Mitochondrial Dysfunction: Cause or Consequence of Vascular Calcification?

Mitochondria are crucial bioenergetics powerhouses and biosynthetic hubs within cells, which can generate and sequester toxic reactive oxygen species (ROS) in response to oxidative stress. Oxidative stress-stimulated ROS production results in ATP depletion and the opening of mitochondrial permeability transition pores, leading to mitochondria dysfunction and cellular apoptosis. Mitochondrial loss of function is also a key driver in the acquisition of a senescence-associated secretory phenotype that drives senescent cells into a pro-inflammatory state. Maintaining mitochondrial homeostasis is crucial for retaining the contractile phenotype of the vascular smooth muscle cells (VSMCs), the most prominent cells of the vasculature. Loss of this contractile phenotype is associated with the loss of mitochondrial function and a metabolic shift to glycolysis. Emerging evidence suggests that mitochondrial dysfunction may play a direct role in vascular calcification and the underlying pathologies including (1) impairment of mitochondrial function by mineral dysregulation i.e., calcium and phosphate overload in patients with end-stage renal disease and (2) presence of increased ROS in patients with calcific aortic valve disease, atherosclerosis, type-II diabetes and chronic kidney disease. In this review, we discuss the cause and consequence of mitochondrial dysfunction in vascular calcification and underlying pathologies; the role of autophagy and mitophagy pathways in preventing mitochondrial dysfunction during vascular calcification and finally we discuss mitochondrial ROS, DRP1, and HIF-1 as potential novel markers and therapeutic targets for maintaining mitochondrial homeostasis in vascular calcification.

Bone loss markers in the earliest Pacific Islanders.

Kingdom of Tonga in Polynesia is one of the most obese nations where metabolic conditions, sedentary lifestyles, and poor quality diet are widespread. These factors can lead to poor musculoskeletal health. However, whether metabolic abnormalities such as osteoporosis occurred in archaeological populations of Tonga is unknown. We employed a microscopic investigation of femur samples to establish whether bone loss afflicted humans in this Pacific region approximately 3000 years ago. Histology, laser confocal microscopy, and synchrotron Fourier-transform infrared microspectroscopy were used to measure bone vascular canal densities, bone porosity, and carbonate and phosphate content of bone composition in eight samples extracted from adult Talasiu males and females dated to 2650 BP. Compared to males, samples from females had fewer vascular canals, lower carbonate and phosphate content, and higher bone porosity. Although both sexes showed evidence of trabecularised cortical bone, it was more widespread in females (35.5%) than males (15.8%). Our data suggest experiences of advanced bone resorption, possibly as a result of osteoporosis. This provides first evidence for microscopic bone loss in a sample of archaeological humans from a Pacific population widely afflicted by metabolic conditions today.

Asymmetric midshaft femur remodeling in an adult male with left sided hip joint ankylosis, Metal Period Nagsabaran, Philippines.

