Patricia Klinger

Chondromodulin 1 stabilizes the chondrocyte phenotype and inhibits endochondral ossification of porcine cartilage repair tissue

OBJECTIVE To investigate the effect of chondromodulin 1 on the phenotype of osteochondral progenitor cells in cartilage repair tissue. METHODS Self-complementary adeno-associated virus (AAV) vectors carrying chondromodulin 1 complementary DNA (AAV-Chm-1) were applied to cartilage lesions in the knee joints of miniature pigs that were treated by the microfracture technique. Alternatively, isolated porcine osteochondral progenitor cells were infected with AAV-Chm-1 or with AAV-GFP control vectors ex vivo prior to being transplanted into cartilage lesions in which the subchondral bone plate was left intact. The quality of the repair tissue and the degree of endochondral ossification were assessed by histochemical and immunohistochemical methods. The effects of chondromodulin 1 overexpression were also analyzed by angiogenesis assays and quantitative reverse transcriptase-polymerase chain reaction. RESULTS AAV-Chm-1-infected cells efficiently produced chondromodulin 1, which had strong antiangiogenic effects, as verified by the inhibition of tube formation of endothelial cells. Gene expression analyses in vitro revealed the cell cycle inhibitor p21WAF1/Cip1 as one target up-regulated by AAV-Chm-1. Direct application of AAV-Chm-1 vectors into microfractured porcine cartilage lesions stimulated chondrogenic differentiation of ingrowing progenitor cells, but significantly inhibited terminal chondrocyte hypertrophy, the invasion of vessel structures, and excessive endochondral ossification, which were otherwise observed in untreated lesions. Indirect gene transfer, with infection of porcine osteochondral progenitor cells by AAV-Chm-1 ex vivo, also supported chondrogenic differentiation of these transplanted cells. AAV-Chm-1-infected cells maintained a chondrocyte-like phenotype and formed a hyaline-like matrix that was superior to that formed by uninfected or AAV-GFP-infected cells. CONCLUSION Our findings indicate that the antiangiogenic factor chondromodulin 1 stabilizes the chondrocyte phenotype by supporting chondrogenesis but inhibiting chondrocyte hypertrophy and endochondral ossification.

Molecular differentiation between osteophytic and articular cartilage – clues for a transient and permanent chondrocyte phenotype

OBJECTIVE To identify the molecular differences between the transient and permanent chondrocyte phenotype in osteophytic and articular cartilage. METHODS Total RNA was isolated from the cartilaginous layer of osteophytes and from intact articular cartilage from knee joints of 15 adult human donors and subjected to cDNA microarray analysis. The differential expression of relevant genes between these two cartilaginous tissues was additionally validated by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry. RESULTS Among 47,000 screened transcripts, 600 transcripts were differentially expressed between osteophytic and articular chondrocytes. Osteophytic chondrocytes were characterized by increased expression of genes involved in the endochondral ossification process [bone gamma-carboxyglutamate protein/osteocalcin (BGLAP), bone morphogenetic protein-8B (BMP8B), collagen type I, alpha 2 (COL1A2), sclerostin (SOST), growth arrest and DNA damage-induced gene 45ß (GADD45ß), runt-related transcription factor 2 (RUNX2)], and genes encoding tissue remodeling enzymes [matrix metallopeptidase (MMP)9, 13, hyaluronan synthase 1 (HAS1)]. Articular chondrocytes expressed increased transcript levels of antagonists and inhibitors of the BMP- and Wnt-signaling pathways [Gremlin-1 (GREM1), frizzled-related protein (FRZB), WNT1 inducible signaling pathway protein-3 (WISP3)], as well as factors that inhibit terminal chondrocyte differentiation and endochondral bone formation [parathyroid hormone-like hormone (PTHLH), sex-determining region Y-box 9 (SOX9), stanniocalcin-2 (STC2), S100 calcium binding protein A1 (S100A1), S100 calcium binding protein B (S100B)]. Immunohistochemistry of tissue sections for GREM1 and BGLAP, the two most prominent differentially expressed genes, confirmed selective detection of GREM1 in articular chondrocytes and that of BGLAP in osteophytic chondrocytes and bone. CONCLUSIONS Osteophytic and articular chondrocytes significantly differ in their gene expression pattern. In articular cartilage, a prominent expression of antagonists inhibiting the BMP- and Wnt-pathway may serve to lock and stabilize the permanent chondrocyte phenotype and thus prevent their terminal differentiation. In contrast, osteophytic chondrocytes express genes with roles in the endochondral ossification process, which may account for their transient phenotype.

