Ming-Ming Jiang

Proteoglycan 4 expression protects against the development of osteoarthritis.

Proteoglycan 4 protects from age-related and posttraumatic osteoarthritis development. Rubbing Arthritis the Wrong Way There are two main forms of joint inflammation (arthritis). Patients with rheumatoid arthritis develop joint inflammation because of an autoimmune reaction, whereas wear and tear is thought to contribute to the development of osteoarthritis. Indeed, most people have at least some symptoms of osteoarthritis by the age of 70. Treatment is mostly limited to reducing symptoms. Now, Ruan et al. provide a new mechanism for a potential new therapy for osteoarthritis. Proteoglycan 4 (lubrican, PRG4) has been previously thought to protect against osteoarthritis by acting as a lubricant between bones in a joint. Here, the authors show that not only does PRG4 protect against the development of osteoarthritis in both aging and trauma mouse models, but that its effect may extend beyond that of a mere lubricant. Indeed, PRG4 has a direct chondroprotective effect in these models, suggesting that it may form the basis for a new disease-altering therapy for osteoarthritis. Osteoarthritis (OA) is a common degenerative condition that afflicts more than 70% of the population between 55 and 77 years of age. Although its prevalence is rising globally with aging of the population, current therapy is limited to symptomatic relief and, in severe cases, joint replacement surgery. We report that intra-articular expression of proteoglycan 4 (Prg4) in mice protects against development of OA. Long-term Prg4 expression under the type II collagen promoter (Col2a1) does not adversely affect skeletal development but protects from developing signs of age-related OA. The protective effect is also shown in a model of posttraumatic OA created by cruciate ligament transection. Moreover, intra-articular injection of helper-dependent adenoviral vector expressing Prg4 protected against the development of posttraumatic OA when administered either before or after injury. Gene expression profiling of mouse articular cartilage and in vitro cell studies show that Prg4 expression inhibits the transcriptional programs that promote cartilage catabolism and hypertrophy through the up-regulation of hypoxia-inducible factor 3α. Analyses of available human OA data sets are consistent with the predictions of this model. Hence, our data provide insight into the mechanisms for OA development and offer a potential chondroprotective approach to its treatment.

Mutations in KAT6B, encoding a histone acetyltransferase, cause Genitopatellar syndrome.

Genitopatellar syndrome (GPS) is a skeletal dysplasia with cerebral and genital anomalies for which the molecular basis has not yet been determined. By exome sequencing, we found de novo heterozygous truncating mutations in KAT6B (lysine acetyltransferase 6B, formerly known as MYST4 and MORF) in three subjects; then by Sanger sequencing of KAT6B, we found similar mutations in three additional subjects. The mutant transcripts do not undergo nonsense-mediated decay in cells from subjects with GPS. In addition, human pathological analyses and mouse expression studies point to systemic roles of KAT6B in controlling organismal growth and development. Myst4 (the mouse orthologous gene) is expressed in mouse tissues corresponding to those affected by GPS. Phenotypic differences and similarities between GPS, the Say-Barber-Biesecker variant of Ohdo syndrome (caused by different mutations of KAT6B), and Rubinstein-Taybi syndrome (caused by mutations in other histone acetyltransferases) are discussed. Together, the data support an epigenetic dysregulation of the limb, brain, and genital developmental programs.

Whole-exome sequencing identifies mutations in the nucleoside transporter gene SLC29A3 in dysosteosclerosis, a form of osteopetrosis

Dysosteosclerosis (DSS) is the form of osteopetrosis distinguished by the presence of skin findings such as red-violet macular atrophy, platyspondyly and metaphyseal osteosclerosis with relative radiolucency of widened diaphyses. At the histopathological level, there is a paucity of osteoclasts when the disease presents. In two patients with DSS, we identified homozygous or compound heterozygous missense mutations in SLC29A3 by whole-exome sequencing. This gene encodes a nucleoside transporter, mutations in which cause histiocytosis-lymphadenopathy plus syndrome, a group of conditions with little or no skeletal involvement. This transporter is essential for lysosomal function in mice. We demonstrate the expression of Slc29a3 in mouse osteoclasts in vivo. In monocytes from patients with DSS, we observed reduced osteoclast differentiation and function (demineralization of calcium surface). Our report highlights the pleomorphic consequences of dysfunction of this nucleoside transporter, and importantly suggests a new mechanism for the control of osteoclast differentiation and function.