OBJECTIVE: This study investigated microstructural changes of the right and left midshaft femur in an archaeological individual afflicted with left-sided hip joint ankylosis to assess whether increased cortical porosity was present as a result of leg disuse. MATERIALS: The individual is a middle-aged adult male excavated from the Metal Period (∼2000 BP) Nagsabaran, Luzon Island, Philippines. METHODS: Following standard examination of femur gross anatomy and differential diagnosis of the hip joint fusion, ∼1 cm thick posterior midshaft femur samples were removed for microstructural examination. Using static histomorphometry, bone multi-cellular unit activity from Haversian canal (vascular pore) density, area, and circularity was reconstructed. Spatial positioning of Haversian canals was mapped using Geographic Information Systems software. Phosphate, carbonate, and carbonate:phosphate ratios were obtained using synchrotron-sourced Fourier transform infrared microspectroscopy. RESULTS: The left femur had greater cortical pore density, with smaller and rounder vascular canals, in addition to lower matrix levels of phosphate and carbonate, when compared to the right femur. CONCLUSIONS: Our data indicate compromised bone tissue in the left femur, and conform to expected bone functional adaptation paradigms of remodeling responses to pathological and biomechanical changes. SIGNIFICANCE: The preservation of this individual's hip abnormality created a unique opportunity to evaluate intra-skeletal bone health asymmetry, which may help other researchers evaluate the presence of limb disuse in archaeological samples. LIMITATIONS: A lack of lower limb data limits our interpretations to femur remodeling only. SUGGESTIONS FOR FURTHER RESEARCH: Future research efforts should aim to examine the presence of remodeling changes in all bones of the lower limb. LAYUNIN: Gamit ang buto ng magkabilang pemur ng isang taong natagpuan sa isang archaeological site na may sakit na ankylosis sa kaliwang balakang, pinag-aralan ang iba't-ibang microstructures galing sa gitnang bahagi o midshaft ng pemur upang malaman kung may makikitang mataas na cortical porosity ang buto dahil hindi ito malimit gamitin. GAMIT: Ang pinag-aaralang buto ay galing sa isang indibidwal na tinatayang middle-age na lalaki na namuhay noong Panahon ng Metal (∼2000 BP) sa Nagsabaran, Cagayan, Republika ng Pilipinas. PAMAMARAAN: Matapos ang unang pagkilatis sa femur at ang pagkilala ng sakit sa balakang, kumuha ng ∼1 sentimetro ng buto galing sa midshaft ng pemur upang lalong mapag-aralan ang kanyang microstructure. Gamit ang static histomorphometry, napag-aralan ang mga naiwang bakas ng multi-cellular unit activity ayon sa kapal, laki at pagkakabilog ng Haversian canal (vascular pore). Gumamit din ng Geographic Information Systems (GIS) software upang mapag-aralan ang kaugnayan ng posisyon ng Haversian canal. Panghuli, gumamit din ng synchroton-sourced Fourier transform infrared (sFTIR) microspectroscopy upang makuha ang bilang ng phosphate, carbonate, at carbonate:phosphate ratio. RESULTA: Napag-alaman na ang kaliwang pemur ay mayroong higit na maraming cortical pores, maliit at mabilog na vascular canals, at mababang bilang ng phosphate, carbonate kung ihahambing sa kanang pemur. KONKLUSYON: Ayon sa aming datos, ang kaliwang pemur ay umaayon sa mga katangian ng isang butong may sakit. Sumunod din ito sa inaasahang bone functional adaptation paradigms of remodeling ng buto dahil may sakit at hindi nagamit. KAHALAGAHAN: Dahil maganda ang pagkakalibing ng buto ng balakang, nagkaroon ng pagkakataong makilatis ang kalusugan ng sinaunang-tao sa pamamagitan ng pag-aaral ng kalusugan ng buto. Dagdag pa, makakatulong din ito upang malaman kung ibang mananaliksik ang pag-aaral ng ibang butong hindi nagagamit mula sa archaeological site. LIMITASYON: Dahil walang nakuhang ibang buto mula sa binti at paa, ang pemur lang ang naimbestigahan. MUNGKAHI PARA SA MGA SUSUNOD NA PAG-AARAL: Kung magkakaroon ng pagkakataon sa susunod, dapat maimbistigahan ang lahat ng buto ng binti (lower limb).

Author Correction: Increased autophagy in EphrinB2-deficient osteocytes is associated with elevated secondary mineralization and brittle bone.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Increased autophagy in EphrinB2-deficient osteocytes is associated with elevated secondary mineralization and brittle bone.

Mineralized bone forms when collagen-containing osteoid accrues mineral crystals. This is initiated rapidly (primary mineralization), and continues slowly (secondary mineralization) until bone is remodeled. The interconnected osteocyte network within the bone matrix differentiates from bone-forming osteoblasts; although osteoblast differentiation requires EphrinB2, osteocytes retain its expression. Here we report brittle bones in mice with osteocyte-targeted EphrinB2 deletion. This is not caused by low bone mass, but by defective bone material. While osteoid mineralization is initiated at normal rate, mineral accrual is accelerated, indicating that EphrinB2 in osteocytes limits mineral accumulation. No known regulators of mineralization are modified in the brittle cortical bone but a cluster of autophagy-associated genes are dysregulated. EphrinB2-deficient osteocytes displayed more autophagosomes in vivo and in vitro, and EphrinB2-Fc treatment suppresses autophagy in a RhoA-ROCK dependent manner. We conclude that secondary mineralization involves EphrinB2-RhoA-limited autophagy in osteocytes, and disruption leads to a bone fragility independent of bone mass.