Solution Structure of the Human Immunodeficiency Virus Type 1 p6 Protein

The human immunodeficiency virus type 1 p6 protein represents a docking site for several cellular and viral binding factors and fulfills major roles in the formation of infectious viruses. To date, however, the structure of this 52-amino acid protein, by far the smallest lentiviral protein known, either in its mature form as free p6 or as the C-terminal part of the Pr55 Gag polyprotein has not been unraveled. We have explored the high resolution structure and folding of p6 by CD and NMR spectroscopy. Under membranous solution conditions, p6 can adopt a helix-flexible helix structure; a short helix-1 (amino acids 14–18) is connected to a pronounced helix-2 (amino acids 33–44) by a flexible hinge region. Thus, p6 can be subdivided into two distinct structural and functional domains; helix-2 perfectly defines the region that binds to the virus budding factor AIP-1/ALIX, indicating that this structure is required for interaction with the endosomal sorting complex required for transport. The PTAP motif at the N terminus, comprising the primary late assembly domain, which is crucial for interaction with another cellular budding factor, Tsg101, does not exhibit secondary structure. However, the adjacent helix-1 may play an indirect role in the specific complex formation between p6 and the binding groove in Tsg101. Moreover, binding studies by NMR demonstrate that helix-2, which also comprises the LXXLF motif required for incorporation of the human immunodeficiency virus type 1 accessory protein Vpr into budding virions, specifically interacts with the Vpr binding region, indicating that under the specific solution conditions used for structure analysis, p6 adopted a functional conformation.

Paracrine effect of transplanted rib chondrocyte spheroids supports formation of secondary cartilage repair tissue

The studys objective was to investigate if transplanted chondrocyte or periosteal cell spheroids have influence on ingrowing bone marrow‐derived cells in a novel cartilage repair approach in miniature pigs. Autologous rib chondrocytes or periosteal cells were cultured as spheroids and press‐fitted into cavities that were milled into large, superficial chondral lesions of the patellar joint surface. Within the milled cavities, the subchondral bone plate was either penetrated or left intact (full‐thickness or partial‐thickness cavities). The transplantation of chondrocyte spheroids into full‐thickness cavities induced the formation of additional secondary repair cartilage that exceeded the original volume of the transplanted spheroids. The resulting continuous tissue was rich in proteoglycans and stained positive for type II collagen. Cell labeling revealed that secondarily invading repair cells did not originate from transplanted spheroids, but rather from arroded bone marrow. However, secondary invasion of repair cells was less pronounced following transplantation of periosteal cells and absent in partial‐thickness cavities. According to in vitro analyses, these observations could be ascribed to the ability of chondrocyte spheroids to secrete relevant amounts of bone morphogenetic protein‐2, which was not detected for periosteal cells. Transplanted chondrocyte spheroids exert a dual function: they provide cells for the repair tissue and have a stimulatory paracrine activity, which promotes ingrowth and chondrogenesis of bone marrow‐derived cells.

The ubiquitin-proteasome system in HIV replication: potential targets for antiretroviral therapy.

