Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells.

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

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.

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.

Dissecting specific Wnt components governing osteogenic differentiation potential by human periodontal ligament stem cells through interleukin-6.

Periodontal ligament stem cells (PDLSCs) play a significant role on periodontal tissue and alveolar bone homeostasis. During inflammation, interleukin (IL)-6 serves as one of key cytokine players controlling tissue reaction as well as alveolar bone tissue remodeling. It is believed that periodontal tissue inflammation causes periodontium degradation, especially alveolar bone. However, in this study, we show that an inflammatory mediator, IL-6, may serve another direction on alveolar bone homeostasis during inflammatory condition. We found that, IL-6 at 10 and 20 ng/mL was not cytotoxic and dose-dependently exerted beneficial effects on osteogenic differentiation of human PDLSCs (hPDLSCs), as demonstrated by increased alkaline phosphatase activity, mRNA expression of osteogenic markers, and matrix mineralization. The presence of physiological and inflammatory level of IL-6, the osteogenic differentiation potential by hPDLSCs was enhanced by several possible mechanisms including transforming growth factor (TGF), Wnt, and Notch pathways. After in-depth and thorough exploration, we found that Wnt pathway serves as key regulator controlling osteogenic differentiation by hPDLSCs amid the IL-6 presentation. Surprisingly, apart from other mesenchymal stem cells, distinct Wnt components are employed by hPDLSCs, and both canonical and non-canonical Wnt pathways are triggered by different mechanisms. Further validation by gene silencing, treatment with recombinant Wnt ligands, and ß-catenin stabilization/translocation confirmed that IL-6 governed the canonical Wnt/ß-catenin pathway via either WNT2B or WNT10B and employed WNT5A to activate the non-canonical Wnt pathway. These findings fulfill the homeostasis pathway governing periodontal tissue and alveolar bone regeneration and may serve for further therapeutic regimen design for restoring the tissues.

Role of Runx2 in Calcific Aortic Valve Disease in Mouse Models.

Background: Calcific aortic valve disease is common in the aging population and is characterized by the histological changes of the aortic valves including extracellular matrix remodeling, osteochondrogenic differentiation, and calcification. Combined, these changes lead to aortic sclerosis, aortic stenosis (AS), and eventually to heart failure. Runt-related transcription factor 2 (Runx2) is a transcription factor highly expressed in the calcified aortic valves. However, its definitive role in the progression of calcific aortic valve disease (CAVD) has not been determined. In this study, we utilized constitutive and transient conditional knockout mouse models to assess the molecular, histological, and functional changes in the aortic valve due to Runx2 depletion. Methods: Lineage tracing studies were performed to determine the provenance of the cells giving rise to Runx2+ osteochondrogenic cells in the aortic valves of LDLr-/- mice. Hyperlipidemic mice with a constitutive or temporal depletion of Runx2 in the activated valvular interstitial cells (aVICs) and sinus wall cells were further investigated. Following feeding with a diabetogenic diet, the mice were examined for changes in gene expression, blood flow dynamics, calcification, and histology. Results: The aVICs and sinus wall cells gave rise to Runx2+ osteochondrogenic cells in diseased mouse aortic valves. The conditional depletion of Runx2 in the SM22α+ aVICs and sinus wall cells led to the decreased osteochondrogenic gene expression in diabetic LDLr-/- mice. The transient conditional depletion of Runx2 in the aVICs and sinus wall cells of LDLr-/-ApoB100 CAVD mice early in disease led to a significant reduction in the aortic peak velocity, mean velocity, and mean gradient, suggesting the causal role of Runx2 on the progression of AS. Finally, the leaflet hinge and sinus wall calcification were significantly decreased in the aortic valve following the conditional and temporal Runx2 depletion, but no significant effect on the valve cusp calcification or thickness was observed. Conclusions: In the aortic valve disease, Runx2 was expressed early and was required for the osteochondrogenic differentiation of the aVICs and sinus wall cells. The transient depletion of Runx2 in the aVICs and sinus wall cells in a mouse model of CAVD with a high prevalence of hemodynamic valve dysfunction led to an improved aortic valve function. Our studies also suggest that leaflet hinge and sinus wall calcification, even in the absence of significant leaflet cusp calcification, may be sufficient to cause significant valve dysfunctions in mice.

Engineered osteoclasts resorb necrotic alveolar bone in anti-RANKL antibody-treated mice.

