Hypoxic regulation of ADAMTS-2 and -3 (a disintegrin and matrix metalloproteinase with thrombospondin motifs 2 and 3) procollagen N proteinases by HIF-1α in endothelial cells.

ADAMTS-2 and ADAMTS-3, known as procollagen amino proteases (PNP), are primarily responsible for processing the amino ends of the fibrillar collagen precursors. ADAMTS-2 is a highly expressed gene in type I collagen-rich tissues, such as skin, bones, tendons, and aorta. ADAMTS-3 is mainly expressed in cartilage, where it colocalizes with type II procollagen and in the nervous system. Studies about ADAMTS-2 and ADAMTS-3 enzymes primarily focused on their collagen processing activity. Knowledge about the transcriptional regulations of these genes is rather limited. Here we analyzed the transcriptional regulations of ADAMTS-2 and ADAMTS-3 genes under chemically induced hypoxic conditions in endothelial cell model, HUVECs. We elucidated that hypoxia is the potent positive regulator of ADAMTS-2 and ADAMTS-3 genes. qRT-PCR and western blotting studies revealed that ADAMTS-2 and ADAMTS-3 expressions were increased at mRNA and protein levels under chemically induced hypoxic conditions in HUVECs. In addition, Transient transfection experiments of ADAMTS-2 and ADAMTS-3 promoter-reporter constructs indicated that low oxygen conditions increased ADAMTS-2 and ADAMTS-3 promoter activities. Furthermore, the DNA-protein interaction assay provided evidence of the functional binding of HIF-1α on bioinformatically determined HRE regions on the ADAMTS-2 and ADAMTS-3 promoters.

Rare and low-frequency coding variants alter human adult height.

Height is a highly heritable, classic polygenic trait with approximately 700 common associated variants identified through genome-wide association studies so far. Here, we report 83 height-associated coding variants with lower minor-allele frequencies (in the range of 0.1-4.8%) and effects of up to 2 centimetres per allele (such as those in IHH, STC2, AR and CRISPLD2), greater than ten times the average effect of common variants. In functional follow-up studies, rare height-increasing alleles of STC2 (giving an increase of 1-2 centimetres per allele) compromised proteolytic inhibition of PAPP-A and increased cleavage of IGFBP-4 in vitro, resulting in higher bioavailability of insulin-like growth factors. These 83 height-associated variants overlap genes that are mutated in monogenic growth disorders and highlight new biological candidates (such as ADAMTS3, IL11RA and NOX4) and pathways (such as proteoglycan and glycosaminoglycan synthesis) involved in growth. Our results demonstrate that sufficiently large sample sizes can uncover rare and low-frequency variants of moderate-to-large effect associated with polygenic human phenotypes, and that these variants implicate relevant genes and pathways.

Biallelic mutation of FBXL7 suggests a novel form of Hennekam syndrome.

Hennekam lymphangiectasia-lymphedema syndrome is an autosomal recessive disorder characterized by congenital lymphedema, intestinal lymphangiectasia, facial dysmorphism, and variable intellectual disability. Known disease genes include CCBE1, FAT4, and ADAMTS3. In a patient with clinically diagnosed Hennekam syndrome but without mutations or copy-number changes in the three known disease genes, we identified a homozygous single-exon deletion affecting FBXL7. Specifically, exon 3, which encodes the F-box domain and several leucine-rich repeats of FBXL7, is eliminated. Our analyses of databases representing >100,000 control individuals failed to identify biallelic loss-of-function variants in FBXL7. Published studies in Drosophila indicate Fbxl7 interacts with Fat, of which human FAT4 is an ortholog, and mutation of either gene yields similar morphological consequences. These data suggest that FBXL7 may be the fourth gene for Hennekam syndrome, acting via a shared pathway with FAT4.

Loss of ADAMTS3 activity causes Hennekam lymphangiectasia-lymphedema syndrome 3.