Isolation, Purification, Generation, and Culture of Osteocytes.

Osteocytes reside within bone matrix and produce both paracrine and endocrine factors that influence the skeleton and other tissues. Despite their abundance and physiological importance, osteocytes have been difficult to study in vitro because they are difficult to extract and purify, and do not retain their phenotype in standard culture conditions. However, new techniques for this purpose are emerging. This chapter will describe three methods we use to study osteocytes: (1) isolating and purifying primary osteocytes from murine bone, with and without hematopoietic-lineage depletion, (2) differentiating cultured osteoblasts (or osteoblast cell lines) until they reach a stage of osteocytic gene expression, and (3) using the Ocy454 osteocyte-like cell line.

Differing Effects of Parathyroid Hormone, Alendronate, and Odanacatib on Bone Formation and on the Mineralization Process in Intracortical and Endocortical Bone of Ovariectomized Rabbits.

Bone is formed by deposition of a collagen-containing matrix (osteoid) that hardens over time as mineral crystals accrue and are modified; this continues until bone remodeling renews that site. Pharmacological agents for osteoporosis differ in their effects on bone remodeling, and we hypothesized that they may differently modify bone mineral accrual. We, therefore, assessed newly formed bone in mature ovariectomized rabbits treated with the anti-resorptive bisphosphonate alendronate (ALN-100µ g/kg/2×/week), the anabolic parathyroid hormone (PTH (1-34)-15µ g/kg/5×/week), or the experimental anti-resorptive odanacatib (ODN 7.5 µM/day), which suppresses bone resorption without suppressing bone formation. Treatments were administered for 10 months commencing 6 months after ovariectomy (OVX). Strength testing, histomorphometry, and synchrotron Fourier-transform infrared microspectroscopy were used to measure bone strength, bone formation, and mineral accrual, respectively, in newly formed endocortical and intracortical bone. In Sham and OVX endocortical and intracortical bone, three modifications occurred as the bone matrix aged: mineral accrual (increase in mineral:matrix ratio), carbonate substitution (increase in carbonate:mineral ratio), and collagen molecular compaction (decrease in amide I:II ratio). ALN suppressed bone formation but mineral accrued normally at those sites where bone formation occurred. PTH stimulated bone formation on endocortical, periosteal, and intracortical bone surfaces, but mineral accrual and carbonate substitution were suppressed, particularly in intracortical bone. ODN treatment did not suppress bone formation, but newly deposited endocortical bone matured more slowly with ODN, and ODN-treated intracortical bone had less carbonate substitution than controls. In conclusion, these agents differ in their effects on the bone matrix. While ALN suppresses bone formation, it does not modify bone mineral accrual in endocortical or intracortical bone. While ODN does not suppress bone formation, it slows matrix maturation. PTH stimulates modelling-based bone formation not only on endocortical and trabecular surfaces, but may also do so in intracortical bone; at this site, new bone deposited contains less mineral than normal.

Anabolic action of parathyroid hormone (PTH) does not compromise bone matrix mineral composition or maturation.

