Toward understanding the cellular control of vertebrate mineralization: The potential role of mitochondria.

This review examines the possible role of mitochondria in maintaining calcium and phosphate ion homeostasis and participating in the mineralization of bone, cartilage and other vertebrate hard tissues. The paper builds on the known structural features of mitochondria and the documented observations in these tissues that the organelles contain calcium phosphate granules. Such deposits in mitochondria putatively form to buffer excessively high cytosolic calcium ion concentrations and prevent metabolic deficits and even cell death. While mitochondria protect cytosolic enzyme systems through this buffering capacity, the accumulation of calcium ions by mitochondria promotes the activity of enzymes of the tricarboxylic acid (TCA/Krebs) cycle, increases oxidative phosphorylation and ATP synthesis, and leads to changes in intramitochondrial pH. These pH alterations influence ion solubility and possibly the transitions and composition in the mineral phase structure of the granules. Based on these considerations, mitochondria are proposed to support the mineralization process by providing a mobile store of calcium and phosphate ions, in smaller cluster or larger granule form, while maintaining critical cellular activities. The rise in the mitochondrial calcium level also increases the generation of citrate and other TCA cycle intermediates that contribute to cell function and the development of extracellular mineral. This paper suggests that another key role of the mitochondrion, along with the effects just noted, is to supply phosphate ions, derived from the breakdown of ATP, to endolysosomes and autophagic vesicles originating in the endoplasmic reticulum and Golgi and at the plasma membrane. These many separate but interdependent mitochondrial functions emphasize the critical importance of this organelle in the cellular control of vertebrate mineralization.

Microbubble cavitation restores Staphylococcus aureus antibiotic susceptibility in vitro and in a septic arthritis model.

Treatment failure in joint infections is associated with fibrinous, antibiotic-resistant, floating and tissue-associated Staphylococcus aureus aggregates formed in synovial fluid (SynF). We explore whether antibiotic activity could be increased against Staphylococcus aureus aggregates using ultrasound-triggered microbubble destruction (UTMD), in vitro and in a porcine model of septic arthritis. In vitro, when bacterially laden SynF is diluted, akin to the dilution achieved clinically with lavage and local injection of antibiotics, amikacin and ultrasound application result in increased bacterial metabolism, aggregate permeabilization, and a 4-5 log decrease in colony forming units, independent of microbubble destruction. Without SynF dilution, amikacin + UTMD does not increase antibiotic activity. Importantly, in the porcine model of septic arthritis, no bacteria are recovered from the SynF after treatment with amikacin and UTMD-ultrasound without UTMD is insufficient. Our data suggest that UTMD + antibiotics may serve as an important adjunct for the treatment of septic arthritis.

GLUT1 is redundant in hypoxic and glycolytic nucleus pulposus cells of the intervertebral disc.

Glycolysis is central to homeostasis of nucleus pulposus (NP) cells in the avascular intervertebral disc. Since the glucose transporter, GLUT1, is a highly enriched phenotypic marker of NP cells, we hypothesized that it is vital for the development and postnatal maintenance of the disc. Surprisingly, primary NP cells treated with 2 well-characterized GLUT1 inhibitors maintained normal rates of glycolysis and ATP production, indicating intrinsic compensatory mechanisms. We showed in vitro that NP cells mitigated GLUT1 loss by rewiring glucose import through GLUT3. Of note, we demonstrated that substrates, such as glutamine and palmitate, did not compensate for glucose restriction resulting from dual inhibition of GLUT1/3, and inhibition compromised long-term cell viability. To investigate the redundancy of GLUT1 function in NP, we generated 2 NP-specific knockout mice: Krt19CreERT Glut1fl/fl and Foxa2Cre Glut1fl/fl. There were no apparent defects in postnatal disc health or development and maturation in mutant mice. Microarray analysis verified that GLUT1 loss did not cause transcriptomic alterations in the NP, supporting that cells are refractory to GLUT1 loss. These observations provide the first evidence to our knowledge of functional redundancy in GLUT transporters in the physiologically hypoxic intervertebral disc and underscore the importance of glucose as the indispensable substrate for NP cells.

