Targeting Synovial Lymphatic Function as a Novel Therapeutic Intervention for Age-Related Osteoarthritis in Mice.

OBJECTIVE: The synovial lymphatic system (SLS) removes catabolic factors from the joint. Vascular endothelial growth factor C (VEGF-C) and its receptor, VEGFR-3, are crucial for lymphangiogenesis. However, their involvement in age-related osteoarthritis (OA) is unknown. This study was undertaken to determine whether the SLS and the VEGF-C/VEGFR-3 pathway contribute to the development and progression of age-related OA, using a murine model of naturally occurring joint disease. METHODS: SLS function was assessed in the knees of young (3-month-old) and aged (19-24-month-old) male and female C57BL/6J mice via a newly established in vivo IVIS-dextran imaging approach, which, in addition to histology, was used to assess the effects of VEGF-C treatment on SLS function and OA pathology in aged mice. RNA-sequencing of synovial tissue was performed to explore molecular mechanisms of the disease in the mouse knee joints. RESULTS: Results showed that aged mice had impaired SLS function, including decreases in joint clearance (mean T1/2 of signal intensity clearance, 2.8 hours in aged mice versus 0.5 hours in young mice; P < 0.0001), synovial influx (mean ± SD 1.7 ± 0.8% in aged mice versus 4.1 ± 1.9% in young mice; P = 0.0004), and lymph node draining capacity (mean ± SD epifluorescence total radiant intensity ([photons/second]/[µW/cm2 ]) 1.4 ± 0.8 in aged mice versus 3.7 ± 1.2 in young mice; P < 0.0001). RNA-sequencing of the synovial tissue showed that Vegf-c and Vegfr3 signaling genes were decreased in the synovium of aged mice. VEGF-C treatment resulted in improvements in SLS function in aged mice, including increased percentage of signal intensity joint clearance (mean ± SD 63 ± 9% in VEGF-C-treated aged mice versus 52 ± 15% in vehicle-treated aged mice; P = 0.012), increased total articular cartilage cross-sectional area (mean ± SD 0.38 ± 0.07 mm2 in VEGF-C-treated aged mice versus 0.26 ± 0.07 mm2 in vehicle-treated aged mice; P < 0.0001), and decreased percentage of matrix metallopeptidase 13-positive staining area within total synovial area in 22-month-old VEGF-C-treated mice versus 22-month-old vehicle-treated mice (mean ± SD decrease 7 ± 2% versus 4 ± 1%; P = 0.0004). CONCLUSION: SLS function is reduced in the knee joints of aged mice due to decreased VEGF-C/VEGFR-3 signaling. VEGF-C treatment attenuates OA joint damage and improves synovial lymphatic drainage in aged mice. The SLS and VEGF-C/VEGFR-3 signaling represent novel physiopathologic mechanisms that could potentially be used as therapeutic targets for age-related OA.

Whole-genome RNA sequencing identifies distinct transcriptomic profiles in impingement cartilage between patients with femoroacetabular impingement and hip osteoarthritis.

Femoroacetabular impingement (FAI) has a strong clinical association with the development of hip osteoarthritis (OA); however, the pathobiological mechanisms underlying the transition from focal impingement to global joint degeneration remain poorly understood. The purpose of this study is to use whole-genome RNA sequencing to identify and subsequently validate differentially expressed genes (DEGs) in femoral head articular cartilage samples from patients with FAI and hip OA secondary to FAI. Thirty-seven patients were included in the study with whole-genome RNA sequencing performed on 10 gender-matched patients in the FAI and OA cohorts and the remaining specimens were used for validation analyses. We identified a total of 3531 DEGs between the FAI and OA cohorts with multiple targets for genes implicated in canonical OA pathways. Quantitative reverse transcription-polymerase chain reaction validation confirmed increased expression of FGF18 and WNT16 in the FAI samples, while there was increased expression of MMP13 and ADAMTS4 in the OA samples. Expression levels of FGF18 and WNT16 were also higher in FAI samples with mild cartilage damage compared to FAI samples with severe cartilage damage or OA cartilage. Our study further expands the knowledge regarding distinct genetic reprogramming in the cartilage between FAI and hip OA patients. We independently validated the results of the sequencing analysis and found increased expression of anabolic markers in patients with FAI and minimal histologic cartilage damage, suggesting that anabolic signaling may be increased in early FAI with a transition to catabolic and inflammatory gene expression as FAI progresses towards more severe hip OA. Clinical significance:Cam-type FAI has a strong clinical association with hip OA; however, the cellular pathophysiology of disease progression remains poorly understood. Several previous studies have demonstrated increased expression of inflammatory markers in FAI cartilage samples, suggesting the involvement of these inflammatory pathways in the disease progression. Our study further expands the knowledge regarding distinct genetic reprogramming in the cartilage between FAI and hip OA patients. In addition to differences in inflammatory gene expression, we also identified differential expression in multiple pathways involved in hip OA progression.