Medication-related osteonecrosis of the jaw (MRONJ) is a serious side effect of antiresorptive medications such as denosumab (humanized anti-RANKL antibody), yet its pathophysiology remains elusive. It has been posited that inhibition of osteoclastic bone resorption leads to the pathological sequelae of dead bone accumulation, impaired new bone formation, and poor wound healing in MRONJ, but this hypothesis has not been definitively tested. We previously engineered myeloid precursors with a conditional receptor activator of nuclear factor kappa-Β intracellular domain (iRANK cells), which differentiate into osteoclasts in response to a chemical inducer of dimerization (CID) independently of RANKL. In this study, we showed that CID-treated iRANK cells differentiated into osteoclasts and robustly resorbed mineralized surfaces even in the presence of anti-RANKL antibody in vitro. We then developed a tooth extraction-triggered MRONJ model in nude mice using anti-RANKL antibody to deplete osteoclasts. This model was used to determine whether reconstitution of engineered osteoclasts within sockets could prevent specific pathological features of MRONJ. Locally delivered iRANK cells successfully differentiated into multinucleated osteoclasts in response to CID treatment in vivo as measured by green fluorescent protein (GFP), tartrate-resistant acid phosphatase (TRAP), carbonic anhydrase II, matrix metallopeptidase 9 (MMP-9), and cathepsin K staining. Sockets treated with iRANK cells + CID had significantly more osteoclasts and less necrotic bone than those receiving iRANK cells alone. These data support the hypothesis that osteoclast deficiency leads to accumulation of necrotic bone in MRONJ.

Adapter Protein RapGEF1 Is Required for ERK1/2 Signaling in Response to Elevated Phosphate in Vascular Smooth Muscle Cells.

The sodium-dependent phosphate transporter, SLC20A1, is required for elevated inorganic phosphate (Pi) induced vascular smooth muscle cell (VSMC) matrix mineralization and phenotype transdifferentiation. Recently, elevated Pi was shown to induce ERK1/2 phosphorylation through SLC20A1 by Pi uptake-independent functions in VSMCs, suggesting a cell signaling response to elevated Pi. Previous studies identified Rap1 guanine nucleotide exchange factor (RapGEF1) as an SLC20A1-interacting protein and RapGEF1 promotes ERK1/2 phosphorylation through Rap1 activation. In this study, we tested the hypothesis that RapGEF1 is a critical component of the SLC20A1-mediated Pi-induced ERK1/2 phosphorylation pathway. Co-localization of SLC20A1 and RapGEF1, knockdown of RapGEF1 with siRNA, and small molecule inhibitors of Rap1, B-Raf, and Mek1/2 were investigated. SLC20A1 and RapGEF1 were co-localized in peri-membranous structures in VSMCs. Knockdown of RapGEF1 and small molecule inhibitors against Rap1, B-Raf, and Mek1/2 eliminated elevated Pi-induced ERK1/2 phosphorylation. Knockdown of RapGEF1 inhibited SM22α mRNA expression and blocked elevated Pi-induced downregulation of SM22α mRNA. Together, these data suggest that RapGEF1 is required for SLC20A1-mediated elevated Pi signaling through a Rap1/B-Raf/Mek1/2 cell signaling pathway, thereby promoting ERK1/2 phosphorylation and inhibiting SM22α gene expression in VSMCs.

Slc20a1/Pit1 and Slc20a2/Pit2 are essential for normal skeletal myofiber function and survival.

Low blood phosphate (Pi) reduces muscle function in hypophosphatemic disorders. Which Pi transporters are required and whether hormonal changes due to hypophosphatemia contribute to muscle function is unknown. To address these questions we generated a series of conditional knockout mice lacking one or both house-keeping Pi transporters Pit1 and Pit2 in skeletal muscle (sm), using the postnatally expressed human skeletal actin-cre. Simultaneous conditional deletion of both transporters caused skeletal muscle atrophy, resulting in death by postnatal day P13. smPit1-/-, smPit2-/- and three allele mutants are fertile and have normal body weights, suggesting a high degree of redundance for the two transporters in skeletal muscle. However, these mice show a gene-dose dependent reduction in running activity also seen in another hypophosphatemic model (Hyp mice). In contrast to Hyp mice, grip strength is preserved. Further evaluation of the mechanism shows reduced ERK1/2 activation and stimulation of AMP kinase in skeletal muscle from smPit1-/-; smPit2-/- mice consistent with energy-stress. Similarly, C2C12 myoblasts show a reduced oxygen consumption rate mediated by Pi transport-dependent and ERK1/2-dependent metabolic Pi sensing pathways. In conclusion, we here show that Pit1 and Pit2 are essential for normal myofiber function and survival, insights which may improve management of hypophosphatemic myopathy.