Conditional Deletion of HIF-2α in Mouse Nucleus Pulposus Reduces Fibrosis and Provides Mild and Transient Protection From Age-Dependent Structural Changes in Intervertebral Disc.

Hypoxia-inducible factors (HIFs) are critical to the development and homeostasis of hypoxic tissues. Although HIF-2α, one of the main HIF-α isoforms, is expressed in nucleus pulposus (NP) cells, its functions remain unknown. We deleted HIF-2α in the NP tissue using a notochord-specific FoxA2Cre allele to study HIF-2α function in the adult intervertebral disc. Unlike observations in HIF-1αcKO mice, fate mapping studies using Rosa26-mTmG reporter showed that HIF-2α loss in NP did not negatively impact cell survival or affect compartment development. Rather, loss of HIF-2α resulted in slightly better attributes of NP morphology in 14-month-old HIF-2αcKO mice as evident from lower scores of degeneration. These 14-month-old HIF-2αcKO mice also exhibited significant reduction in NP tissue fibrosis and lower collagen turnover in the annulus fibrosis (AF) compartment. Imaging-Fourier transform-infrared (FTIR) analyses showed decreased collagen and protein content in the NP and maintained chondroitin sulfate levels in 14-month-old HIF-2αcKO . Mechanistically, global transcriptomic analysis showed enrichment of differentially expressed genes with Gene Ontology (GO) terms related to metabolic processes and cell development, molecular functions concerned with histone and protein binding, and associated pathways, including oxidative stress. Noteworthy, these morphological differences were not apparent in 24-month-old HIF-2αcKO , indicating that aging is the dominant factor in governing disc health. Together these data suggest that loss of HIF-2α in the NP compartment is not detrimental to the intervertebral disc development but rather mitigates NP tissue fibrosis and offers mild but transient protection from age-dependent early degenerative changes. © 2022 American Society for Bone and Mineral Research (ASBMR).

Abcc6 Null Mice-a Model for Mineralization Disorder PXE Shows Vertebral Osteopenia Without Enhanced Intervertebral Disc Calcification With Aging.

Chronic low back pain is a highly prevalent health condition intricately linked to intervertebral disc degeneration. One of the prominent features of disc degeneration that is commonly observed with aging is dystrophic calcification. ATP-binding cassette sub-family C member 6 (ABCC6), a presumed ATP efflux transporter, is a key regulator of systemic levels of the mineralization inhibitor pyrophosphate (PPi). Mutations in ABCC6 result in pseudoxanthoma elasticum (PXE), a progressive human metabolic disorder characterized by mineralization of the skin and elastic tissues. The implications of ABCC6 loss-of-function on pathological mineralization of structures in the spine, however, are unknown. Using the Abcc6 -/- mouse model of PXE, we investigated age-dependent changes in the vertebral bone and intervertebral disc. Abcc6 -/- mice exhibited diminished trabecular bone quality parameters at 7 months, which remained significantly lower than the wild-type mice at 18 months of age. Abcc6 -/- vertebrae showed increased TRAP staining along with decreased TNAP staining, suggesting an enhanced bone resorption as well as decreased bone formation. Surprisingly, however, loss of ABCC6 resulted only in a mild, aging disc phenotype without evidence of dystrophic mineralization. Finally, we tested the utility of oral K3Citrate to treat the vertebral phenotype since it is shown to regulate hydroxyapatite mechanical behavior. The treatment resulted in inhibition of the osteoclastic response and an early improvement in mechanical properties of the bone underscoring the promise of potassium citrate as a therapeutic agent. Our data suggest that although ectopic mineralization is tightly regulated in the disc, loss of ABCC6 compromises vertebral bone quality and dysregulates osteoblast-osteoclast coupling.