Transcriptional regulation of cyclophilin D by BMP/Smad signaling and its role in osteogenic differentiation.

Cyclophilin D (CypD) promotes opening of the mitochondrial permeability transition pore (MPTP) which plays a key role in both cell physiology and pathology. It is, therefore, beneficial for cells to tightly regulate CypD and MPTP but little is known about such regulation. We have reported before that CypD is downregulated and MPTP deactivated during differentiation in various tissues. Herein, we identify BMP/Smad signaling, a major driver of differentiation, as a transcriptional regulator of the CypD gene, Ppif. Using osteogenic induction of mesenchymal lineage cells as a BMP/Smad activation-dependent differentiation model, we show that CypD is in fact transcriptionally repressed during this process. The importance of such CypD downregulation is evidenced by the negative effect of CypD 'rescue' via gain-of-function on osteogenesis both in vitro and in a mouse model. In sum, we characterized BMP/Smad signaling as a regulator of CypD expression and elucidated the role of CypD downregulation during cell differentiation.

IKKß-NF-κB signaling in adult chondrocytes promotes the onset of age-related osteoarthritis in mice.

Canonical nuclear factor κB (NF-κB) signaling mediated by homo- and heterodimers of the NF-κB subunits p65 (RELA) and p50 (NFKB1) is associated with age-related pathologies and with disease progression in posttraumatic models of osteoarthritis (OA). Here, we established that NF-κB signaling in articular chondrocytes increased with age, concomitant with the onset of spontaneous OA in wild-type mice. Chondrocyte-specific expression of a constitutively active form of inhibitor of κB kinase ß (IKKß) in young adult mice accelerated the onset of the OA-like phenotype observed in aging wild-type mice, including degenerative changes in the articular cartilage, synovium, and menisci. Both in vitro and in vivo, chondrocytes expressing activated IKKß had a proinflammatory secretory phenotype characterized by markers typically associated with the senescence-associated secretory phenotype (SASP). Expression of these factors was differentially regulated by p65, which contains a transactivation domain, and p50, which does not. Whereas the loss of p65 blocked the induction of genes encoding SASP factors in chondrogenic cells treated with interleukin-1ß (IL-1ß) in vitro, the loss of p50 enhanced the IL-1ß­induced expression of some SASP factors. The loss of p50 further exacerbated cartilage degeneration in mice with chondrocyte-specific IKKß activation. Overall, our data reveal that IKKß-mediated activation of p65 can promote OA onset and that p50 may limit cartilage degeneration in settings of joint inflammation including advanced age.

Development of Bisphosphonate-Conjugated Antibiotics to Overcome Pharmacodynamic Limitations of Local Therapy: Initial Results with Carbamate Linked Sitafloxacin and Tedizolid.

The use of local antibiotics to treat bone infections has been questioned due to a lack of clinical efficacy and emerging information about Staphylococcus aureus colonization of the osteocyte-lacuno canalicular network (OLCN). Here we propose bisphosphonate-conjugated antibiotics (BCA) using a "target and release" approach to deliver antibiotics to bone infection sites. A fluorescent bisphosphonate probe was used to demonstrate bone surface labeling adjacent to bacteria in a S. aureus infected mouse tibiae model. Bisphosphonate and hydroxybisphosphonate conjugates of sitafloxacin and tedizolid (BCA) were synthesized using hydroxyphenyl and aminophenyl carbamate linkers, respectively. The conjugates were adequately stable in serum. Their cytolytic activity versus parent drug on MSSA and MRSA static biofilms grown on hydroxyapatite discs was established by scanning electron microscopy. Sitafloxacin O-phenyl carbamate BCA was effective in eradicating static biofilm: no colony formation units (CFU) were recovered following treatment with 800 mg/L of either the bisphosphonate or α-hydroxybisphosphonate conjugated drug (p < 0.001). In contrast, the less labile tedizolid N-phenyl carbamate linked BCA had limited efficacy against MSSA, and MRSA. CFU were recovered from all tedizolid BCA treatments. These results demonstrate the feasibility of BCA eradication of S. aureus biofilm on OLCN bone surfaces and support in vivo drug development of a sitafloxacin BCA.