PiT-2, a type III sodium-dependent phosphate transporter, protects against vascular calcification in mice with chronic kidney disease fed a high-phosphate diet.

PiT-2, a type III sodium-dependent phosphate transporter, is a causative gene for the brain arteriolar calcification in people with familial basal ganglion calcification. Here we examined the effect of PiT-2 haploinsufficiency on vascular calcification in uremic mice using wild-type and global PiT-2 heterozygous knockout mice. PiT-2 haploinsufficiency enhanced the development of vascular calcification in mice with chronic kidney disease fed a high-phosphate diet. No differences were observed in the serum mineral biomarkers and kidney function between the wild-type and PiT-2 heterozygous knockout groups. Micro computed tomography analyses of femurs showed that haploinsufficiency of PiT-2 decreased trabecular bone mineral density in uremia. In vitro, sodium-dependent phosphate uptake was decreased in cultured vascular smooth muscle cells isolated from PiT-2 heterozygous knockout mice compared with those from wild-type mice. PiT-2 haploinsufficiency increased phosphate-induced calcification of cultured vascular smooth muscle cells compared to the wild-type. Furthermore, compared to wild-type vascular smooth muscle cells, PiT-2 deficient vascular smooth muscle cells had lower osteoprotegerin levels and increased matrix calcification, which was attenuated by osteoprotegerin supplementation. Thus, PiT-2 in vascular smooth muscle cells protects against phosphate-induced vascular calcification and may be a therapeutic target in the chronic kidney disease population.

Role of smooth muscle cells in vascular calcification: implications in atherosclerosis and arterial stiffness.

Vascular calcification is associated with a significant increase in all-cause mortality and atherosclerotic plaque rupture. Calcification has been determined to be an active process driven in part by vascular smooth muscle cell (VSMC) transdifferentiation within the vascular wall. Historically, VSMC phenotype switching has been viewed as binary, with the cells able to adopt a physiological contractile phenotype or an alternate 'synthetic' phenotype in response to injury. More recent work, including lineage tracing has however revealed that VSMCs are able to adopt a number of phenotypes, including calcific (osteogenic, chondrocytic, and osteoclastic), adipogenic, and macrophagic phenotypes. Whilst the mechanisms that drive VSMC differentiation are still being elucidated it is becoming clear that medial calcification may differ in several ways from the intimal calcification seen in atherosclerotic lesions, including risk factors and specific drivers for VSMC phenotype changes and calcification. This article aims to compare and contrast the role of VSMCs in driving calcification in both atherosclerosis and in the vessel media focusing on the major drivers of calcification, including aging, uraemia, mechanical stress, oxidative stress, and inflammation. The review also discusses novel findings that have also brought attention to specific pro- and anti-calcifying proteins, extracellular vesicles, mitochondrial dysfunction, and a uraemic milieu as major determinants of vascular calcification.

Increased Calcific Aortic Valve Disease in response to a diabetogenic, procalcific diet in the LDLr-/-ApoB100/100 mouse model.