Since the discovery of HIV approximately 20 years ago, more than 60 million individuals have been infected, and AIDS still remains one of the most devastating diseases humankind has ever faced. Unfortunately, there is little hope that an effective vaccine will be developed in the near future. Current antiretroviral treatment is based on drugs that either target the viral enzymes (protease and reverse transcriptase) or the attachment and entry of the virus. Although the introduction of highly active antiretroviral therapy in the mid-1990s has led to a profound reduction in HIV-related morbidity and mortality, the complete eradication of the virus from infected individuals has never been achieved. In addition, these antiviral drugs can induce serious adverse effects, particularly when administered in combination over prolonged treatment periods. A further drawback to these treatments is that with the high mutation rate of HIV, drug-resistant mutants are evolving, particularly when antiretroviral treatment only suppresses virus replication to marginal levels in latently infected cells making up the virus reservoirs in vivo. Cellular genes have much lower mutation rates, and drug-mediated modulation of specific cellular pathways represents an attractive antiviral strategy. Recent findings showing that proteasome inhibitors interfere with budding, maturation and infectivity of HIV have triggered intensive investigation of the hitherto unappreciated function of the ubiquitin–proteasome system in HIV replication. It was also observed that, like several other retroviruses, HIV-1 virions contain a small amount of mono-ubiquitinylated Gag proteins. Currently, two E3-type ubiquitin ligases, in addition to one E3-like protein, have been identified as regulators of HIV budding. These ligases might represent interesting targets for therapeutic intervention.

DYNC2LI1 mutations broaden the clinical spectrum of dynein-2 defects

Skeletal ciliopathies are a heterogeneous group of autosomal recessive osteochondrodysplasias caused by defects in formation, maintenance and function of the primary cilium. Mutations in the underlying genes affect the molecular motors, intraflagellar transport complexes (IFT), or the basal body. The more severe phenotypes are caused by defects of genes of the dynein-2 complex, where mutations in DYNC2H1, WDR34 and WDR60 have been identified. In a patient with a Jeune-like phenotype we performed exome sequencing and identified compound heterozygous missense and nonsense mutations in DYNC2LI1 segregating with the phenotype. DYNC2LI1 is ubiquitously expressed and interacts with DYNC2H1 to form the dynein-2 complex important for retrograde IFT. Using DYNC2LI1 siRNA knockdown in fibroblasts we identified a significantly reduced cilia length proposed to affect cilia function. In addition, depletion of DYNC2LI1 induced altered cilia morphology with broadened ciliary tips and accumulation of IFT-B complex proteins in accordance with retrograde IFT defects. Our results expand the clinical spectrum of ciliopathies caused by defects of the dynein-2 complex.

Wnt Inhibitory Factor 1 Deficiency Uncouples Cartilage and Bone Destruction in Tumor Necrosis Factor α–Mediated Experimental Arthritis

OBJECTIVE Wnt signaling plays a pivotal role in skeletal development and in the control of cartilage and bone turnover. We have recently shown that the secreted Wnt antagonist Wnt inhibitory factor 1 (WIF-1) is mainly expressed in the upper layers of epiphyseal and articular cartilage and, to a lesser extent, in bone. Nevertheless, WIF-1(-/-) mice develop normally. In light of these findings, we undertook this study to analyze the role of WIF-1 in arthritis. METHODS Expression analyses for WIF-1 were performed by real-time reverse transcription-polymerase chain reaction (RT-PCR). WIF-1(-/-) and tumor necrosis factor (TNF)-transgenic mice were crossbred, and the progression of arthritis in TNF-transgenic WIF-1(-/-) mice and littermate controls was evaluated. Structural joint damage was analyzed by histologic staining, histomorphometry, and micro-computed tomography. Wnt/β-catenin signaling was investigated by real-time RT-PCR and immunofluorescence on primary chondrocytes. RESULTS WIF-1 expression was repressed by TNFα in chondrocytes and osteoblasts and down-regulated in experimental arthritis and in articular cartilage from patients with rheumatoid arthritis. WIF-1 deficiency partially protected TNF-transgenic mice against bone erosion and loss of trabecular bone, probably as a result of less osteoclast activity. In contrast, arthritis-related cartilage damage was aggravated by WIF-1 deficiency, while overexpression of WIF-1 attenuated cartilage degradation in TNF-transgenic mice. In chondrocytes, TNFα stimulated canonical Wnt signaling, which could be blocked by WIF-1, indicating a direct effect of TNFα and WIF-1 on Wnt signaling in this system. CONCLUSION These data suggest that WIF-1 may take part in the fine-tuning of cartilage and bone turnover, promoting the balance of cartilage versus bone anabolism.