Isolation and Culture of Neonatal Mouse Calvarial Osteoblasts.

This chapter describes the isolation and culture of neonatal mouse calvarial osteoblasts. This primary cell population is obtained by sequential enzymatic digestion of the calvarial bone matrix and is capable of differentiating in vitro into mature osteoblasts that deposit a collagen extracellular matrix and form mineralized bone nodules. Maturation of the cultures can be monitored by gene expression analyses and staining for the presence of alkaline phosphatase or matrix mineralization. This culture system, therefore, provides a powerful model in which to test how various experimental conditions, such as the manipulation of gene expression, may affect osteoblast maturation and/or function.

NF-κB activation persists into the remodeling phase of tendon healing and promotes myofibroblast survival.

Although inflammation is necessary during the early phases of tissue repair, persistent inflammation contributes to fibrosis. Acute tendon injuries often heal through a fibrotic mechanism, which impedes regeneration and functional recovery. Because inflammation mediated by nuclear factor κB (NF-κB) signaling is implicated in this process, we examined the spatial, temporal, and cell type-specific activation profile of canonical NF-κB signaling during tendon healing. NF-κB signaling was maintained through all phases of tendon healing in mice, including the remodeling phase, and tenocytes and myofibroblasts from the Scleraxis (Scx) lineage were the predominant populations that retained NF-κB activation into the late stages of repair. We confirmed persistent NF-κB activation in myofibroblasts in human tendon scar tissue. Deleting the canonical NF-κB kinase, IKKß, in Scx-lineage cells in mice increased apoptosis and the deposition of the matrix protein periostin during the late stages of tendon repair, suggesting that persistent NF-κB signaling may facilitate myofibroblast survival and fibrotic progression. Consistent with this, myofibroblasts in human tendon scar samples displayed enhanced prosurvival signaling compared to control tissue. Together, these data suggest that NF-κB may contribute to fibrotic tendon healing through both inflammation-dependent and inflammation-independent functions, such as NF-κB-mediated cell survival.

Inhibition of the Prostaglandin EP-1 Receptor in Periosteum Progenitor Cells Enhances Osteoblast Differentiation and Fracture Repair.

Fracture healing is a complex and integrated process that involves mesenchymal progenitor cell (MPC) recruitment, proliferation and differentiation that eventually results in bone regeneration. Prostaglandin E2 (PGE2) is an important regulator of bone metabolism and has an anabolic effect on fracture healing. Prior work from our laboratory showed EP1-/- mice have enhanced fracture healing, stronger cortical bones, higher trabecular bone volume and increased in vivo bone formation. We also showed that bone marrow MSCs from EP1-/- mice exhibit increased osteoblastic differentiation in vitro. In this study we investigate the changes in the periosteal derived MPCs (PDMPCs), which are crucial for fracture repair, upon EP1 deletion. EP1-/- PDMPCs exhibit increased numbers of total (CFU-F) and osteoblastic colonies (CFU-O) as well as enhanced osteoblastic and chondrogenic differentiation. Moreover, we tested the possible therapeutic application of a specific EP1 receptor antagonist to accelerate fracture repair. Our findings showed that EP1 antagonist administration to wild type mice in the early stages of repair similarly resulted in enhanced CFU-F, CFU-O, and osteoblast differentiation in PDMPCs and resulted in enhanced fracture callus formation at 10 days post fracture and increased bone volume and improved biomechanical healing of femur fractures at 21 days post fracture.

Chondrocyte-Specific RUNX2 Overexpression Accelerates Post-traumatic Osteoarthritis Progression in Adult Mice.