Long-term treatment with senolytic drugs Dasatinib and Quercetin ameliorates age-dependent intervertebral disc degeneration in mice.

Intervertebral disc degeneration is highly prevalent within the elderly population and is a leading cause of chronic back pain and disability. Due to the link between disc degeneration and senescence, we explored the ability of the Dasatinib and Quercetin drug combination (D + Q) to prevent an age-dependent progression of disc degeneration in mice. We treated C57BL/6 mice beginning at 6, 14, and 18 months of age, and analyzed them at 23 months of age. Interestingly, 6- and 14-month D + Q cohorts show lower incidences of degeneration, and the treatment results in a significant decrease in senescence markers p16INK4a, p19ARF, and SASP molecules IL-6 and MMP13. Treatment also preserves cell viability, phenotype, and matrix content. Although transcriptomic analysis shows disc compartment-specific effects of the treatment, cell death and cytokine response pathways are commonly modulated across tissue types. Results suggest that senolytics may provide an attractive strategy to mitigating age-dependent disc degeneration.

The role of HIF proteins in maintaining the metabolic health of the intervertebral disc.

The physiologically hypoxic intervertebral disc and cartilage rely on the hypoxia-inducible factor (HIF) family of transcription factors to mediate cellular responses to changes in oxygen tension. During homeostatic development, oxygen-dependent prolyl hydroxylases, circadian clock proteins and metabolic intermediates control the activities of HIF1 and HIF2 in these tissues. Mechanistically, HIF1 is the master regulator of glycolytic metabolism and cytosolic lactate levels. In addition, HIF1 regulates mitochondrial metabolism by promoting flux through the tricarboxylic acid cycle, inhibiting downsteam oxidative phosphorylation and controlling mitochondrial health through modulation of the mitophagic pathway. Accumulation of metabolic intermediates from HIF-dependent processes contribute to intracellular pH regulation in the disc and cartilage. Namely, to prevent changes in intracellular pH that could lead to cell death, HIF1 orchestrates a bicarbonate buffering system in the disc, controlled by carbonic anhydrase 9 (CA9) and CA12, sodium bicarbonate cotransporters and an intracellular H+/lactate efflux mechanism. In contrast to HIF1, the role of HIF2 remains elusive; in disorders of the disc and cartilage, its function has been linked to both anabolic and catabolic pathways. The current knowledge of hypoxic cell metabolism and regulation of HIF1 activity provides a strong basis for the development of future therapies designed to repair the degenerative disc.

Hypoxia and Hypoxia-Inducible Factor-1α Regulate Endoplasmic Reticulum Stress in Nucleus Pulposus Cells: Implications of Endoplasmic Reticulum Stress for Extracellular Matrix Secretion.

Endoplasmic reticulum (ER) stress is shown to promote nucleus pulposus (NP) cell apoptosis and intervertebral disc degeneration. However, little is known about ER stress regulation by the hypoxic disc microenvironment and its contribution to extracellular matrix homeostasis. NP cells were cultured under hypoxia (1% partial pressure of oxygen) to assess ER stress status, and gain-of-function and loss-of-function approaches were used to assess the role of hypoxia-inducible factor (HIF)-1α in this pathway. In addition, the contribution of ER stress induction on the NP cell secretome was assessed by a nontargeted quantitative proteomic analysis by sequential windowed data independent acquisition of the total high-resolution mass spectra-mass spectrometry. NP cells exhibited a lower ER stress burden under hypoxia. Knockdown of HIF-1α increased C/EBP homologous protein, protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) levels, whereas HIF-1α stabilization decreased the expression of ER stress markers Ddit3, Hsp5a, Atf6, and Eif2a. Interestingly, ER stress inducers tunicamycin and thapsigargin induced HIF-1α activity under hypoxia while promoting the unfolded protein response. NP cell secretome analysis demonstrated an impact of ER stress induction on extracellular matrix secretion, with decreases in collagens and cell adhesion-related proteins. Moreover, analysis of transcriptomic data of NP tissues from aged mice and degenerated human discs showed higher levels of unfolded protein response markers and decreased levels of matrix components. Our study shows, for the first time, that hypoxia and HIF-1α attenuate ER stress responses in NP cells, and ER stress promotes inefficient extracellular matrix secretion under hypoxia.