The Transient Chondrocyte Phenotype in Human Osteophytic Cartilage A Role of Pigment Epithelium-Derived Factor?

Objective: To identify factors that are responsible for the phenotypic differences between transient chondrocytes within human osteophytes prone to endochondral ossification and permanent chondrocytes within articular cartilage persisting for decades. Methods: Differential gene expression of chondrocytes from human osteophytes or from articular cartilage was detected by cDNA microarray analysis. The expression of pigment epithelium-derived factor (PEDF), one of the most impressively differentially expressed genes, was validated by quantitative reverse transcriptase polymerase chain reaction as well as immunohistochemistry. The mode of action of PEDF was explored by cell viability assays and by detecting target genes. Results: PEDF mRNA expression was upregulated by 118.5-fold (P = 0.01) in human osteophytic cartilage compared with articular cartilage, which was reflected by strong immunostaining for PEDF in the cartilaginous layer of osteophytes but largely negative staining in articular cartilage. Elevated levels of PEDF in osteophytes were associated with enhanced apoptosis. PEDF increased the expression of the proapoptotic factor FasL and induced cell death in cell culture. Osteochondral progenitor cells were more responsive to PEDF than differentiated articular chondrocytes. Conclusions: The induction of the proapoptotic factor PEDF within the osteophyte cartilage suggests a molecular concept for the transient chondrocyte phenotype that arises from progenitor cells and is prone to terminal differentiation and cell death.

Clinical relevance of systematic phenotyping and exome sequencing in patients with short stature

PurposeShort stature is a common condition of great concern to patients and their families. Mostly genetic in origin, the underlying cause often remains elusive due to clinical and genetic heterogeneity.MethodsWe systematically phenotyped 565 patients where common nongenetic causes of short stature were excluded, selected 200 representative patients for whole-exome sequencing, and analyzed the identified variants for pathogenicity and the affected genes regarding their functional relevance for growth.ResultsBy standard targeted diagnostic and phenotype assessment, we identified a known disease cause in only 13.6% of the 565 patients. Whole-exome sequencing in 200 patients identified additional mutations in known short-stature genes in 16.5% of these patients who manifested only part of the symptomatology. In 15.5% of the 200 patients our findings were of significant clinical relevance. Heterozygous carriers of recessive skeletal dysplasia alleles represented 3.5% of the cases.ConclusionA combined approach of systematic phenotyping, targeted genetic testing, and whole-exome sequencing allows the identification of the underlying cause of short stature in at least 33% of cases, enabling physicians to improve diagnosis, treatment, and genetic counseling. Exome sequencing significantly increases the diagnostic yield and consequently care in patients with short stature.

Deletion of the oxygen-dependent degradation domain results in impaired transcriptional activity of hypoxia-inducible factors.

Hypoxia-inducible factors (HIF1α/HIF2α) are key transcription factors that promote angiogenesis. The overexpression of degradation-resistant HIF mutants is considered a promising pro-angiogenic therapeutic tool. We compared the transcriptional activity of HIF1α/HIF2α mutants that obtained their resistance to oxygen-dependent degradation either by deletion of their entire oxygen-dependent degradation (ODD) domain or by replacement of prolyl residues that are crucial for oxygen-dependent degradation. Although all HIF mutants translocated into the nucleus, HIF1α and HIF2α mutants inclosing the point mutations were significantly more effective in trans-activating the target gene VEGF and in inducing tube formation of endothelial cells than mutants lacking the complete ODD domain.