RUNX2 is a transcription factor critical for chondrocyte maturation and normal endochondral bone formation. It promotes the expression of factors catabolic to the cartilage extracellular matrix and is upregulated in human osteoarthritic cartilage and in murine articular cartilage following joint injury. To date, in vivo studies of RUNX2 overexpression in cartilage have been limited to forced expression in osteochondroprogenitor cells preventing investigation into the effects of chondrocyte-specific RUNX2 overexpression in postnatal articular cartilage. Here, we used the Rosa26Runx2 allele in combination with the inducible Col2a1CreERT2 transgene or the inducible AcanCreERT2 knock-in allele to achieve chondrocyte-specific RUNX2 overexpression (OE) during embryonic development or in the articular cartilage of adult mice, respectively. RUNX2 OE was induced at embryonic day 13.5 (E13.5) for all developmental studies. Histology and in situ hybridization analyses suggest an early onset of chondrocyte hypertrophy and accelerated terminal maturation in the limbs of the RUNX2 OE embryos compared to control embryos. For all postnatal studies, RUNX2 OE was induced at 2 months of age. Surprisingly, no histopathological signs of cartilage degeneration were observed even 6 months following induction of RUNX2 OE. Using the meniscal/ligamentous injury (MLI), a surgical model of knee joint destabilization and meniscal injury, however, we found that RUNX2 OE accelerates the progression of cartilage degeneration following joint trauma. One month following MLI, the numbers of MMP13-positive and TUNEL-positive chondrocytes were significantly greater in the articular cartilage of the RUNX2 OE joints compared to control joints and 2 months following MLI, histomorphometry and Osteoarthritis Research Society International (OARSI) scoring revealed decreased cartilage area in the RUNX2 OE joints. Collectively, these results suggest that although RUNX2 overexpression alone may not be sufficient to initiate the OA degenerative process, it may predetermine the rate of OA onset and/or progression following traumatic joint injury. © 2019 American Society for Bone and Mineral Research.

Teriparatide (human PTH1-34) compensates for impaired fracture healing in COX-2 deficient mice.

Genetic ablation of cyclooxygenase-2 (COX-2) in mice is known to impair fracture healing. To determine if teriparatide (human PTH1-34) can promote healing of Cox-2-deficient fractures, we performed detailed in vivo analyses using a murine stabilized tibia fracture model. Periosteal progenitor cell proliferation as well as bony callus formation was markedly reduced in Cox-2-/- mice at day 10 post-fracture. Remarkably, intermittent PTH1-34 administration increased proliferation of periosteal progenitor cells, restored callus formation on day 7, and enhanced bone formation on days 10, 14 and 21 in Cox-2-deficient mice. PTH1-34 also increased biomechanical torsional properties at days 10 or 14 in all genotypes, consistent with enhanced bony callus formation by radiologic examinations. To determine the effects of intermittent PTH1-34 for callus remodeling, TRAP staining was performed. Intermittent PTH1-34 treatment increased the number of TRAP positive cells per total callus area on day 21 in Cox-2-/- fractures. Taken together, the present findings indicate that intermittent PTH1-34 treatment could compensate for COX-2 deficiency and improve impaired fracture healing in Cox-2-deficient mice.

Aging does not alter tendon mechanical properties during homeostasis, but does impair flexor tendon healing.

Aging is an important factor in disrupted homeostasis of many tissues. While an increased incidence of tendinopathy and tendon rupture are observed with aging, it is unclear whether this is due to progressive changes in tendon cell function and mechanics over time, or an impaired repair reaction from aged tendons in response to insult or injury. In the present study, we examined changes in the mechanical properties of Flexor Digitorum Longus (FDL), Flexor Carpi Ulnaris (FCU), and tail fascicles in both male and female C57Bl/6 mice between 3 and 27 months of age to better understand the effects of sex and age on tendon homeostasis. No change in max load at failure was observed in any group over the course of aging, although there were significant decreases in toe and linear stiffness in female mice from 3 to 15 months, and 3 to 27 months. No changes in cell proliferation were observed with aging, although an observable decrease in cellularity occurred in 31-month old tendons. Given that aging did not dramatically alter tendon mechanical homeostasis we hypothesized that a disruption in tendon homeostasis, via acute injury would result in an impaired healing response. Significant decreases in max load, stiffness, and yield load were observed in repairs of 22-month old mice, relative to 4-month old mice. No changes in cell proliferation were observed between young and aged, however, a dramatic loss of bridging collagen extracellular matrix was observed in aged repairs suggest that matrix production, but not cell proliferation leads to impaired tendon healing with aging. Results © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2716-2724, 2017.

PGE2 Receptor Subtype 1 (EP1) Regulates Mesenchymal Stromal Cell Osteogenic Differentiation by Modulating Cellular Energy Metabolism.