Intermittent administration of parathyroid hormone (PTH) is used to stimulate bone formation in patients with osteoporosis. A reduction in the degree of matrix mineralisation has been reported during treatment, which may reflect either production of undermineralised matrix or a greater proportion of new matrix within the bone samples assessed. To explore these alternatives, high resolution synchrotron-based Fourier Transform Infrared Microspectroscopy (sFTIRM) coupled with calcein labelling was used in a region of non-remodelling cortical bone to determine bone composition during anabolic PTH treatment compared with region-matched samples from controls. 8week old male C57BL/6 mice were treated with vehicle or 50µg/kg PTH, 5 times/week for 4weeks (n=7-9/group). Histomorphometry confirmed greater trabecular and periosteal bone formation and 3-point bending tests confirmed greater femoral strength in PTH-treated mice. Dual calcein labels were used to match bone regions by time-since-mineralisation (bone age) and composition was measured by sFTIRM in six 15µm2 regions at increasing depth perpendicular to the most immature bone on the medial periosteal edge; this allowed in situ measurement of progressive changes in bone matrix during its maturation. The sFTIRM method was validated in vehicle-treated bones where the expected progressive increases in mineral:matrix ratio and collagen crosslink type ratio were detected with increasing bone maturity. We also observed a gradual increase in carbonate content that strongly correlated with an increase in longitudinal stretch of the collagen triple helix (amide I:amide II ratio). PTH treatment did not alter the progressive changes in any of these parameters from the periosteal edge through to the more mature bone. These data provide new information about how the bone matrix matures in situ and confirm that bone deposited during PTH treatment undergoes normal collagen maturation and normal mineral accrual.

RARγ is a negative regulator of osteoclastogenesis.

Vitamin A is known to influence post-natal bone content, with excess intake being associated with reduced bone mineral density and increased fracture risk. Despite this, the roles retinoids play in regulating osteoclastogenesis, particularly in vivo, remain unresolved. This study therefore aimed to determine the effect of loss of retinoic acid receptors (RAR)α or RARγ on bone mass (analyzed by histomorphometry and dual-energy X-ray absorptiometry) and osteoclastogenesis in mice in vivo. RARγ null mice had significantly less trabecular bone at 8 weeks of age compared to wildtype littermates. In contrast, no change in trabecular bone mass was detected in RARα null mice at this age. Further histomorphometric analysis revealed a significantly greater osteoclast surface in bones from 8-week-old RARγ null male mice. This in vivo effect was cell lineage autonomous, and was associated with increased osteoclastogenesis in vitro from hematopoietic cells obtained from 8-week-old RARγ null male mice. The use of highly selective agonists in RANKL-induced osteoclast differentiation of wild type mouse whole bone marrow cells and RAW264.7 cells in vitro showed a stronger inhibitory effect of RARγ than RARα agonists, suggesting that RARγ is a more potent inhibitor of osteoclastogenesis. Furthermore, NFAT activation was also more strongly inhibited by RARγ than RARα agonists. While RARα and RARγ antagonists did not significantly affect osteoclast numbers in vitro, larger osteoclasts were observed in cultures stimulated with the antagonists, suggesting increased osteoclast fusion. Further investigation into the effect of retinoids in vivo revealed that oral administration of 5mg/kg/day ATRA for 10 days protected against bone loss induced by granulocyte colony-stimulating factor (G-CSF) by inhibiting the pro-osteoclastogenic action of G-CSF. Collectively, our data indicates a physiological role for RARγ as a negative regulator of osteoclastogenesis in vivo and in vitro, and reveals distinct influences of RARα and RARγ in bone structure regulation.

Ubiquitous expression of the Pik3caH1047R mutation promotes hypoglycemia, hypoinsulinemia, and organomegaly.

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K, are among the most common mutations found in human cancer and have also recently been implicated in a range of overgrowth syndromes in humans. We have used a novel inducible "exon-switch" approach to knock in the constitutively active Pik3ca(H1047R) mutation into the endogenous Pik3ca gene of the mouse. Ubiquitous expression of the Pik3ca(H1047R) mutation throughout the body resulted in a dramatic increase in body weight within 3 weeks of induction (mutant 150 ± 5%; wild-type 117 ± 3%, mean ± sem), which was associated with increased organ size rather than adiposity. Severe metabolic effects, including a reduction in blood glucose levels to 59 ± 4% of baseline (11 days postinduction) and undetectable insulin levels, were also observed. Pik3ca(H1047R) mutant mice died earlier (median survival 46.5 d post-mutation induction) than wild-type control mice (100% survival > 250 days). Although deletion of Akt2 increased median survival by 44%, neither organ overgrowth, nor hypoglycemia were rescued, indicating that both the growth and metabolic functions of constitutive PI3K activity can be Akt2 independent. This mouse model demonstrates the critical role of PI3K in the regulation of both organ size and glucose metabolism at the whole animal level.

