Coexisting Primary Sclerosing Cholangitis and Autoimmune Hepatitis: Overlapping Challenges in Diagnosis and Treatment.

Introduction: Hepatobiliary overlap syndromes describe the coinciding presentation of more than one immune-mediated biliary and liver disease in a single patient and present complex challenges in diagnosis and treatment. We report a case of ulcerative colitis with primary sclerosing cholangitis and autoimmune hepatitis overlap syndrome responsive to vancomycin. Case Presentation: The patient is a 30-year-old female with known ulcerative pancolitis and autoimmune hepatitis. She presented to the emergency department with a constellation of gastrointestinal symptoms, including diffuse lower abdominal pain, bloody diarrhea, and nausea with bilious vomiting. Subsequent imaging revealed the additional diagnosis of primary sclerosing cholangitis, and she was diagnosed with overlap syndrome. Multiple treatment regimens were trialed with minimal improvement. She eventually achieved normalization of both clinical status and biochemical markers after the addition of vancomycin. Conclusion: Vancomycin is an underutilized therapy; its potential role in primary sclerosing cholangitis and autoimmune hepatitis overlap syndrome has not been previously reported.

Extraenteric Malignant Gastrointestinal Neuroectodermal Tumor-A Clinicopathologic and Molecular Genetic Study of 11 Cases.

Malignant gastrointestinal neuroectodermal tumors (MGNETs), also known as "gastrointestinal clear cell sarcoma-like tumors", are very rare, aggressive sarcomas characterized by enteric location, distinctive pathologic features, and EWSR1/FUS::ATF1/CREB1 fusions. Despite identical genetics, the clinicopathologic features of MGNET are otherwise quite different from those of clear cell sarcoma of soft parts. Only exceptional extraenteric MGNET (E-MGNET) has been reported. We report a series of 11 E-MGNETs, the largest to date. Cases diagnosed with MGNET and occurring in nonintestinal locations were retrieved. A clinical follow-up was obtained. The tumors occurred in 3 men and 8 women (range, 14-70 years of age; median, 33 years) and involved the soft tissues of the neck (3), shoulder (1), buttock (2), orbit (1), tongue/parapharyngeal space (1), urinary bladder (1), and falciform ligament/liver (1). Tumors showed morphologic features of enteric MGNET (small, relatively uniform, round to ovoid cells with round, regular nuclei containing small nucleoli growing in multinodular and vaguely lobular patterns, with solid, pseudoalveolar, and pseudopapillary architecture). Immunohistochemical results were S100 protein (11/11), SOX10 (11/11), synaptophysin (3/10), CD56 (7/9), CD117 (3/9), DOG1 (0/4), ALK (4/8), chromogranin A (0/10), HMB-45 (0/11), Melan-A (0/11), tyrosinase (0/4), and MiTF (0/11). Next-generation sequencing results were EWSR1::ATF1 (7 cases), EWSR1::CREB1 (3 cases), and EWSR1::PBX1 (1 case). The EWSR1::PBX1-positive tumor was similar to other cases, including osteoclast-like giant cells, and negative for myoepithelial markers. A clinical follow-up (range, 10-70 months; median, 34 months) showed 4 patients dead of disease (10.5, 12, 25, and 64 months after diagnosis), 1 patient alive with extensive metastases (43 months after diagnosis), 1 patient alive with persistent local disease (11 months after diagnosis), and 4 alive without disease (10, 47, 53, and 70 months after diagnosis). One case is too recent for the follow-up. The clinicopathologic and molecular genetic features of rare E-MGNET are essentially identical to those occurring in intestinal locations. Otherwise, typical E-MGNET may harbor EWSR1::PBX1, a finding previously unreported in this tumor type. As in enteric locations, the behavior of E-MGNET is aggressive, with metastases and/or death from disease in at least 50% of patients. E-MGNET should be distinguished from clear cell sarcoma of soft parts and other tumors with similar fusions. ALK expression appears to be a common feature of tumors harboring EWSR1/FUS::ATF1/CREB1 fusion but is unlikely to predict the therapeutic response to ALK inhibition. Future advances in our understanding of these unusual tumors will hopefully lead to improved nomenclature.

