathologic analysis, histochemical staining for tartrate-resistant acid phosphatase (TRAP), and immunohistochemical analysis for osteoclast and macrophage antigens, including CD68, colony stimulating factor-1 receptor (CSF-1R), cathepsin K (cat-K), receptor activator of nuclear factor-κB (RANK), and osteoprotegerin (OPG). Lesions were classified according to the AHA system, and further grouped as calcified or non-calcified (with necrotic cores or suture granulomas). Multinucleated giant cells morphologically similar to osteoclasts were frequently seen, sometimes exhibited morphologic evidence of polarization, were closely associated with regions of calcification, fibrosis, or granulomatous tissue, and also appeared to be associated with neovascularization and regions of intraplaque hemorrhage. TRAP-positive cells often expressed the osteoclast-associated antigens cat-K, RANK, and OPG. Calcification typically occurred at the base of plaque or in necrotic cores in various morphologies, including a fine powdery pattern, a diffuse pattern of larger deposits near cholesterol clefts and necrotic centers, and nodular forms. Regions of frank ossification were rarely observed. CONCLUSION: OLCs are frequently found in plaque, and co-localize with sub-regions of cholesterol deposition, mineralization, and necrotic and foreign debris. True bone tissue is rare in carotid plaque, although more common in other arteries. Our findings suggest that arterial OLCs might degrade mineral deposits, prevent formation of calcification or both and therefore counterbalance the activity of the osteoblast-like cells in atherosclerosis.

Comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins.

Background: Ever since their discovery, induced pluripotent stem cells (iPSCs) have been extensively differentiated into a large variety of cell types. However, a limited amount of work has been dedicated to differentiating iPSCs into osteoclasts. While several differentiation protocols have been published, it remains unclear which protocols or differentiation methods are preferrable regarding the differentiation of osteoclasts. Methods: In this study we compare the osteoclastogenesis capacity of a peripheral blood mononuclear cell (PBMC)-derived iPSC line to a fibroblast-derived iPSC line in conjunction with either embryoid body-based or monolayer-based differentiation strategies. Both cell lines and differentiation protocols were investigated regarding their ability to generate osteoclasts and their inherent robustness and ease of use. The ability of both cell lines to remain undifferentiated while propagating using a feeder-free system was assessed using alkaline phosphatase staining. This was followed by evaluating mesodermal differentiation and the characterization of hematopoietic progenitor cells using flow cytometry. Finally, osteoclast yield and functionality based on resorptive activity, Cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression were assessed. Results were validated using qRT-PCR throughout the differentiation stages. Results: Embryoid-body based differentiation yielded CD45+, CD14+, CD11b+ subpopulations which in turn differentiated into osteoclasts which demonstrated TRAP positivity, Cathepsin K expression and mineral resorptive capabilities. This was regardless of which iPSC line was used. Monolayer-based differentiation yielded lower quantities of hematopoietic cells that were mostly CD34+ and did not subsequently differentiate into osteoclasts. Conclusions: The outcome of this study demonstrates the successful differentiation of osteoclasts from iPSCs in conjunction with the embryoid-based differentiation method, while the monolayer-based method did not yield osteoclasts. No differences were observed regarding osteoclast differentiation between the PBMC and fibroblast-derived iPSC lines.

Comparison of osteoclast differentiation protocols from human induced pluripotent stem cells of different tissue origins.

BACKGROUND: Ever since their discovery, induced pluripotent stem cells (iPSCs) have been extensively differentiated into a large variety of cell types. However, a limited amount of work has been dedicated to differentiating iPSCs into osteoclasts. While several differentiation protocols have been published, it remains unclear which protocols or differentiation methods are preferable regarding the differentiation of osteoclasts. METHODS: In this study, we compared the osteoclastogenesis capacity of a peripheral blood mononuclear cell (PBMC)-derived iPSC line to a fibroblast-derived iPSC line in conjunction with either embryoid body-based or monolayer-based differentiation strategies. Both cell lines and differentiation protocols were investigated regarding their ability to generate osteoclasts and their inherent robustness and ease of use. The ability of both cell lines to remain undifferentiated while propagating using a feeder-free system was assessed using alkaline phosphatase staining. This was followed by evaluating mesodermal differentiation and the characterization of hematopoietic progenitor cells using flow cytometry. Finally, osteoclast yield and functionality based on resorptive activity, Cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression were assessed. The results were validated using qRT-PCR throughout the differentiation stages. RESULTS: Embryoid body-based differentiation yielded CD45+, CD14+, CD11b+ subpopulations which in turn differentiated into osteoclasts which demonstrated TRAP positivity, Cathepsin K expression and mineral resorptive capabilities. This was regardless of which iPSC line was used. Monolayer-based differentiation yielded lower quantities of hematopoietic cells that were mostly CD34+ and did not subsequently differentiate into osteoclasts. CONCLUSIONS: The outcome of this study demonstrates the successful differentiation of osteoclasts from iPSCs in conjunction with the embryoid-based differentiation method, while the monolayer-based method did not yield osteoclasts. No differences were observed regarding osteoclast differentiation between the PBMC and fibroblast-derived iPSC lines.