Sox9 deletion causes severe intervertebral disc degeneration characterized by apoptosis, matrix remodeling, and compartment-specific transcriptomic changes.

SOX9 plays an important role in chondrocyte differentiation and, in the developing axial skeleton, maintains the notochord and the demarcation of intervertebral disc compartments. Diminished expression is linked to campomelic dysplasia, resulting in severe scoliosis and progressive disc degeneration. However, the specific functions of SOX9 in the adult spinal column and disc are largely unknown. Accordingly, employing a strategy to conditionally delete Sox9 in Acan-expressing cells (AcanCreERT2Sox9fl/fl), we delineated these functions in the adult intervertebral disc. AcanCreERT2Sox9fl/fl mice (Sox9cKO) showed extensive and progressive remodeling of the extracellular matrix in nucleus pulposus (NP) and annulus fibrosus (AF), consistent with human disc degeneration. Progressive degeneration of the cartilaginous endplates (EP) was also evident in Sox9cKO mice, and it preceded morphological changes seen in the NP and AF compartments. Fate mapping using tdTomato reporter, EdU chase, and quantitative immunohistological studies demonstrated that SOX9 is crucial for disc cell survival and phenotype maintenance. Microarray analysis showed that Sox9 regulated distinct compartment-specific transcriptomic landscapes, with prominent contributions to the ECM, cytoskeleton-related, and metabolic pathways in the NP and ion transport, the cell cycle, and signaling pathways in the AF. In summary, our work provides new insights into disc degeneration in Sox9cKO mice at the cellular, molecular, and transcriptional levels, underscoring tissue-specific roles of this transcription factor. Our findings may direct future cell therapies targeting SOX9 to mitigate disc degeneration.

Comparison of inbred mouse strains shows diverse phenotypic outcomes of intervertebral disc aging.

Intervertebral disc degeneration presents a wide spectrum of clinically degenerative disc phenotypes; however, the contribution of genetic background to the degenerative outcomes has not been established. We characterized the spinal phenotype of 3 mouse strains with varying cartilage-regenerative potential at 6 and 23 months: C57BL/6, LG/J and SM/J. All strains showed different aging phenotypes. Importantly, LG/J mice showed an increased prevalence of dystrophic disc calcification in caudal discs with aging. Quantitative-histological analyses of LG/J and SM/J caudal discs evidenced accelerated degeneration compared to BL6, with cellular disorganization and cell loss together with fibrosis of the NP, respectively. Along with the higher grades of disc degeneration, SM/J, at 6M, also differed the most in terms of NP gene expression compared to other strains. Moreover, although we found common DEGs between BL6 and LG/J aging, most of them were divergent between the strains. Noteworthy, the common DEGs altered in both LG/J and BL6 aging were associated with inflammatory processes, response to stress, cell differentiation, cell metabolism and cell division. Results suggested that disc calcification in LG/J resulted from a dystrophic calcification process likely aggravated by cell death, matrix remodelling, changes in calcium/phosphate homeostasis and cell transformation. Lastly, we report 7 distinct phenotypes of human disc degeneration based on transcriptomic profiles, that presented similar pathways and DEGs found in aging mouse strains. Together, our results suggest that disc aging and degeneration depends on the genetic background and involves changes in various molecular pathways, which might help to explain the diverse phenotypes seen during disc disease.