OBJECTIVE: Calcific aortic valve disease (CAVD) is a major cause of aortic stenosis (AS) and cardiac insufficiency. Patients with type II diabetes mellitus (T2DM) are at heightened risk for CAVD, and their valves have greater calcification than nondiabetic valves. No drugs to prevent or treat CAVD exist, and animal models that might help identify therapeutic targets are sorely lacking. To develop an animal model mimicking the structural and functional features of CAVD in people with T2DM, we tested a diabetogenic, procalcific diet and its effect on the incidence and severity of CAVD and AS in the, LDLr-/-ApoB100/100 mouse model. RESULTS: LDLr-/-ApoB100/100 mice fed a customized diabetogenic, procalcific diet (DB diet) developed hyperglycemia, hyperlipidemia, increased atherosclerosis, and obesity when compared with normal chow fed LDLr-/-ApoB100/100 mice, indicating the development of T2DM and metabolic syndrome. Transthoracic echocardiography revealed that LDLr-/-ApoB100/100 mice fed the DB diet had 77% incidence of hemodynamically significant AS, and developed thickened aortic valve leaflets and calcification in both valve leaflets and hinge regions. In comparison, normal chow (NC) fed LDLr-/-ApoB100/100 mice had 38% incidence of AS, thinner valve leaflets and very little valve and hinge calcification. Further, the DB diet fed mice with AS showed significantly impaired cardiac function as determined by reduced ejection fraction and fractional shortening. In vitro mineralization experiments demonstrated that elevated glucose in culture medium enhanced valve interstitial cell (VIC) matrix calcium deposition. CONCLUSIONS: By manipulating the diet we developed a new model of CAVD in T2DM, hyperlipidemic LDLr-/-ApoB100/100 that shows several important functional, and structural features similar to CAVD found in people with T2DM and atherosclerosis including AS, cardiac dysfunction, and inflamed and calcified thickened valve cusps. Importantly, the high AS incidence of this diabetic model may be useful for mechanistic and translational studies aimed at development of novel treatments for CAVD.

Loss of PiT-2 results in abnormal bone development and decreased bone mineral density and length in mice.

Normal bone mineralization requires phosphate oversaturation in bone matrix vesicles, as well as normal regulation of phosphate metabolism via the interplay among bone, intestine, and kidney. In turn, derangement of phosphate metabolism greatly affects bone function and structure. The type III sodium-dependent phosphate transporters, PiT-1 and PiT-2, are believed to be important in tissue phosphate metabolism and physiological bone formation, but their requirement and molecular roles in bone remain poorly investigated. In order to decipher the role of PiT-2 in bone, we examined normal bone development, growth, and mineralization in global PiT-2 homozygous knockout mice. PiT-2 deficiency resulted in reduced vertebral column, femur, and tibia length as well as mandibular dimensions. Micro-computed tomography analysis revealed that bone mineral density in the mandible, femur, and tibia were decreased, indicating that maintenance of bone function and structure is impaired in both craniofacial and long bones of PiT-2 deficient mice. Both cortical and trabecular thickness and mineral density were reduced in PiT-2 homozygous knockout mice compared with wild-type mice. These results suggest that PiT-2 is involved in normal bone development and growth and plays roles in cortical and trabecular bone metabolism feasibly by regulating local phosphate transport and mineralization processes in the bone. Further studies that evaluate bone cell-specific loss of PiT-2 are now warranted and may yield insight into complex mechanisms of bone development and growth, leading to identification of new therapeutic options for patients with bone diseases.

Osteoclast precursors do not express CD68: results from CD68 promoter-driven RANK transgenic mice.

Macrophages and osteoclasts are thought to derive from CD68 lineage marker-positive common myeloid precursors. We used the CD68 promoter to drive an inducible receptor activator of NF-κB (iRANK) construct that selectively activates RANK signaling in myeloid cells in vivo. The cytoplasmic portion of RANK was fused to a mutant FK506 binding domain, which selectively binds the chemical inducer of dimerization AP20187 and initiates signaling. iRANK mRNA was expressed in macrophages isolated from peritoneal cavity, spleen-, and bone marrow-derived myeloid cells. Unexpectedly, AP20187 did not induce osteoclast formation in spleen- and bone marrow-derived myeloid cells. However, AP20187-dependent RANK signaling induced ERK1/2 phosphorylation and mRNA expression of MMP9 and CathepsinK in peritoneal macrophages. Importantly, CD68 was not expressed until day 3 and day 5 in bone marrow and spleen myeloid cells, respectively. Contrary to dogma, osteoclast precursors do not express the lineage marker CD68.

Vascular calcification in CKD-MBD: Roles for phosphate, FGF23, and Klotho.

Vascular calcification (VC) is highly prevalent in aging, diabetes mellitus, and chronic kidney disease (CKD). VC is a strong predictor of cardiovascular morbidity and mortality in the CKD population. Complex pathological mechanisms are involved in the development of VC, including osteochondrogenic differentiation and apoptosis of vascular smooth muscle cells, instability and release of extracellular vesicles loaded calcium and phosphate, and elastin degradation. Elevated serum phosphate is a late manifestation of CKD, and has been shown to accelerate mineral deposition in both the vessel wall and heart valves. α-Klotho and fibroblast growth factor 23 (FGF23) are emerging factors in CKD-mineral and bone disorder (CKD-MBD) and are thought to be involved in the pathogenesis of uremic VC. There are discordant reports regarding the biomedical effects of FGF23 on VC. In contrast, mounting evidence supports a well-supported protective role for α-Klotho on VC. Further studies are warranted to elucidate potential roles of FGF23 and α-Klotho in VC and to determine where and how they are synthesized in normal and disease conditions. A thorough systemic evaluation of the biomedical interplay of phosphate, FGF23, and α-Klotho may potentially lead to new therapeutic options for patients with CKD-MBD.