Quantitative imaging of murine osteoarthritic cartilage by phase-contrast micro–computed tomography

OBJECTIVEnThe mouse is an optimal model organism in which gene-environment interactions can be used to study the pathogenesis of osteoarthritis (OA). The gold standard for arthritis research in mice is based on histopathology and immunohistochemistry, which are labor-intensive, prone to sampling bias and technical variability, and limited in throughput. The aim of this study was to develop a new technique that assesses mouse cartilage by integrating quantitative volumetric imaging techniques.nnnMETHODSnA novel mouse model of OA was generated by cruciate ligament transection (CLT) and evaluated by histopathology and immunohistochemistry. Knee joint specimens were then imaged using a new technique that combines high-resolution micro-computed tomography (micro-CT) and phase-contrast optics followed by quantitative analyses. A comparative analysis was also performed in a previously established mouse model of OA generated by destabilization of the medial meniscus (DMM).nnnRESULTSnPhase-contrast micro-CT achieved cellular resolution of chondrocytes and quantitative assessment of parameters such as articular cartilage volume and surface area. In mouse models of OA generated by either CLT or DMM, we showed that phase-contrast micro-CT distinguished control and OA cartilage by providing quantitative measures with high reproducibility and minimal variability. Features of OA at the cellular or tissue level could also be observed in images generated by phase-contrast micro-CT.nnnCONCLUSIONnWe established an imaging technology that comprehensively assessed and quantified the 2-dimensional and 3-dimensional changes of articular cartilage. Application of this technology will facilitate the rapid and high-throughput assessment of genetic and therapeutic models of OA in mice.

Pain, motor and gait assessment of murine osteoarthritis in a cruciate ligament transection model

OBJECTIVEnThe major complaint of Osteoarthritis (OA) patients is pain. However, due to the nature of clinical studies and the limitation of animal studies, few studies have linked function impairment and behavioral changes in OA animal models to cartilage loss and histopathology. Our objective was to study surrogate markers of functional impairment in relation to cartilage loss and pathological changes in a post-traumatic mouse model of OA.nnnMETHODnWe performed a battery of functional analyses in a mouse model of OA generated by cruciate ligament transection (CLT). The changes in functional analyses were linked to histological changes graded by OARSI standards, histological grading of synovitis, and volumetric changes of the articular cartilage and osteophytes quantified by phase contrast micro-computed tomography (μCT).nnnRESULTSnOA generated by CLT led to decreased time on rotarod, delayed response on hotplate analysis, and altered gait starting from 4 weeks after surgery. Activity in open field analysis did not change at 4, 8, or 12 weeks after CLT. The magnitude of behavioral changes was directly correlated with higher OARSI histological scores of OA, synovitis in the knee joints, cartilage volume loss, and osteophyte formation.nnnCONCLUSIONnOur findings link functional analyses to histological grading, synovitis, comprehensive three-dimensional assessment of cartilage volume and osteophyte formation. This serves as a reference for a mouse model in predicting outcomes of OA treatment.

E-selectin ligand 1 regulates bone remodeling by limiting bioactive TGF-β in the bone microenvironment

TGF-β is abundantly produced in the skeletal system and plays a crucial role in skeletal homeostasis. E-selectin ligand-1 (ESL-1), a Golgi apparatus-localized protein, acts as a negative regulator of TGF-β bioavailability by attenuating maturation of pro–TGF-β during cartilage homeostasis. However, whether regulation of intracellular TGF-β maturation by ESL-1 is also crucial during bone homeostasis has not been well defined. Here, we show that Esl-1−/− mice exhibit a severe osteopenia with elevated bone resorption and decreased bone mineralization. In primary culture, Esl-1−/− osteoclast progenitors show no difference in osteoclastogenesis. However, Esl-1−/− osteoblasts show delayed differentiation and mineralization and stimulate osteoclastogenesis more potently in the osteoblast–osteoclast coculture, suggesting that ESL-1 primarily acts in osteoblasts to regulate bone homeostasis. In addition, Esl-1−/− calvaria exhibit an elevated mature TGF-β/pro–TGF-β ratio, with increased expression of TGF-β downstream targets (plasminogen activator inhibitor-1, parathyroid hormone-related peptide, connective tissue growth factor, and matrix metallopeptidase 13, etc.) and a key regulator of osteoclastogenesis (receptor activator of nuclear factor κB ligand). Moreover, in vivo treatment with 1D11, a pan–TGF-β antibody, significantly improved the low bone mass of Esl-1−/− mice, suggesting that elevated TGF-β signaling is the major cause of osteopenia in Esl-1−/− mice. In summary, our study identifies ESL-1 as an important regulator of bone remodeling and demonstrates that the modulation of TGF-β maturation is pivotal in the maintenance of a homeostatic bone microenvironment and for proper osteoblast–osteoclast coupling.

Restoration of the serum level of SERPINF1 does not correct the bone phenotype in Serpinf1 null mice

Osteogenesis imperfecta (OI) is a group of genetic disorders characterized by bone fragility and deformity. OI type VI is unique owing to the mineralization defects observed in patient biopsies. Furthermore, it has been reported to respond less well to standard therapy with bisphosphonates [1]. Others and we have previously identified SERPINF1 mutations in patients with OI type VI. SERPINF1 encodes pigment epithelium derived factor (PEDF), a secreted collagen-binding glycoprotein that is absent in the sera of patients with OI type VI. Serpinf1 null mice show increased osteoid and decreased bone mass, and thus recapitulate the OI type VI phenotype. We tested whether restoration of circulating PEDF in the blood could correct the phenotype of OI type VI in the context of protein replacement. To do so, we utilized a helper-dependent adenoviral vector (HDAd) to express human SERPINF1 in the mouse liver and assessed whether PEDF secreted from the liver was able to rescue the bone phenotype observed in Serpinf1(-/-) mice. We confirmed that expression of SERPINF1 in the liver restored the serum level of PEDF. We also demonstrated that PEDF secreted from the liver was biologically active by showing the expected metabolic effects of increased adiposity and impaired glucose tolerance in Serpinf1(-/-) mice. Interestingly, overexpression of PEDF in vitro increased mineralization with a concomitant increase in the expression of bone gamma-carboxyglutamate protein, alkaline phosphatase and collagen, type I, alpha I, but the increased serum PEDF level did not improve the bone phenotype of Serpinf1(-/-) mice. These results suggest that PEDF may function in a context-dependent and paracrine fashion in bone homeostasis.