Chordoma.

CONTEXT.­: Chordomas are uncommon malignant neoplasms with notochordal differentiation encountered by neuropathologists, bone/soft tissue pathologists, and general surgical pathologists. These lesions most commonly arise in the axial skeleton. Optimal therapy typically involves complete surgical resection, which is often technically difficult owing to the anatomic location, leading to a high rate of recurrence. Lesions have been generally resistant to radiation and chemotherapy; however, experimental studies involving targeted therapy and immunotherapy are currently underway. OBJECTIVE.­: To summarize the clinical and pathologic findings of the various types of chordoma (conventional chordoma, dedifferentiated chordoma, and poorly differentiated chordoma), the differential diagnosis, and recent advances in molecular pathogenesis and therapeutic modalities that are reliant on accurate diagnosis. DATA SOURCES.­: Literature review based on PubMed searches containing the term "chordoma" that address novel targeted and immunomodulatory therapeutic modalities; ongoing clinical trials involved in treating chordoma with novel therapeutic modalities identified through the Chordoma Foundation and ClinicalTrials.gov; and the authors' practice experience combined with various authoritative texts concerning the subject. CONCLUSIONS.­: Chordoma is a clinically and histologically unique malignant neoplasm, and numerous diagnostic considerations must be excluded to establish the correct diagnosis. Treatment options have largely been centered on surgical excision with marginal results; however, novel therapeutic options including targeted therapy and immunotherapy are promising means to improve prognosis.

Association of Increased Serum Lipopolysaccharide, But Not Microbial Dysbiosis, With Obesity-Related Osteoarthritis.

OBJECTIVE: To test the hypothesis that an altered gut microbiota (dysbiosis) plays a role in obesity-associated osteoarthritis (OA). METHODS: Stool and blood samples were collected from 92 participants with a body mass index (BMI) ≥30 kg/m2 , recruited from the Johnston County Osteoarthritis Project. OA patients (n = 50) had hand and knee OA (Kellgren/Lawrence [K/L] grade ≥2 or arthroplasty). Controls (n = 42) had no hand OA and a K/L grade of 0-1 for the knees. Compositional analysis of stool samples was carried out by 16S ribosomal RNA amplicon sequencing. Alpha- and beta-diversity and differences in taxa relative abundances were determined. Blood samples were used for multiplex cytokine analysis and measures of lipopolysaccharide (LPS) and LPS binding protein. Germ-free mice were gavaged with patient- or control-pooled fecal samples and fed a 40% fat, high-sucrose diet for 40 weeks. Knee OA was evaluated histologically. RESULTS: On average, OA patients were slightly older than the controls, consisted of more women, and had a higher mean BMI, higher mean Western Ontario and McMaster Universities Osteoarthritis Index pain score, and higher mean K/L grade. There were no significant differences in α- or ß-diversity or genus level composition between patients and controls. Patients had higher plasma levels of osteopontin (P = 0.01) and serum LPS (P < 0.0001) compared to controls. Mice transplanted with patient or control microbiota exhibited a significant difference in α-diversity (P = 0.02) and ß-diversity, but no differences in OA severity were observed. CONCLUSION: The lack of differences in the gut microbiota, but increased serum LPS levels, suggest the possibility that increased intestinal permeability allowing for greater absorption of LPS, rather than a dysbiotic microbiota, may contribute to the development of OA associated with obesity.

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.

Polyphenols suppress inducible oxidative stress in human osteoarthritic and bovine chondrocytes.

Reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in chondrocyte senescence and cartilage aging, pathogenesis of osteoarthritis (OA), and rheumatoid arthritis. Naturally occurring polyphenolic compounds (PPCs), such as curcumin (turmeric), resveratrol (grape), and epigallocatechin-3-gallate (EGCG) (green tea), have been known for their anti-inflammatory and chondroprotective effects. However, the potential protective effects of these PPCs against oxidative stress in chondrocytes are unclear. To investigate this, bovine articular chondrocytes and human osteoarthritic chondrocytes were pre-treated with PPCs at varying concentrations, and then exposed to hydrogen peroxide (H2O2) as an ROS inducer or S-nitroso-N-acetylpenicillamine (SNAP) as a NO donor. Alternatively, chondrocytes were co-treated with polyphenols and H2O2. Intracellular ROS/NO were measured using a fluorescent dye technique (H2DCF-DA for ROS; DAF-FM for NO). Our findings showed that PPC pre-/co-treatment inhibited both H2O2-induced ROS and SNAP-induced NO at different concentrations in both bovine chondrocytes and human osteoarthritic chondrocytes. Curcumin also increased glutathione peroxidase activity in the presence of H2O2 in bovine chondrocytes. Taken together, these findings indicate that PPCs are capable of suppressing oxidative stress- induced responses in chondrocytes, which may have potential therapeutic value for OA clinical application.

Impaired Annulus Fibrosus Development and Vertebral Fusion Cause Severe Scoliosis in Mice with Deficiency of c-Jun NH2-Terminal Kinases 1 and 2.

Mitogen-activated protein kinases, including c-Jun NH2-terminal kinase (JNK), play an important role in the development and function of a large variety of tissues. The skeletal phenotype of JNK1 and JNK2 double-knockout (dKO) mice (JNK1fl/flCol2-Cre/JNK2-/-) and control genotypes were analyzed at different embryonic and postnatal stages. JNK1/2 dKO mice displayed a severe scoliotic phenotype beginning during development that was grossly apparent around weaning age. Alcian blue staining at embryonic day 17.5 showed abnormal fusion of the posterior spinal elements. In adult mice, fusion of vertebral bodies and of spinous and transverse processes was noted by micro-computed tomography, Alcian blue/Alizarin red staining, and histology. The long bones developed normally, and histologic sections of growth plate and articular cartilage revealed no significant abnormalities. Histologic sections of the vertebral column at embryonic days 15.5 and 17.5 revealed an abnormal organization of the annulus fibrosus in the dKOs, with chondrocyte-like cells and fusion of dorsal processes. Spinal sections in 10-week-old dKO mice showed replacement of intervertebral disk structures (annulus fibrosus and nucleus pulposus) by cartilage and bone tissues, with cells staining for markers of hypertrophic chondrocytes, including collagen X and runt-related transcription factor 2. These findings demonstrate a requirement for both JNK1 and JNK2 in the normal development of the axial skeleton. Loss of JNK signaling results in abnormal endochondral bone formation and subsequent severe scoliosis.

Time between anterior cruciate ligament injury and reconstruction and cartilage metabolism six-months following reconstruction.

BACKGROUND: To determine the association between time from injury to ACL reconstruction (TimeInjury-ACLR) and biochemical markers of cartilage metabolism and inflammation six months following ACL reconstruction (ACLR). METHODS: Individuals with a unilateral ACL injury were enrolled at initial presentation in the orthopedic clinic; blood was collected six months following ACLR. Enzyme-linked immunosorbent assays were used to analyze the ratio of serum concentrations of type-II collagen breakdown (C2C) to synthesis (CPII), plasma matrix metalloproteinase-3 (MMP-3), interleukin-6 (IL-6), and serum aggrecan neoepitope (ARGS). We used separate linear regressions to assess associations between biochemical markers and TimeInjury-ACLR. RESULTS: Twenty-two participants (50% females, mean [SD], age 21.9 [4.5] years old; BMI 23.8 [2.6] kg/m2) completed the study. TimeInjury-ACLR ranged from nine to 67days (31.0 [14.4days]). Greater TimeInjury-ACLR predicted greater serum C2C:CPII ratios six months following ACLR (C2C:CPII=0.15 [0.02], R2=0.213, P=0.030). Males (R2=0.733, P=0.001) but not females (R2=0.030, P=0.609) demonstrated a significant association between greater C2C:CPII and TimeInjury-ACLR at the six-month follow-up exam. TimeInjury-ACLR did not associate with IL-6, MMP-3, or ARGS at six months. CONCLUSIONS: Greater time between injury and ACL reconstruction was associated with greater serum C2C:CPII six months following ACLR in males but not females, and IL-6, MMP-3, and ARGS levels were not associated with TimeInjury-ACLR in males or females. The time between ACL injury and ACLR may affect collagen metabolism in males and should be further investigated in a larger study along with other patient-relevant outcomes.