Mesenchymal stromal cells (MSCs) are multipotent progenitors capable of differentiation into osteoblasts and can potentially serve as a source for cell-based therapies for bone repair. Many factors have been shown to regulate MSC differentiation into the osteogenic lineage such as the Cyclooxygenase-2 (COX2)/Prostaglandin E2 (PGE2) signaling pathway that is critical for bone repair. PGE2 binds four different receptors EP1-4. While most studies focus on the role PGE2 receptors EP2 and EP4 in MSC differentiation, our study focuses on the less studied, receptor subtype 1 (EP1) in MSC function. Recent work from our laboratory showed that EP1-/- mice have enhanced fracture healing, stronger cortical bones, higher trabecular bone volume and increased in vivo bone formation, suggesting that EP1 is a negative regulator of bone formation. In this study, the regulation of MSC osteogenic differentiation by EP1 receptor was investigated using EP1 genetic deletion in EP1-/- mice. The data suggest that EP1 receptor functions to maintain MSCs in an undifferentiated state. Loss of the EP1 receptor changes MSC characteristics and permits stem cells to undergo more rapid osteogenic differentiation. Notably, our studies suggest that EP1 receptor regulates MSC differentiation by modulating MSC bioenergetics, preventing the shift to mitochondrial oxidative phosphorylation by maintaining high Hif1α activity. Loss of EP1 results in inactivation of Hif1α, increased oxygen consumption rate and thus increased osteoblast differentiation. J. Cell. Biochem. 118: 4383-4393, 2017. © 2017 Wiley Periodicals, Inc.

Daily oral consumption of hydrolyzed type 1 collagen is chondroprotective and anti-inflammatory in murine posttraumatic osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease for which there are no disease modifying therapies. Thus, strategies that offer chondroprotective or regenerative capability represent a critical unmet need. Recently, oral consumption of a hydrolyzed type 1 collagen (hCol1) preparation has been reported to reduce pain in human OA and support a positive influence on chondrocyte function. To evaluate the tissue and cellular basis for these effects, we examined the impact of orally administered hCol1 in a model of posttraumatic OA (PTOA). In addition to standard chow, male C57BL/6J mice were provided a daily oral dietary supplement of hCol1 and a meniscal-ligamentous injury was induced on the right knee. At various time points post-injury, hydroxyproline (hProline) assays were performed on blood samples to confirm hCol1 delivery, and joints were harvested for tissue and molecular analyses were performed, including histomorphometry, OARSI and synovial scoring, immunohistochemistry and mRNA expression studies. Confirming ingestion of the supplements, serum hProline levels were elevated in experimental mice administered hCol1. In the hCol1 supplemented mice, chondroprotective effects were observed in injured knee joints, with dose-dependent increases in cartilage area, chondrocyte number and proteoglycan matrix at 3 and 12 weeks post-injury. Preservation of cartilage and increased chondrocyte numbers correlated with reductions in MMP13 protein levels and apoptosis, respectively. Supplemented mice also displayed reduced synovial hyperplasia that paralleled a reduction in Tnf mRNA, suggesting an anti-inflammatory effect. These findings establish that in the context of murine knee PTOA, daily oral consumption of hCol1 is chondroprotective, anti-apoptotic in articular chondrocytes, and anti-inflammatory. While the underlying mechanism driving these effects is yet to be determined, these findings provide the first tissue and cellular level information explaining the already published evidence of symptom relief supported by hCol1 in human knee OA. These results suggest that oral consumption of hCol1 is disease modifying in the context of PTOA.

BMP-TAK1 (MAP3K7) Induces Adipocyte Differentiation Through PPARγ Signaling.

BMPs have been shown to promote adipocyte differentiation through SMAD-dependent signaling. However, the role of TGF-ß-activated kinase 1 (TAK1) in non-canonical BMP signaling in adipocyte differentiation remains unclear. Here, we show that TAK1 inhibition decreases lipid accumulation in C3H10T1/2 mesenchymal stem cells (MSCs) induced to differentiate into adipocytes. TAK1 knockdown by siRNA further confirms that TAK1 is required for adipocyte commitment of MSCs. Additionally, TAK1 knockdown inhibits adipogenesis of 3T3-L1 preadipocytes, indicating that TAK1 is not only needed for adipocyte commitment, but also required for adipocyte terminal differentiation. Furthermore, TAK1 ablation specifically in adipocytes reduced high fat diet-induced weight gain and improved glucose tolerance. Mechanistically, we demonstrate that TAK1 is required for PPARγ transactivation and promotes PPARγ transcriptional activity synergistically with TAK1 binding protein 1 (TAB1). Collectively, our results demonstrate that TAK1 plays a critical role in BMP-mediated adipocyte differentiation. J. Cell. Biochem. 118: 204-210, 2017. © 2016 Wiley Periodicals, Inc.