Hypoxic Regulation of Mitochondrial Metabolism and Mitophagy in Nucleus Pulposus Cells Is Dependent on HIF-1α-BNIP3 Axis.

Nucleus pulposus (NP) cells reside in an avascular and hypoxic microenvironment of the intervertebral disc and are predominantly glycolytic due to robust HIF-1 activity. It is generally thought that NP cells contain few functional mitochondria compared with cells that rely on oxidative metabolism. Consequently, the contribution of mitochondria to NP cell metabolism and the role of hypoxia and HIF-1 in mitochondrial homeostasis is poorly understood. Using mitoQC reporter mice, we show for the first time to our knowledge that NP cell mitochondria undergo age-dependent mitophagy in vivo. Mechanistically, in vitro studies suggest that, under hypoxic conditions, mitochondria in primary NP cells undergo HIF-1α-dependent fragmentation, controlled by modulating the levels of key proteins DRP1 and OPA1 that are involved in mitochondrial fission and fusion, respectively. Seahorse assays and steady state metabolic profiling coupled with [1-2-13 C]-glucose flux analysis revealed that in hypoxia, HIF-1α regulated metabolic flux through coordinating glycolysis and the mitochondrial TCA cycle interactions, thereby controlling the overall biosynthetic capacity of NP cells. We further show that hypoxia and HIF-1α trigger mitophagy in NP cells through the mitochondrial translocation of BNIP3, an inducer of receptor-mediated mitophagy. Surprisingly, however, loss of HIF-1α in vitro and analysis of NP-specific HIF-1α null mice do not show a decrease in mitophagic flux in NP cells but a compensatory increase in NIX and PINK1-Parkin pathways with higher mitochondrial number. Taken together, our studies provide novel mechanistic insights into the complex interplay between hypoxia and HIF-1α signaling on the mitochondrial metabolism and quality control in NP cells. © 2020 American Society for Bone and Mineral Research.

Arp2/3 inactivation causes intervertebral disc and cartilage degeneration with dysregulated TonEBP-mediated osmoadaptation.

Extracellular matrix and osmolarity influence the development and homeostasis of skeletal tissues through Rho GTPase-mediated alteration of the actin cytoskeleton. This study investigated whether the actin-branching Arp2/3 complex, a downstream effector of the Rho GTPases Cdc42 and Rac1, plays a critical role in maintaining the health of matrix-rich and osmotically loaded intervertebral discs and cartilage. Mice with constitutive intervertebral disc- and cartilage-specific deletion of the critical Arp2/3 subunit Arpc2 (Col2-Cre; Arpc2fl/fl) developed chondrodysplasia and spinal defects. Since these mice did not survive to adulthood, we generated mice with inducible Arpc2 deletion in disc and cartilage (Acan-CreERT2; Arpc2fl/fl). Inactivation of Arp2/3 at skeletal maturity resulted in growth plate closure, loss of proteoglycan content in articular cartilage, and degenerative changes in the intervertebral disc at 1 year of age. Chondrocytes with Arpc2 deletion showed compromised cell spreading on both collagen and fibronectin. Pharmacological inhibition of Cdc42 and Arp2/3 prevented the osmoadaptive transcription factor TonEBP/NFAT5 from recruiting cofactors in response to a hyperosmolarity challenge. Together, these findings suggest that Arp2/3 plays a critical role in cartilaginous tissues through the regulation of cell-extracellular matrix interactions and modulation of TonEBP-mediated osmoadaptation.

Lactate Efflux From Intervertebral Disc Cells Is Required for Maintenance of Spine Health.