Biochemical markers of cartilage metabolism are associated with walking biomechanics 6-months following anterior cruciate ligament reconstruction.

The purpose of our study was to determine the association between biomechanical outcomes of walking gait (peak vertical ground reaction force [vGRF], vGRF loading rate [vGRF-LR], and knee adduction moment [KAM]) 6 months following anterior cruciate ligament reconstruction (ACLR) and biochemical markers of serum type-II collagen turnover (collagen type-II cleavage product to collagen type-II C-propeptide [C2C:CPII]), plasma degenerative enzymes (matrix metalloproteinase-3 [MMP-3]), and a pro-inflammatory cytokine (interleukin-6 [IL-6]). Biochemical markers were evaluated within the first 2 weeks (6.5 ± 3.8 days) following ACL injury and again 6 months following ACLR in eighteen participants. All peak biomechanical outcomes were extracted from the first 50% of the stance phase of walking gait during a 6-month follow-up exam. Limb symmetry indices (LSI) were used to normalize the biomechanical outcomes in the ACLR limb to that of the contralateral limb (ACLR/contralateral). Bivariate correlations were used to assess associations between biomechanical and biochemical outcomes. Greater plasma MMP-3 concentrations after ACL injury and at the 6-month follow-up exam were associated with lesser KAM LSI. Lesser KAM was associated with greater plasma IL-6 at the 6-month follow-up exam. Similarly, lesser vGRF-LR LSI was associated with greater plasma MMP-3 concentrations at the 6-month follow-up exam. Lesser peak vGRF LSI was associated with higher C2C:CPII after ACL injury, yet this association was not significant after accounting for walking speed. Therefore, lesser biomechanical loading in the ACLR limb, compared to the contralateral limb, 6 months following ACLR may be related to deleterious joint tissue metabolism that could influence future cartilage breakdown. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2288-2297, 2017.

A mouse model for chronic pain-induced increase in ethanol consumption.

Chronic pain conditions are often comorbid with alcohol abuse. "Self-medication" with alcohol introduces a host of problems associated with the abuse of alcohol which over time has the potential of exacerbating the painful condition. Despite the prevalence of chronic pain being associated with alcohol abuse, rodent models which mimic the comorbid conditions are lacking. In this study, we model osteoarthritis (OA) in C57BL/6J mice by surgically destabilizing the medial meniscus (DMM). Sham-operated mice served as controls. Thirteen weeks after surgery, DMM but not sham-operated mice exhibited pronounced incapacitance of the surgically manipulated hind limb compared with the nonsurgically manipulated hind limb. At this time, the mice were exposed to the 2-bottle ethanol choice, beginning with 2.5% with a gradual increasing to 20%. Compared with sham controls, DMM mice consumed more EtOH and preferred EtOH over water at the 20% EtOH concentration. Histological analysis verified that the DMM mice exhibited significant damage to the articular cartilage and osteophyte growth compared with sham controls and these measures of the severity of OA correlated with the amount of ethanol intake. Thus, the combination of the DMM model of OA with the enhanced two-bottle ethanol choice is a potential preclinical approach in mice by which the basis of the comorbid association of alcohol abuse and chronic pain conditions can be explored.

Context-specific protection of TGFα null mice from osteoarthritis.

Transforming growth factor alpha (TGFα) is a growth factor involved in osteoarthritis (OA). TGFα induces an OA-like phenotype in articular chondrocytes, by inhibiting matrix synthesis and promoting catabolic factor expression. To better understand TGFα's potential as a therapeutic target, we employed two in vivo OA models: (1) post-traumatic and (2) aging related OA. Ten-week old and six-month old male Tgfa null mice and their heterozygous (control) littermates underwent destabilization of the medial meniscus (DMM) surgery. Disease progression was assessed histologically using the Osteoarthritis Research Society International (OARSI) scoring system. As well, spontaneous disease progression was analyzed in eighteen-month-old Tgfa null and heterozygous mice. Ten-week old Tgfa null mice were protected from OA progression at both seven and fourteen weeks post-surgery. No protection was seen however in six-month old null mice after DMM surgery, and no differences were observed between genotypes in the aging model. Thus, young Tgfa null mice are protected from OA progression in the DMM model, while older mice are not. In addition, Tgfa null mice are equally susceptible to spontaneous OA development during aging. Thus, TGFα might be a valuable therapeutic target in some post-traumatic forms of OA, however its role in idiopathic disease is less clear.