EphrinB2 signaling in osteoblasts promotes bone mineralization by preventing apoptosis.

Cells that form bone (osteoblasts) express both ephrinB2 and EphB4, and previous work has shown that pharmacological inhibition of the ephrinB2/EphB4 interaction impairs osteoblast differentiation in vitro and in vivo. The purpose of this study was to determine the role of ephrinB2 signaling in the osteoblast lineage in the process of bone formation. Cultured osteoblasts from mice with osteoblast-specific ablation of ephrinB2 showed delayed expression of osteoblast differentiation markers, a finding that was reproduced by ephrinB2, but not EphB4, RNA interference. Microcomputed tomography, histomorphometry, and mechanical testing of the mice lacking ephrinB2 in osteoblasts revealed a 2-fold delay in bone mineralization, a significant reduction in bone stiffness, and a 50% reduction in osteoblast differentiation induced by anabolic parathyroid hormone (PTH) treatment, compared to littermate sex- and age-matched controls. These defects were associated with significantly lower mRNA levels of late osteoblast differentiation markers and greater levels of osteoblast and osteocyte apoptosis, indicated by TUNEL staining and transmission electron microscopy of bone samples, and a 2-fold increase in annexin V staining and 7-fold increase in caspase 8 activation in cultured ephrinB2 deficient osteoblasts. We conclude that osteoblast differentiation and bone strength are maintained by antiapoptotic actions of ephrinB2 signaling within the osteoblast lineage.

Regulation of cortical and trabecular bone mass by communication between osteoblasts, osteocytes and osteoclasts.

The size and strength of bone is determined by two fundamental processes. One process, bone remodelling, renews the skeleton throughout life. In this process existing bone is resorbed by osteoclasts and replaced, in the same location, by osteoblasts. The other process is bone modelling, where bone formation and resorption occur at different sites so that the shape of bone is changed. Recent data suggests that both remodelling and modelling are controlled by signals between the cells that carry out these two processes. Osteoclasts both resorb bone, and provide inhibitory and stimulatory signals, including cardiotrophin-1 and sphingosine-1-kinase, to the osteoblast lineage thereby regulating their differentiation and activity on both trabecular and cortical surfaces. In addition, the osteoblast lineage, including osteoblast progenitors, matrix-producing osteoblasts, bone lining cells, and matrix-embedded osteocytes, produce both inhibitory and stimulatory factors that stimulate osteoclast differentiation. We will discuss the roles of osteoblast- and osteocyte-derived RANKL, and paracrine, autocrine and endocrine factors, such as ephrinB2, the IL-6/gp130 family of cytokines, parathyroid hormone, and its related peptide, PTHrP. These factors not only stimulate RANKL production, but also stimulate osteoblast differentiation and activity. This review will focus on recent data, generated from pharmacological and genetic studies of mouse models and what these data reveal about these pathways at different stages of osteoblast differentiation and their impact on both bone remodelling and modelling in trabecular and cortical bone.

The primary function of gp130 signaling in osteoblasts is to maintain bone formation and strength, rather than promote osteoclast formation.

Interleukin-6 (IL-6) family cytokines act via gp130 in the osteoblast lineage to stimulate the formation of osteoclasts (bone resorbing cells) and the activity of osteoblasts (bone forming cells), and to inhibit expression of the osteocyte protein, sclerostin. We report here that a profound reduction in trabecular bone mass occurs both when gp130 is deleted in the entire osteoblast lineage (Osx1Cre gp130 f/f) and when this deletion is restricted to osteocytes (DMP1Cre gp130 f/f). This was caused not by an alteration in osteoclastogenesis, but by a low level of bone formation specific to the trabecular compartment. In contrast, cortical diameter increased to maintain ultimate bone strength, despite a reduction in collagen type 1 production. We conclude that osteocytic gp130 signaling is required for normal trabecular bone mass and proper cortical bone composition.