Primary lymphedema is due to developmental and/or functional defects in the lymphatic system. It may affect any part of the body, with predominance for the lower extremities. Twenty-seven genes have already been linked to primary lymphedema, either isolated, or as part of a syndrome. The proteins that they encode are involved in VEGFR3 receptor signaling. They account for about one third of all primary lymphedema cases, underscoring the existence of additional genetic factors. We used whole-exome sequencing to investigate the underlying cause in a non-consanguineous family with two children affected by lymphedema, lymphangiectasia and distinct facial features. We discovered bi-allelic missense mutations in ADAMTS3. Both were predicted to be highly damaging. These amino acid substitutions affect well-conserved residues in the prodomain and in the peptidase domain of ADAMTS3. In vitro, the mutant proteins were abnormally processed and sequestered within cells, which abolished proteolytic activation of pro-VEGFC. VEGFC processing is also affected by CCBE1 mutations that cause the Hennekam lymphangiectasia-lymphedema syndrome syndrome type1. Our data identifies ADAMTS3 as a novel gene that can be mutated in individuals affected by the Hennekam syndrome. These patients have distinctive facial features similar to those with mutations in CCBE1. Our results corroborate the recent in vitro and murine data that suggest a close functional interaction between ADAMTS3 and CCBE1 in triggering VEGFR3 signaling, a cornerstone for the differentiation and function of lymphatic endothelial cells.

Chondrocytic ephrin B2 promotes cartilage destruction by osteoclasts in endochondral ossification.

The majority of the skeleton arises by endochondral ossification, whereby cartilaginous templates expand and are resorbed by osteoclasts then replaced by osteoblastic bone formation. Ephrin B2 is a receptor tyrosine kinase expressed by osteoblasts and growth plate chondrocytes that promotes osteoblast differentiation and inhibits osteoclast formation. We investigated the role of ephrin B2 in endochondral ossification using Osx1Cre-targeted gene deletion. Neonatal Osx1Cre.Efnb2(Δ/Δ) mice exhibited a transient osteopetrosis demonstrated by increased trabecular bone volume with a high content of growth plate cartilage remnants and increased cortical thickness, but normal osteoclast numbers within the primary spongiosa. Osteoclasts at the growth plate had an abnormal morphology and expressed low levels of tartrate-resistant acid phosphatase; this was not observed in more mature bone. Electron microscopy revealed a lack of sealing zones and poor attachment of Osx1Cre.Efnb2(Δ/Δ) osteoclasts to growth plate cartilage. Osteoblasts at the growth plate were also poorly attached and impaired in their ability to deposit osteoid. By 6 months of age, trabecular bone mass, osteoclast morphology and osteoid deposition by Osx1Cre.Efnb2(Δ/Δ) osteoblasts were normal. Cultured chondrocytes from Osx1Cre.Efnb2(Δ/Δ) neonates showed impaired support of osteoclastogenesis but no significant change in Rankl (Tnfsf11) levels, whereas Adamts4 levels were significantly reduced. A population of ADAMTS4(+) early hypertrophic chondrocytes seen in controls was absent from Osx1Cre.Efnb2(Δ/Δ) neonates. This suggests that Osx1Cre-expressing cells, including hypertrophic chondrocytes, are dependent on ephrin B2 for their production of cartilage-degrading enzymes, including ADAMTS4, and this might be required for attachment of osteoclasts and osteoblasts to the cartilage surface during endochondral ossification.

The "elusive DMOAD": Aggrecanase inhibition from laboratory to clinic.

From the time of their discovery in 1999, the aggrecanases, and ADAMTS-5 in particular, have been heavily investigated as targets for disease-modifying osteoarthritis drug (DMOAD) development. Here, we provide a brief narrative review of the discovery efforts to target these enzymes, and how this led to the current ongoing programmes that hold promise for the future. We discuss a comparison of inhibition of collagen breakdown versus inhibition of aggrecan breakdown. We then summarise existing programmes that target ADAMTS-5, including small molecule inhibitors, monoclonal neutralising antibodies and nanobodies, and gene editing technologies. We also briefly discuss the potential analgesic effects this strategy may offer in addition to its joint-protective effects.

Secreted Metalloproteinase ADAMTS-3 Inactivates Reelin.