Maintenance of glycolytic metabolism is postulated to be required for health of the spinal column. In the hypoxic tissues of the intervertebral disc and glycolytic cells of vertebral bone, glucose is metabolized into pyruvate for ATP generation and reduced to lactate to sustain redox balance. The rise in intracellular H+ /lactate concentrations are balanced by plasma-membrane monocarboxylate transporters (MCTs). Using MCT4 null mice and human tissue samples, complemented with genetic and metabolic approaches, we determine that H+ /lactate efflux is critical for maintenance of disc and vertebral bone health. Mechanistically, MCT4 maintains glycolytic and tricarboxylic acid (TCA) cycle flux and intracellular pH homeostasis in the nucleus pulposus compartment of the disc, where hypoxia-inducible factor 1α (HIF-1α) directly activates an intronic enhancer in SLC16A3. Ultimately, our results provide support for research into lactate as a diagnostic biomarker for chronic, painful, disc degeneration. © 2019 American Society for Bone and Mineral Research.

Differential Effect of Long-Term Systemic Exposure of TNFα on Health of the Annulus Fibrosus and Nucleus Pulposus of the Intervertebral Disc.

The inflammatory cytokine tumor necrosis factor alpha (TNFα) is considered to play a key role in the pathogenesis of intervertebral disc disease. To evaluate the importance of this cytokine we examined the inflammatory environment and spinal phenotype of 9-month-old human TNFα overexpressing transgenic (hTNFα-TG) mice. The mice evidenced increased circulating levels of interleukin-1ß (IL-1ß), IL-2, keratinocyte chemoattractant/human growth-regulated oncogene (KC/GRO), and monocyte chemoattractant protein-1 (MCP-1) along with thinning of the cortical and trabecular vertebral bone. Surprisingly, although the nucleus pulposus (NP) of these mice was intact and healthy, the caudal annulus fibrosus (AF) evidenced robust cell death and immune cell infiltration. Despite these differences, there were no obvious alterations in the collagen or aggrecan content in the NP and AF. However, there was a reduction in cartilage oligomeric matrix protein (COMP), suggesting destabilization of the AF matrix. Microarray analysis of the NP from hTNFα-TG mice cells revealed minimal changes in global gene expression. These findings lend support to the notion that NP tissue is isolated from systemic inflammation. In contrast, the severe AF phenotype suggests that systemic inflammation interferes with AF health, predisposing discs to herniation as opposed to directly causing NP degeneration. © 2020 American Society for Bone and Mineral Research.

TonEBP-deficiency accelerates intervertebral disc degeneration underscored by matrix remodeling, cytoskeletal rearrangements, and changes in proinflammatory gene expression.

The tonicity-responsive enhancer binding protein (TonEBP) plays an important role in intervertebral disc and axial skeleton embryogenesis. However, the contribution of this osmoregulatory transcription factor in postnatal intervertebral disc homeostasis is not known in vivo. Here, we show for the first time that TonEBP-deficient mice have pronounced age-related degeneration of the intervertebral disc with annular and endplate herniations. Using FTIR-imaging spectroscopy, quantitative immunohistochemistry, and tissue-specific transcriptomic analysis, we provide morphological and molecular evidence that the overall phenotype is driven by a replacement of water-binding proteoglycans with fibrocartilaginous matrix. Whereas TonEBP deficiency in the AF compartment caused tissue fibrosis associated with alterations in actin cytoskeleton and adhesion molecules, predominant changes in pro-inflammatory pathways were seen in the NP compartment of mutants, underscoring disc compartment-specific effects. Additionally, TonEBP-deficient mice presented with compromised trabecular bone properties of vertebrae. These results provide the first in vivo support to the long-held hypothesis that TonEBP is crucial for postnatal homeostasis of the spine and controls a multitude of functions in addition to cellular osmoadaptation.

Nucleus pulposus primary cilia alter their length in response to changes in extracellular osmolarity but do not control TonEBP-mediated osmoregulation.