Cyclophilin D Knock-Out Mice Show Enhanced Resistance to Osteoporosis and to Metabolic Changes Observed in Aging Bone.

Pathogenic factors associated with aging, such as oxidative stress and hormone depletion converge on mitochondria and impair their function via opening of the mitochondrial permeability transition pore (MPTP). The MPTP is a large non-selective pore regulated by cyclophilin D (CypD) that disrupts mitochondrial membrane integrity. MPTP involvement has been firmly established in degenerative processes in heart, brain, and muscle. Bone has high energy demands and is therefore expected to be highly sensitive to mitochondrial dysfunction. Despite this, the role of mitochondria and the MPTP in bone maintenance and bone pathology has not been elucidated. Our goal was to determine whether mitochondria are impaired in aging bone and to see if protecting mitochondria from MPTP opening via CypD deletion protects against bone loss. We found that bone mass, strength, and formation progressively decline over the course of 18 months in C57BL/6J mice. Using metabolomics and electron microscopy, we determined that oxidative metabolism is impaired in aging bone leading to a glycolytic shift, imbalance in nucleotides, and decreased NAD+/NADH ratio. Mitochondria in osteocytes appear swollen which is a major marker of MPTP opening. CypD deletion by CypD knockout mouse model (CypD KO) protects against bone loss in 13- and 18-month-old mice and prevents decline in bone formation and mitochondrial changes observed in wild type C57BL/6J mice. Together, these data demonstrate that mitochondria are impaired in aging bone and that CypD deletion protects against this impairment to prevent bone loss. This implicates CypD-regulated MPTP and mitochondrial dysfunction in the impairment of bone cells and in aging-related bone loss. Our findings suggest mitochondrial metabolism as a new target for bone therapeutics and inhibition of CypD as a novel strategy against bone loss.

CCN1 Regulates Chondrocyte Maturation and Cartilage Development.

WNT/ß-CATENIN signaling is involved in multiple aspects of skeletal development, including chondrocyte differentiation and maturation. Although the functions of ß-CATENIN in chondrocytes have been extensively investigated through gain-of-function and loss-of-function mouse models, the precise downstream effectors through which ß-CATENIN regulates these processes are not well defined. Here, we report that the matricellular protein, CCN1, is induced by WNT/ß-CATENIN signaling in chondrocytes. Specifically, we found that ß-CATENIN signaling promotes CCN1 expression in isolated primary sternal chondrocytes and both embryonic and postnatal cartilage. Additionally, we show that, in vitro, CCN1 overexpression promotes chondrocyte maturation, whereas inhibition of endogenous CCN1 function inhibits maturation. To explore the role of CCN1 on cartilage development and homeostasis in vivo, we generated a novel transgenic mouse model for conditional Ccn1 overexpression and show that cartilage-specific CCN1 overexpression leads to chondrodysplasia during development and cartilage degeneration in adult mice. Finally, we demonstrate that CCN1 expression increases in mouse knee joint tissues after meniscal/ligamentous injury (MLI) and in human cartilage after meniscal tear. Collectively, our data suggest that CCN1 is an important regulator of chondrocyte maturation during cartilage development and homeostasis.

Suppressive Effects of Insulin on Tumor Necrosis Factor-Dependent Early Osteoarthritic Changes Associated With Obesity and Type 2 Diabetes Mellitus.