The secreted glycoprotein Reelin regulates embryonic brain development and adult brain functions. It has been suggested that reduced Reelin activity contributes to the pathogenesis of several neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease; however, noninvasive methods that can upregulate Reelin activity in vivo have yet to be developed. We previously found that the proteolytic cleavage of Reelin within Reelin repeat 3 (N-t site) abolishes Reelin activity in vitro, but it remains controversial as to whether this effect occurs in vivo Here we partially purified the enzyme that mediates the N-t cleavage of Reelin from the culture supernatant of cerebral cortical neurons. This enzyme was identified as a disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3). Recombinant ADAMTS-3 cleaved Reelin at the N-t site. ADAMTS-3 was expressed in excitatory neurons in the cerebral cortex and hippocampus. N-t cleavage of Reelin was markedly decreased in the embryonic cerebral cortex of ADAMTS-3 knock-out (KO) mice. Importantly, the amount of Dab1 and the phosphorylation level of Tau, which inversely correlate with Reelin activity, were significantly decreased in the cerebral cortex of ADAMTS-3 KO mice. Conditional KO mice, in which ADAMTS-3 was deficient only in the excitatory neurons of the forebrain, showed increased dendritic branching and elongation in the postnatal cerebral cortex. Our study shows that ADAMTS-3 is the major enzyme that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. Therefore, inhibition of ADAMTS-3 may be an effective treatment for neuropsychiatric and neurodegenerative disorders.SIGNIFICANCE STATEMENT ADAMTS-3 was identified as the protease that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. ADAMTS-3 was expressed in the excitatory neurons of the embryonic and postnatal cerebral cortex and hippocampus. Cleavage by ADAMTS-3 is the major contributor of Reelin inactivation in vivo Tau phosphorylation was decreased and dendritic branching and elongation was increased in ADAMTS-3-deficient mice. Therefore, inhibition of ADAMTS-3 upregulates Reelin activity and may be a potential therapeutic strategy for the prevention or treatment of neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease.

Genetic variants in the metzincin metallopeptidase family genes predict melanoma survival.

Metzincins are key molecules in the degradation of the extracellular matrix and play an important role in cellular processes such as cell migration, adhesion, and cell fusion of malignant tumors, including cutaneous melanoma (CM). We hypothesized that genetic variants of the metzincin metallopeptidase family genes would be associated with CM-specific survival (CMSS). To test this hypothesis, we first performed Cox proportional hazards regression analysis to evaluate the associations between genetic variants of 75 metzincin metallopeptidase family genes and CMSS using the dataset from the genome-wide association study (GWAS) from The University of Texas MD Anderson Cancer Center (MDACC) which included 858 non-Hispanic white patients with CM, and then validated using the dataset from the Harvard GWAS study which had 409 non-Hispanic white patients with invasive CM. Four independent SNPs (MMP16 rs10090371 C>A, ADAMTS3 rs788935 T>C, TLL2 rs10882807 T>C and MMP9 rs3918251 A>G) were identified as predictors of CMSS, with a variant-allele attributed hazards ratio (HR) of 1.73 (1.32-2.29, 9.68E-05), 1.46 (1.15-1.85, 0.002), 1.68 (1.31-2.14, 3.32E-05) and 0.67 (0.51-0.87, 0.003), respectively, in the meta-analysis of these two GWAS studies. Combined analysis of risk genotypes of these four SNPs revealed a decreased CMSS in a dose-response manner as the number of risk genotypes increased (Ptrend < 0.001). An improvement was observed in the prediction model (area under the curve [AUC] = 81.4% vs. 78.6%), when these risk genotypes were added to the model containing non-genotyping variables. Our findings suggest that these genetic variants may be promising prognostic biomarkers for CMSS.

An additional case of Hennekam lymphangiectasia-lymphedema syndrome caused by loss-of-function mutation in ADAMTS3.

Hennekam lymphangiectasia-lymphedema syndrome (HKLLS) is a genetically heterogeneous lymphatic dysplasia with characteristic of facial dysmorphism, neurocognitive impairments, and abnormalities of the pericardium, intestinal tract, and extremities. It is an autosomal recessive condition caused by biallelic mutations in CCBE1 (collagen- and calcium-binding epidermal growth factor domain-containing protein 1) (HKLLS1; OMIM 235510) or FAT4 (HKLLS2; OMIM 616006). CCBE1 acts via ADAMTS3 (a disintegrin and metalloprotease with thrombospondin motifs-3 protease) to enhance vascular endothelial growth factor C signaling. There is report of one family supporting mutations in ADAMTS3 as causative for the phenotype labeled as HKLLS3. Here, we report an additional case of HKLLS that appears to be associated with homozygous nonsense mutation of ADAMTS3.

Disruption of murine Adamtsl4 results in zonular fiber detachment from the lens and in retinal pigment epithelium dedifferentiation.