The nucleus pulposus (NP) cells adapt to their physiologically hyperosmotic microenvironment through Tonicity-responsive enhancer binding protein (TonEBP/nuclear factor of activated T-cell5 [NFAT5])-mediated osmoregulation. Primary cilia in different organs serve diverse roles including osmosensing, but its contribution to NP cell osmoadaptive response is unknown. A high percentage of cultured primary NP cells possessed primary cilia that changed length in response to osmotic stimuli. Stable silencing of Intraflagellar Transport 88 (Ift88) or Kinesin Family Member 3 A (Kif3a) to inhibit the formation of primary cilia did not affect hyperosmotic upregulation of TonEBP. While ShKif3a blocked hyperosmotic increase of TonEBP-Transactivation Domain (TAD) activity, overall the knockdown of either gene did not alter the hyperosmotic status of proximal promoter activities and transcription of key TonEBP targets. On the other hand, a small decrease in TonEBP level under hypoosmotic condition was attenuated by Ift88 or Kif3a knockdown. Noteworthy, none of the TonEBP target genes were responsive to hypoosmotic stimulus in control and Ift88 or Kif3a knockdown cells, suggesting the primary role of TonEBP in the hyperosmotic adaptation of NP cells. Similarly, in Kif3a null mouse embryonic fibroblasts (MEFs), the overall TonEBP-dependent hyperosmotic responses were preserved. Unlike NP cells, TonEBP targets were responsive to hypoosmolarity in wild-type MEFs, and these responses remained intact in Kif3a null MEFs. Together, these results suggest that primary cilia are dispensable for TonEBP-dependent osmoadaptive response.

Equine or porcine synovial fluid as a novel ex vivo model for the study of bacterial free-floating biofilms that form in human joint infections.

Bacterial invasion of synovial joints, as in infectious or septic arthritis, can be difficult to treat in both veterinary and human clinical practice. Biofilms, in the form of free-floating clumps or aggregates, are involved with the pathogenesis of infectious arthritis and periprosthetic joint infection (PJI). Infection of a joint containing an orthopedic implant can additionally complicate these infections due to the presence of adherent biofilms. Because of these biofilm phenotypes, bacteria within these infected joints show increased antimicrobial tolerance even at high antibiotic concentrations. To date, animal models of PJI or infectious arthritis have been limited to small animals such as rodents or rabbits. Small animal models, however, yield limited quantities of synovial fluid making them impractical for in vitro research. Herein, we describe the use of ex vivo equine and porcine models for the study of synovial fluid induced biofilm aggregate formation and antimicrobial tolerance. We observed Staphylococcus aureus and other bacterial pathogens adapt the same biofilm aggregate phenotype with significant antimicrobial tolerance in both equine and porcine synovial fluid, analogous to human synovial fluid. We also demonstrate that enzymatic dispersal of synovial fluid aggregates restores the activity of antimicrobials. Future studies investigating the interaction of bacterial cell surface proteins with host synovial fluid proteins can be readily carried out in equine or porcine ex vivo models to identify novel drug targets for treatment of prevention of these difficult to treat infectious diseases.

NFAT5/TonEBP controls early acquisition of notochord phenotypic markers, collagen composition, and sonic hedgehog signaling during mouse intervertebral disc embryogenesis.

High osmolarity, bound water, and hydrostatic pressure contribute to notochord mechanics and its morphogenesis into the nucleus pulposus (NP) compartment of the intervertebral disc. Indeed, the osmoadaptive transcription factor, nuclear factor of activated T-cells 5 (NFAT5 aka TonEBP), is robustly expressed by resident cells of the notochord and NP. Nevertheless, the molecular mechanisms that drive notochord osmoregulation and the functions of NFAT5 in disc embryogenesis remain largely unexplored. In this study, we show that deletion of NFAT5 in mice results in delayed vertebral column development and a reduced NP aspect ratio in the caudal spine. This phenotype is associated with lower levels of the T-box transcription factor, Brachyury, delayed expression of notochord phenotypic markers, and decreased collagen II deposition in the perinotochordal sheath and condensing mesenchyme. In addition, NFAT5 mutants showed a stage-dependent dysregulation of sonic hedgehog (Shh) signaling with non-classical expression of Gli1. Generation of mice with notochord-specific deletion of IFT88 (ShhcreERT2;Ift88f/f) supported this mode of Gli1 regulation. Using isolated primary NP cells and bioinformatics approaches, we further show that Ptch1 and Smo expression is controlled by NFAT5 in a cell autonomous manner. Altogether, our results demonstrate that NFAT5 contributes to notochord and disc embryogenesis through its regulation of hallmark notochord phenotypic markers, extracellular matrix, and Shh signaling.