SLC20A2 Deficiency in Mice Leads to Elevated Phosphate Levels in Cerbrospinal Fluid and Glymphatic Pathway-Associated Arteriolar Calcification, and Recapitulates Human Idiopathic Basal Ganglia Calcification.

Idiopathic basal ganglia calcification is a brain calcification disorder that has been genetically linked to autosomal dominant mutations in the sodium-dependent phosphate co-transporter, SLC20A2. The mechanisms whereby deficiency of Slc20a2 leads to basal ganglion calcification are unknown. In the mouse brain, we found that Slc20a2 was expressed in tissues that produce and/or regulate cerebrospinal fluid, including choroid plexus, ependyma and arteriolar smooth muscle cells. Haploinsufficient Slc20a2 +/- mice developed age-dependent basal ganglia calcification that formed in glymphatic pathway-associated arterioles. Slc20a2 deficiency uncovered phosphate homeostasis dysregulation characterized by abnormally high cerebrospinal fluid phosphate levels and hydrocephalus, in addition to basal ganglia calcification. Slc20a2 siRNA knockdown in smooth muscle cells revealed increased susceptibility to high phosphate-induced calcification. These data suggested that loss of Slc20a2 led to dysregulated phosphate homeostasis and enhanced susceptibility of arteriolar smooth muscle cells to elevated phosphate-induced calcification. Together, dysregulated cerebrospinal fluid phosphate and enhanced smooth muscle cell susceptibility may predispose to glymphatic pathway-associated arteriolar calcification.

Runx2 deletion in smooth muscle cells inhibits vascular osteochondrogenesis and calcification but not atherosclerotic lesion formation.

AIMS: Vascular smooth muscle cells (SMCs) are major precursors contributing to osteochondrogenesis and calcification in atherosclerosis. Runt-related transcription factor-2 (Runx2) has been found essential for SMC differentiation to an osteochondrogenic phenotype and subsequent calcification in vitro. A recent study using a conditional targeting allele that produced a truncated Runx2 protein in SMCs of ApoE-/- mice showed reduced vascular calcification, likely occurring via reduction of receptor activator of nuclear factor-κB ligand (RANKL), macrophage infiltration, and atherosclerotic lesion formation. Using an improved conditional Runx2 knockout mouse model, we have elucidated new roles for SMC-specific Runx2 in arterial intimal calcification (AIC) without effects on atherosclerotic lesion size. METHODS AND RESULTS: We used an improved targeting construct to generate LDLr-/- mice with floxed-Runx2 alleles ( LDLr-/- :Runx2 f/f ) such that Cre-mediated recombination ( LDLr-/- :Runx2 ΔSM ) does not produce functional truncated Runx2 protein, thereby avoiding off-target effects. We found that both LDLr-/- :Runx2 f/f and LDLr-/- :Runx2 ΔSM mice fed with a high fat diet developed atherosclerosis. SMC-specific Runx2 deletion did not significantly reduce atherosclerotic lesion size, macrophage number, or expression of RANKL, MCP-1, and CCR2. However, it significantly reduced AIC by 50%. Mechanistically, Sox9 and type II collagen were unaltered in vessels of LDLr-/- :Runx2 ΔSM mice compared to LDLr-/- :Runx2 f/f counterparts, while type X collagen, MMP13 and the osteoblastic marker osteocalcin were significantly reduced. CONCLUSIONS: SMC autonomous Runx2 is required for SMC differentiation towards osteoblast-like cells, SMC-derived chondrocyte maturation and AIC in atherosclerotic mice. These effects were independent of systemic lipid metabolism, RANKL expression, macrophage infiltration, and atheromatous lesion progression.

Osteopontin protects against high phosphate-induced nephrocalcinosis and vascular calcification.