Human gene mutations have revealed that a significant number of ADAMTS (a disintegrin-like and metalloproteinase (reprolysin type) with thrombospondin type 1 motifs) proteins are necessary for normal ocular development and eye function. Mutations in human ADAMTSL4, encoding an ADAMTS-like protein which has been implicated in fibrillin microfibril biogenesis, cause ectopia lentis (EL) and EL et pupillae. Here, we report the first ADAMTSL4 mouse model, tvrm267, bearing a nonsense mutation in Adamtsl4. Homozygous Adamtsl4(tvrm267) mice recapitulate the EL phenotype observed in humans, and our analysis strongly suggests that ADAMTSL4 is required for stable anchorage of zonule fibers to the lens capsule. Unexpectedly, homozygous Adamtsl4(tvrm267) mice exhibit focal retinal pigment epithelium (RPE) defects primarily in the inferior eye. RPE dedifferentiation was indicated by reduced pigmentation, altered cellular morphology and a reduction in RPE-specific transcripts. Finally, as with a subset of patients with ADAMTSL4 mutations, increased axial length, relative to age-matched controls, was observed and was associated with the severity of the RPE phenotype. In summary, the Adamtsl4(tvrm267) model provides a valuable tool to further elucidate the molecular basis of zonule formation, the pathophysiology of EL and ADAMTSL4 function in the maintenance of the RPE.

Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-ß signaling as primary targets.

A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2, 3, and 14 are collectively named procollagen N-proteinases (pNPs) because of their specific ability to cleave the aminopropeptide of fibrillar procollagens. Several reports also indicate that they could be involved in other biological processes, such as blood coagulation, development, and male fertility, but the potential substrates associated with these activities remain unknown. Using the recently described N-terminal amine isotopic labeling of substrate approach, we analyzed the secretomes of human fibroblasts and identified 8, 17, and 22 candidate substrates for ADAMTS2, 3, and 14, respectively. Among these newly identified substrates, many are components of the extracellular matrix and/or proteins related to cell signaling such as latent TGF-ß binding protein 1, TGF-ß RIII, and dickkopf-related protein 3. Candidate substrates for the 3 ADAMTS have been biochemically validated in different contexts, and the implication of ADAMTS2 in the control of TGF-ß activity has been further demonstrated in human fibroblasts. Finally, the cleavage site specificity was assessed showing a clear and unique preference for nonpolar or slightly hydrophobic amino acids. This work shows that the activities of the pNPs extend far beyond the classically reported processing of the aminopropeptide of fibrillar collagens and that they should now be considered as multilevel regulators of matrix deposition and remodeling.-Bekhouche, M., Leduc, C., Dupont, L., Janssen, L., Delolme, F., Vadon-Le Goff, S., Smargiasso, N., Baiwir, D., Mazzucchelli, G., Zanella-Cleon, I., Dubail, J., De Pauw, E., Nusgens, B., Hulmes, D. J. S., Moali, C., Colige, A. Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-ß signaling as primary targets.

Development of human neutralizing antibody to ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2).

ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2), members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, are considered to play a key role in aggrecan degradation of articular cartilage in human osteoarthritis. Here, we developed a neutralizing antibody to these aggrecanases by screening human combinatorial antibody library. Among the five candidate antibodies, one antibody was immunoreactive with both ADAMTS4 and ADAMTS5, showing no or negligible cross-reactivity with 10 different related metalloproteinases of the ADAMTS, ADAM (a disintegrin and metalloproteinase) and MMP (matrix metalloproteinase) gene families. This antibody almost completely and partially inhibited aggrecanase activity of ADAMTS4 and ADAMTS5, respectively. It also suppressed the aggrecanase activity derived from interleukin-1-stimulated osteoarthritic chondrocytes. These data demonstrate that the antibody is specific to ADAMTS4 and ADAMTS5 and inhibits their aggrecanase activity at molecular and cellular levels, and suggest that this antibody may be useful for treatment of pathological conditions such as osteoarthritis.

Elevated expression of periostin in human osteoarthritic cartilage and its potential role in matrix degradation via matrix metalloproteinase-13.

We investigated the role of periostin, an extracellular matrix protein, in the pathophysiology of osteoarthritis (OA). In OA, dysregulated gene expression and phenotypic changes in articular chondrocytes culminate in progressive loss of cartilage from the joint surface. The molecular mechanisms underlying this process are poorly understood. We examined periostin expression by immunohistochemical analysis of lesional and nonlesional cartilage from human and rodent OA knee cartilage. In addition, we used small interfering (si)RNA and adenovirus transduction of chondrocytes to knock down and up-regulate periostin levels, respectively, and analyzed its effect on matrix metalloproteinase (MMP)-13, a disintegrin and MMP with thrombospondin motifs (ADAMTS)-4, and type II collagen expression. We found high periostin levels in human and rodent OA cartilage. Periostin increased MMP-13 expression dose [1-10 µg/ml (EC50 0.5-1 µg/ml)] and time (24-72 h) dependently, significantly enhanced expression of ADAMTS4 mRNA, and promoted cartilage degeneration through collagen and proteoglycan degradation. Periostin induction of MMP-13 expression was inhibited by CCT031374 hydrobromide, an inhibitor of the canonical Wnt/ß-catenin signaling pathway. In addition, siRNA-mediated knockdown of endogenous periostin blocked constitutive MMP-13 expression. These findings implicate periostin as a catabolic protein that promotes cartilage degeneration in OA by up-regulating MMP-13 through canonical Wnt signaling.