Arginase overexpression in neurons and its effect on traumatic brain injury

Arginine is a semi-essential amino acid which serves as a substrate for nitric oxide (NO) production by nitric oxide synthase (NOS) and a precursor for various metabolites including ornithine, creatine, polyamines, and agmatine. Arginase competes with nitric oxide synthase for substrate arginine to produce orthinine and urea. There is contradictory evidence in the literature on the role of nitric oxide in the pathophysiology of traumatic brain injury (TBI). These contradictory perspectives are likely due to different NOS isoforms - endothelial (eNOS), inducible (iNOS) and neuronal (nNOS) which are expressed in the central nervous system. Of these, the role of nNOS in acute injury remains less clear. This study aimed to employ a genetic approach by overexpressing arginase isoforms specifically in neurons using a Thy-1 promoter to manipulate cell autonomous NO production in the context of TBI. The hypothesis was that increased arginase would divert arginine from pathological NO production. We generated 2 mouse lines that overexpress arginase I (a cytoplasmic enzyme) or arginase II (a mitochondrial enzyme) in neurons of FVB mice. We found that two-weeks after induction of controlled cortical injury, overexpressing arginase I but not arginase II in neurons significantly reduced contusion size and contusion index compared to wild-type (WT) mice. This study establishes enhanced neuronal arginase levels as a strategy to affect the course of TBI and provides support for the potential role of neuronal NO production in this condition.

Whole-Exome Sequencing Identifies an Intronic Cryptic Splice Site in SERPINF1 Causing Osteogenesis Imperfecta Type VI: WES FINDS INTRONIC CRYPTIC SPLICE SITE IN SERPINF1 CAUSING OI TYPE VI

The heritable disorder osteogenesis imperfecta (OI) is characterized by bone fragility and low bone mass. OI type VI is an autosomal recessive form of the disorder with moderate to severe bone fragility. OI type VI is caused by mutations in the serpin peptidase inhibitor, clade F, member 1 (SERPINF1), the gene coding for pigment epithelium‐derived factor (PEDF). Here, we report a patient with OI type VI caused by a novel homozygous intronic variant in SERPINF1 identified by whole‐exome sequencing (WES). The mutation was not identified using a low bone mass gene panel based on next‐generation sequencing. This variant creates a novel consensus splice donor site (AGGC to AGGT) in intron 4. Analysis of cDNA generated from fibroblasts revealed retention of a 32‐bp intronic fragment between exons 4 and 5 in the cDNA, a result of alternative splicing from the novel splice‐donor site. As a result, the aberrant insertion of this intronic fragment generated a frameshift pathogenic variant and induced nonsense‐mediated decay. Furthermore, gene expression by quantitative PCR showed SERPINF1 expression was dramatically reduced in patient fibroblasts, and PEDF level was also significantly reduced in the patients plasma. In conclusion, we report a novel homozygous variant that generates an alternative splice‐donor in intron 4 of SERPINF1 which gives rise to severe bone fragility. The work also demonstrates clinical utility of WES analysis, and consideration of noncoding variants, in the diagnostic setting of rare bone diseases.

Combinatorial Prg4 and Il-1ra gene therapy protects against hyperalgesia and cartilage degeneration in post-traumatic osteoarthritis

Osteoarthritis (OA) is a degenerative disease of synovial joints characterized by progressive loss of articular cartilage, subchondral bone remodeling, and intra-articular inflammation with synovitis that results in chronic pain and motor impairment. Despite the economic and health impacts, current medical therapies are targeted at symptomatic relief of OA and fail to alter its progression. Given the complexity of OA pathogenesis, we hypothesized that a combinatorial gene therapy approach, designed to inhibit inflammation with interleukin-1 receptor antagonist (IL-1Ra) while promoting chondroprotection using lubricin (PRG4), would improve preservation of the joint compared to monotherapy alone. Employing two surgical techniques to model mild, moderate and severe posttraumatic OA, we found that combined delivery of helper-dependent adenoviruses (HDVs), expressing IL-1Ra and PRG4, preserved articular cartilage better than either monotherapy in both models as demonstrated by preservation of articular cartilage volume and surface area. This improved protection was associated with increased expression of proanabolic and cartilage matrix genes together with decreased expression of catabolic genes and inflammatory mediators. In addition to improvements in joint tissues, this combinatorial gene therapy prolonged protection against thermal hyperalgesia compared to either monotherapy. Taken together, our results show that a combinatorial strategy is superior to monotherapeutic approaches for treatment of posttraumatic OA.