Platelet-Rich Plasma Inhibits Mechanically Induced Injury in Chondrocytes.

PURPOSE: To investigate the effect of platelet-rich plasma (PRP) on mechanically injured chondrocytes. METHODS: PRP from bovine whole blood was activated to prepare platelet-rich plasma releasate (PRPr). Bovine articular chondrocytes were subjected to 16%, 0.5-Hz biaxial cyclic tensile strain (CTS) for 48 hours and cultured for another 24 hours without cell stretching as an in vitro model of mechanically injured chondrocytes. Culture medium in the 3 PRP- and CTS-treated groups was supplemented with 10% PRPr at the start of CTS, after 24 hours of CTS, and after 48 hours of CTS, respectively. Gene expression levels of type II collagen, aggrecan, matrix metalloproteinase (MMP)-3, MMP-13, inducible nitric oxide synthase, and cyclooxygenase 2 were quantitatively evaluated. Changes in the content of nitric oxide (NO), prostaglandin E2 (PGE2), MMP-3, and tissue inhibitor of metalloproteinase 1 in the culture medium were also measured. RESULTS: PRPr increased type II collagen and aggrecan messenger RNA expression; diminished CTS-dependent up-regulation of MMP-3, inducible nitric oxide synthase, and cyclooxygenase 2 gene expression; and reduced CTS-induced overproduction of NO and PGE2 when PRPr was applied early at the start of CTS. The addition of PRPr after 24 hours of CTS only inhibited MMP-3 gene up-regulation and the increase of NO and PGE2 induced by CTS. These changes were not observed when PRPr was supplemented after 48 hours of CTS. PRPr mitigated the increased MMP-3 production and decreased tissue inhibitor of metalloproteinase 1 secretion resulting from CTS in a time-dependent manner. CONCLUSIONS: PRP treatment ameliorated multiple CTS-mediated catabolic and inflammatory responses in chondrocytes. More beneficial effects were observed with early PRP application. CLINICAL RELEVANCE: Intra-articular PRP injections at the beginning of strenuous exercises may be used to protect chondrocytes from mechanical injury, thus preventing joints from increased wear.

Time-series transcriptional profiling yields new perspectives on susceptibility to murine osteoarthritis.

OBJECTIVE: Chronological age is a powerful epidemiologic risk factor for osteoarthritis (OA), a multifactorial disease that is characterized by articular cartilage (AC) degradation. It is unclear from a molecular perspective how aging interacts with OA to produce this risk to AC integrity. To address this key question, we used in vivo time-course analysis of OA development and murine interstrain variability in natural susceptibility to OA to examine changes in non-OA-prone CBA mice versus OA-prone STR/Ort mice, which develop disease that bears significant histologic resemblance to human OA. Through global transcriptome profiling, we attempted to discover the molecular signature linked with both OA vulnerability and progression. METHODS: Affymetrix Mouse Gene 1.0 ST Array profiles were generated from AC samples derived from CBA and STR/Ort mice at 3 different ages, corresponding to the stages prior to, at, and late after the natural onset of OA in the STR/Ort mice. RESULTS: We found that the OA in STR/Ort mice exhibited a molecular phenotype resembling human OA, and we pinpointed a central role of NF-κB signaling and the emergence of an immune-related signature in OA cartilage over time. We discovered that, strikingly, young healthy AC has a highly expressed skeletal muscle gene expression program, which is switched off during maturation, but is intriguingly retained in AC during OA development in STR/Ort mice. CONCLUSION: This study is the first to show that AC chondrocytes share a high-abundance gene-expression program with skeletal muscle. We show that failure to switch this program off, as well as the restoration of this program, is associated with inappropriate expression of NF-κB signaling pathways, skeletal muscle-related genes, and induction and/or progression of OA.