Tendinosis develops from age- and oxygen tension-dependent modulation of Rac1 activity.

Age-related tendon degeneration (tendinosis) is characterized by a phenotypic change in which tenocytes display characteristics of fibrochondrocytes and mineralized fibrochondrocytes. As tendon degeneration has been noted in vivo in areas of decreased tendon vascularity, we hypothesized that hypoxia is responsible for the development of the tendinosis phenotype, and that these effects are more pronounced in aged tenocytes. Hypoxic (1% O2 ) culture of aged, tendinotic, and young human tenocytes resulted in a mineralized fibrochondrocyte phenotype in aged tenocytes, and a fibrochondrocyte phenotype in young and tendinotic tenocytes. Investigation of the molecular mechanism responsible for this phenotype change revealed that the fibrochondrocyte phenotype in aged tenocytes occurs with decreased Rac1 activity in response to hypoxia. In young hypoxic tenocytes, however, the fibrochondrocyte phenotype occurs with concomitant decreased Rac1 activity coupled with increased RhoA activity. Using pharmacologic and adenoviral manipulation, we confirmed that these hypoxic effects on the tenocyte phenotype are linked directly to the activity of RhoA/Rac1 GTPase in in vitro human cell culture and tendon explants. These results demonstrate that hypoxia drives tenocyte phenotypic changes, and provide a molecular insight into the development of human tendinosis that occurs with aging.

A New Understanding of the Role of IL-1 in Age-Related Intervertebral Disc Degeneration in a Murine Model.

Increased cytokine expression, in particular interleukin-1ß (IL-1ß), is considered a hallmark of intervertebral disc degeneration. However, the causative relationship between IL-1 and age-dependent degeneration has not been established. To investigate the role of IL-1 in driving age-related disc degeneration, we studied the spine phenotype of global IL-1α/ß double knockout (IL-1KO) mice at 12 and 20 months. Multiplex ELISA analysis of blood revealed significant reductions in the concentrations of IFN-γ, IL-5, IL-15, TNF-α, IP-10, and a trend of reduced concentrations of IL-10, macrophage inflammatory protein 1α (MIP-1α), keratinocyte chemoattractant/human growth-regulated oncogene (KC/GRO), and IL-6. However, the circulating level of MIP-2, a neutrophil chemoattractant, was increased in the IL-1KO. The alterations in systemic cytokine levels coincided with altered bone morphology-IL-1KO mice exhibited significantly thicker caudal cortical bone at 12 and 20 months. Despite these systemic inflammatory and bony changes, IL-1 deletion only minimally affected disc health. Both wild-type (WT) and IL-1KO mice showed age-dependent disc degeneration. Unexpectedly, rather than protecting the animals from degeneration, the aging phenotype was more pronounced in IL-1KO animals: knockout mice evidenced significantly more degenerative changes in the annulus fibrosis (AF) together with alterations in collagen type and maturity. At 20 months, there were no changes in nucleus pulposus (NP) extracellular matrix composition or cellular marker expression; however, the IL-1KO NP cells occupied a smaller proportion of the NP compartment that those of WT controls. Taken together, these results show that IL-1 deletion altered the systemic inflammatory environment and vertebral bone morphology. However, instead of protecting discs from age-related disc degeneration, global IL-1 deletion amplified the degenerative phenotype. © 2019 American Society for Bone and Mineral Research.