Pathologic calcification is a significant cause of increased morbidity and mortality in patients with chronic kidney disease. The precise mechanisms of ectopic calcification are not fully elucidated, but it is known to be caused by an imbalance of procalcific and anticalcific factors. In the chronic kidney disease population, an elevated phosphate burden is both highly prevalent and a known risk factor for ectopic calcification. Here we tested whether osteopontin, an inhibitor of calcification, protects against high phosphate load-induced nephrocalcinosis and vascular calcification. Osteopontin knockout mice were placed on a high phosphate diet for 11 weeks. Osteopontin deficiency together with phosphate overload caused uremia, nephrocalcinosis characterized by substantial renal tubular and interstitial calcium deposition, and marked vascular calcification when compared with control mice. Although the osteopontin-deficient mice did not exhibit hypercalcemia or hyperphosphatemia, they did show abnormalities in the mineral metabolism hormone fibroblast growth factor-23. Thus, endogenous osteopontin plays a critical role in the prevention of phosphate-induced nephrocalcinosis and vascular calcification in response to high phosphate load. A better understanding of osteopontin's role in phosphate-induced calcification will hopefully lead to better biomarkers and therapies for this disease, especially in patients with chronic kidney disease and other at-risk populations.

Slc20a2 deficiency results in fetal growth restriction and placental calcification associated with thickened basement membranes and novel CD13 and lamininα1 expressing cells.

The essential nutrient phosphorus must be taken up by the mammalian embryo during gestation. The mechanism(s) and key proteins responsible for maternal to fetal phosphate transport have not been identified. Established parameters for placental phosphate transport match those of the type III phosphate transporters, Slc20a1 and Slc20a2. Both members are expressed in human placenta, and their altered expression is linked to preeclampsia. In this study, we tested the hypothesis that Slc20a2 is required for placental function. Indeed, complete deficiency of Slc20a2 in either the maternal or embryonic placental compartment results in fetal growth restriction. We found that Slc20a2 null mice can reproduce, but are subviable; ∼50% are lost prior to weaning age. We also observed that 23% of Slc20a2 deficient females develop pregnancy complications at full term, with tremors and placental abnormalities including abnormal vascular structure, increased basement membrane deposition, abundant calcification, and accumulation of novel CD13 and lamininα1 positive cells. Together these data support that Slc20a2 deficiency impacts both maternal and neonatal health, and Slc20a2 is required for normal placental function. In humans, decreased levels of placental Slc20a1 and Slc20a2 have been correlated with early onset preeclampsia, a disorder that can manifest from placental dysfunction. In addition, preterm placental calcification has been associated with poor pregnancy outcomes. We surveyed placental calcification in human preeclamptic placenta samples, and detected basement membrane-associated placental calcification as well as a comparable lamininα1 positive cell type, indicating that similar mechanisms may underlie both human and mouse placental calcification.

Runx2 Expression in Smooth Muscle Cells Is Required for Arterial Medial Calcification in Mice.

Arterial medial calcification (AMC) is a hallmark of aging, diabetes, and chronic kidney disease. Smooth muscle cell (SMC) transition to an osteogenic phenotype is a common feature of AMC, and is preceded by expression of runt-related transcription factor 2 (Runx2), a master regulator of bone development. Whether SMC-specific Runx2 expression is required for osteogenic phenotype change and AMC remains unknown. We therefore created an improved targeting construct to generate mice with floxed Runx2 alleles (Runx2(f/f)) that do not produce truncated Runx2 proteins after Cre recombination, thereby preventing potential off-target effects. SMC-specific deletion using SM22-recombinase transgenic allele mice (Runx2(ΔSM)) led to viable mice with normal bone and arterial morphology. After vitamin D overload, arterial SMCs in Runx2(f/f) mice expressed Runx2, underwent osteogenic phenotype change, and developed severe AMC. In contrast, vitamin D-treated Runx2(ΔSM) mice had no Runx2 in blood vessels, maintained SMC phenotype, and did not develop AMC. Runx2 deletion did not affect serum calcium, phosphate, fibroblast growth factor-23, or alkaline phosphatase levels. In vitro, Runx2(f/f) SMCs calcified to a much greater extent than those derived from Runx2(ΔSM) mice. These data indicate a critical role of Runx2 in SMC osteogenic phenotype change and mineral deposition in a mouse model of AMC, suggesting that Runx2 and downstream osteogenic pathways in SMCs may be useful therapeutic targets for treating or preventing AMC in high-risk patients.