An in vivo investigation of the initiation and progression of subchondral cysts in a rodent model of secondary osteoarthritis.

INTRODUCTION: Subchondral bone cysts (SBC) have been identified in patients with knee osteoarthritis (OA) as a cause of greater pain, loss of cartilage and increased chance of joint replacement surgery. Few studies monitor SBC longitudinally, and clinical research using three-dimensional imaging techniques, such as magnetic resonance imaging (MRI), is limited to retrospective analyses as SBC are identified within an OA patient cohort. The purpose of this study was to use dual-modality, preclinical imaging to monitor the initiation and progression of SBC occurring within an established rodent model of knee OA. METHODS: Eight rodents underwent anterior cruciate ligament transection and partial medial meniscectomy (ACLX) of the right knee. In vivo 9.4 T MRI and micro-computed tomography (micro-CT) scans were performed consecutively prior to ACLX and 4, 8, and 12 weeks post-ACLX. Resultant images were co-registered using anatomical landmarks, which allowed for precise tracking of SBC size and composition throughout the study. The diameter of the SBC was measured, and the volumetric bone mineral density (vBMD) was calculated within the bone adjacent to SBC. At 12 weeks, the ACLX and contralateral knees were processed for histological analysis, immunohistochemistry, and Osteoarthritis Research Society International (OARSI) pathological scoring. RESULTS: At 4 weeks post-ACLX, 75% of the rodent knees had at least 1 cyst that formed in the medial tibial plateau; by 12 weeks all ACLX knees contained SBC. Imaging data revealed that the SBC originate in the presence of a subchondral bone plate breach, with evolving composition over time. The diameter of the SBC increased significantly over time (P = 0.0033) and the vBMD significantly decreased at 8 weeks post-ACLX (P = 0.033). Histological analysis demonstrated positive staining for bone resorption and formation surrounding the SBC, which were consistently located beneath the joint surface with the greatest cartilage damage. Trabecular bone adjacent the SBC lacked viable osteocytes and, combined with bone marrow changes, indicated osteonecrosis. CONCLUSIONS: This study provides insight into the mechanisms leading to SBC formation in knee OA. The expansion of these lesions is due to stress-induced bone resorption from the incurred mechanical instability. Therefore, we suggest these lesions can be more accurately described as a form of OA-induced osteonecrosis, rather than 'subchondral cysts'.

Genome-wide analyses of gene expression during mouse endochondral ossification.

BACKGROUND: Endochondral ossification is a complex process involving a series of events that are initiated by the establishment of a chondrogenic template and culminate in its replacement through the coordinated activity of osteoblasts, osteoclasts and endothelial cells. Comprehensive analyses of in vivo gene expression profiles during these processes are essential to obtain a complete understanding of the regulatory mechanisms involved. METHODOLOGY/PRINCIPAL FINDINGS: To address these issues, we completed a microarray screen of three zones derived from manually segmented embryonic mouse tibiae. Classification of genes differentially expressed between each respective zone, functional categorization as well as characterization of gene expression patterns, cytogenetic loci, signaling pathways and functional motifs both confirmed reported data and provided novel insights into endochondral ossification. Parallel comparisons of the microdissected tibiae data set with our previously completed micromass culture screen further corroborated the suitability of micromass cultures for modeling gene expression in chondrocyte development. The micromass culture system demonstrated striking similarities to the in vivo microdissected tibiae screen; however, the micromass system was unable to accurately distinguish gene expression differences in the hypertrophic and mineralized zones of the tibia. CONCLUSIONS/SIGNIFICANCE: These studies allow us to better understand gene expression patterns in the growth plate and endochondral bones and provide an important technical resource for comparison of gene expression in diseased or experimentally-manipulated cartilages. Ultimately, this work will help to define the genomic context in which genes are expressed in long bones and to understand physiological and pathological ossification.

Regulation of gene expression by PI3K in mouse growth plate chondrocytes.