OBJECTIVE: Obesity is a state of chronic inflammation that is associated with insulin resistance and type 2 diabetes mellitus (DM), as well as an increased risk of osteoarthritis (OA). This study was undertaken to define the links between obesity-associated inflammation, insulin resistance, and OA, by testing the hypotheses that 1) tumor necrosis factor (TNF) is critical in mediating these pathologic changes in OA, and 2) insulin has direct effects on the synovial joint that are compromised by insulin resistance. METHODS: The effects of TNF and insulin on catabolic gene expression were determined in fibroblast-like synoviocytes (FLS) isolated from human OA synovium. Synovial TNF expression and OA progression were examined in 2 mouse models, high-fat (HF) diet-fed obese mice with type 2 DM and TNF-knockout mice. Insulin resistance was investigated in synovium from patients with type 2 DM. RESULTS: Insulin receptors (IRs) were abundant in both mouse and human synovial membranes. Human OA FLS were insulin responsive, as indicated by the dose-dependent phosphorylation of IRs and Akt. In cultures of human OA FLS with exogenous TNF, the expression and release of MMP1, MMP13, and ADAMTS4 by FLS were markedly increased, whereas after treatment with insulin, these effects were selectively inhibited by >50%. The expression of TNF and its abundance in the synovium were elevated in samples from obese mice with type 2 DM. In TNF-knockout mice, increases in osteophyte formation and synovial hyperplasia associated with the HF diet were blunted. The synovium from OA patients with type 2 DM contained markedly more macrophages and showed elevated TNF levels as compared to the synovium from OA patients without diabetes. Moreover, insulin-dependent phosphorylation of IRs and Akt was blunted in cultures of OA FLS from patients with type 2 DM. CONCLUSION: TNF appears to be involved in mediating the advanced progression of OA seen in type 2 DM. While insulin plays a protective, antiinflammatory role in the synovium, insulin resistance in patients with type 2 DM may impair this protective effect and promote the progression of OA.

Development of antisense oligonucleotide (ASO) technology against Tgf-ß signaling to prevent scarring during flexor tendon repair.

Flexor tendons (FT) in the hand provide near frictionless gliding to facilitate hand function. Upon injury and surgical repair, satisfactory healing is hampered by fibrous adhesions between the tendon and synovial sheath. In the present study we used antisense oligonucleotides (ASOs), specifically targeted to components of Tgf-ß signaling, including Tgf-ß1, Smad3 and Ctgf, to test the hypothesis that local delivery of ASOs and suppression of Tgf-ß1 signaling would enhance murine FT healing by suppressing adhesion formation while maintaining strength. ASOs were injected in to the FT repair site at 2, 6 and 12 days post-surgery. ASO treatment suppressed target gene expression through 21 days. Treatment with Tgf-ß1, Smad3 or Ctgf ASOs resulted in significant improvement in tendon gliding function at 14 and 21 days, relative to control. Consistent with a decrease in adhesions, Col3a1 expression was significantly decreased in Tgf-ß1, Smad3 and Ctgf ASO treated tendons relative to control. Smad3 ASO treatment enhanced the maximum load at failure of healing tendons at 14 days, relative to control. Taken together, these data support the use of ASO treatment to improve FT repair, and suggest that modulation of the Tgf-ß1 signaling pathway can reduce adhesions while maintaining the strength of the repair.

Primary murine growth plate and articular chondrocyte isolation and cell culture.

The ability to isolate primary chondrocytes from wild-type and genetically altered mice has provided tremendous advances in the understanding of signaling networks that regulate chondrocytes in health and disease. Isolation of chondrocytes from both growth plate tissues and articular cartilage has been challenging due to the cells being embedded within a highly organized tissue matrix. Here we describe highly reproducible methods for the isolation of pure populations of growth plate chondrocytes from the murine sternum and ribs and articular chondrocytes from the knee joint.

Loss of the PGE2 receptor EP1 enhances bone acquisition, which protects against age and ovariectomy-induced impairments in bone strength.

PGE2 exerts anabolic and catabolic effects on bone through the discrete actions of four prostanoid receptors (EP1-4). We have previously demonstrated that loss EP1 accelerates fracture repair by enhancing bone formation. In the present study we defined the role of EP1 in bone maintenance and homeostasis during aging and in response to ovariectomy. The femur and L4 vertebrae of wild type (WT) and EP1(-/-) mice were examined at 2-months, 6-months, and 1-year of age, and in WT and EP1(-/-) mice following ovariectomy (OVX) or sham surgery. Bone volume fraction, trabecular architecture and mechanical properties were maintained during aging in EP1(-/-) mice to a greater degree than age-matched WT mice. Moreover, significant increases in bone formation rate (BFR) (+60%) and mineral apposition rate (MAR) (+50%) were observed in EP1(-/-), relative to WT, while no change in osteoclast number and osteoclast surface were observed. Following OVX, loss of EP1 was protective against bone loss in both femur and L4 vertebrae, with increased bone volume/total volume (BV/TV) (+32% in femur) and max load at failure (+10% in femur) relative to WT OVX, likely resulting from the increased bone formation rate that was observed in these mice. Taken together these studies identify inhibition of EP1 as a potential therapeutic approach to suppress bone loss in aged or post-menopausal patients.