BACKGROUND: Endochondral ossification, the process through which long bones are formed, involves chondrocyte proliferation and hypertrophic differentiation in the cartilage growth plate. In a previous publication we showed that pharmacological inhibition of the PI3K signaling pathway results in reduced endochondral bone growth, and in particular, shortening of the hypertrophic zone in a tibia organ culture system. In this current study we aimed to investigate targets of the PI3K signaling pathway in hypertrophic chondrocytes. METHODOLOGY/PRINCIPAL FINDINGS: Through the intersection of two different microarray analyses methods (classical single gene analysis and GSEA) and two different chondrocyte differentiation systems (primary chondrocytes treated with a pharmacological inhibitor of PI3K and microdissected growth plates), we were able to identify a high number of genes grouped in GSEA functional categories regulated by the PI3K signaling pathway. Genes such as Phlda2 and F13a1 were down-regulated upon PI3K inhibition and showed increased expression in the hypertrophic zone compared to the proliferative/resting zone of the growth plate. In contrast, other genes including Nr4a1 and Adamts5 were up-regulated upon PI3K inhibition and showed reduced expression in the hypertrophic zone. Regulation of these genes by PI3K signaling was confirmed by quantitative RT-PCR. We focused on F13a1 as an interesting target because of its known role in chondrocyte hypertrophy and osteoarthritis. Mouse E15.5 tibiae cultured with LY294002 (PI3K inhibitor) for 6 days showed decreased expression of factor XIIIa in the hypertrophic zone compared to control cultures. CONCLUSIONS/SIGNIFICANCE: Discovering targets of signaling pathways in hypertrophic chondrocytes could lead to targeted therapy in osteoarthritis and a better understanding of the cartilage environment for tissue engineering.

The role of Akt1 in terminal stages of endochondral bone formation: angiogenesis and ossification.

Longitudinal bone growth is the result of endochondral bone formation which takes place in the growth plate. The rate of chondrocyte proliferation and hypertrophy, vascular invasion with the formation of primary ossification centers and cartilage replacement by bone tissue are all important processes required for normal growth. We have shown a role for the PI3K signaling pathway in chondrocyte hypertrophy and bone growth in tibia explant cultures. In this current study, we aimed to investigate the role of Akt1, an important target of PI3K, in endochondral ossification. Akt1 KO mice showed reduced size compared to their littermates throughout life, but the largest difference in body size was observed around 1 week of age. Focusing on this specific developmental stage, we discovered delayed secondary ossification in the long bones of Akt1 KO mice. A delay in formation of a structure resembling a secondary ossification center was also seen in tibia organ cultures treated with the PI3K inhibitor LY294002. The expression of matrix metalloproteinase-14 (MMP-14), the main protease responsible for development of secondary ossification centers, was decreased in the epiphysis of Akt1 KO mice, possibly explaining the delay in secondary ossification centers seen in the Akt1 KO mice. Bone mineral density (BMD) and bone mineral content (BMC) measured in the proximal tibia of 1-year-old mice were decreased in Akt1 KO mice, suggesting that the original delay in ossification might affect bone quality in older animals.

The PI3K pathway regulates endochondral bone growth through control of hypertrophic chondrocyte differentiation.

BACKGROUND: The majority of our bones develop through the process of endochondral ossification that involves chondrocyte proliferation and hypertrophic differentiation in the cartilage growth plate. A large number of growth factors and hormones have been implicated in the regulation of growth plate biology, however, less is known about the intracellular signaling pathways involved. PI3K/Akt has been identified as a major regulator of cellular proliferation, differentiation and death in multiple cell types. RESULTS AND DISCUSSION: Employing an organ culture system of embryonic mouse tibiae and LY294002, a pharmacological inhibitor of PI3K, we show that inhibition of the pathway results in significant growth reduction, demonstrating that PI3K is required for normal endochondral bone growth in vitro. PI3K inhibition reduces the length of the proliferating and particularly of the hypertrophic zone. Studies with organ cultures and primary chondrocytes in micromass culture show delayed hypertrophic differentiation of chondrocytes and increased apoptosis in the presence of LY294002. Surprisingly, PI3K inhibition had no strong effect on IGF1-induced bone growth, but partially blocked the anabolic effects of C-type natriuretic peptide. CONCLUSION: Our data demonstrate an essential role of PI3K signaling in chondrocyte differentiation and as a consequence of this, in the endochondral bone growth process.