Metalloproteases meprin α and meprin ß are C- and N-procollagen proteinases important for collagen assembly and tensile strength.

Type I fibrillar collagen is the most abundant protein in the human body, crucial for the formation and strength of bones, skin, and tendon. Proteolytic enzymes are essential for initiation of the assembly of collagen fibrils by cleaving off the propeptides. We report that Mep1a(-/-) and Mep1b(-/-) mice revealed lower amounts of mature collagen I compared with WT mice and exhibited significantly reduced collagen deposition in skin, along with markedly decreased tissue tensile strength. While exploring the mechanism of this phenotype, we found that cleavage of full-length human procollagen I heterotrimers by either meprin α or meprin ß led to the generation of mature collagen molecules that spontaneously assembled into collagen fibrils. Thus, meprin α and meprin ß are unique in their ability to process and release both C- and N-propeptides from type I procollagen in vitro and in vivo and contribute to the integrity of connective tissue in skin, with consequent implications for inherited connective tissue disorders.

Shedding of syndecan-4 promotes immune cell recruitment and mitigates cardiac dysfunction after lipopolysaccharide challenge in mice.

Inflammation is central to heart failure progression. Innate immune signaling increases expression of the transmembrane proteoglycan syndecan-4 in cardiac myocytes and fibroblasts, followed by shedding of its ectodomain. Circulating shed syndecan-4 is increased in heart failure patients, however the pathophysiological and molecular consequences associated with syndecan-4 shedding remain poorly understood. Here we used lipopolysaccharide (LPS) challenge to investigate the effects of syndecan-4 shedding in the heart. Wild-type mice (10mg/kg, 9h) and cultured neonatal rat cardiomyocytes and fibroblasts were subjected to LPS challenge. LPS increased cardiac syndecan-4 mRNA without altering full-length protein. Elevated levels of shedding fragments in the myocardium and blood from the heart confirmed syndecan-4 shedding in vivo. A parallel upregulation of ADAMTS1, ADAMTS4 and MMP9 mRNA suggested these shedding enzymes to be involved. Echocardiography revealed reduced ejection fraction, diastolic tissue velocity and prolonged QRS duration in mice unable to shed syndecan-4 (syndecan-4 KO) after LPS challenge. In line with syndecan-4 shedding promoting immune cell recruitment, expression of immune cell markers (CD8, CD11a, F4/80) and adhesion receptors (Icam1, Vcam1) were attenuated in syndecan-4 KO hearts after LPS. Cardiomyocytes and fibroblasts exposed to shed heparan sulfate-substituted syndecan-4 ectodomains showed increased Icam1, Vcam1, TNFα and IL-1ß expression and NF-κB-activation, suggesting direct regulation of immune cell recruitment pathways. In cardiac fibroblasts, shed ectodomains regulated expression of extracellular matrix constituents associated with collagen synthesis, cross-linking and turnover. Higher syndecan-4 levels in the coronary sinus vs. the radial artery of open heart surgery patients suggested that syndecan-4 is shed from the human heart. Our data demonstrate that shedding of syndecan-4 ectodomains is part of the cardiac innate immune response, promoting immune cell recruitment, extracellular matrix remodeling and mitigating cardiac dysfunction in response to LPS.

Low density lipoprotein receptor-related protein 1 (LRP1)-mediated endocytic clearance of a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4): functional differences of non-catalytic domains of ADAMTS-4 and ADAMTS-5 in LRP1 binding.

Degradation of the cartilage proteoglycan aggrecan is an early event in the development of osteoarthritis, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and ADAMTS-5 are considered to be the major aggrecan-degrading enzymes. We have recently found that ADAMTS-5 is rapidly endocytosed via low density lipoprotein receptor-related protein 1 (LRP1) and degraded by chondrocytes. Here we report that this regulatory mechanism also applies to ADAMTS-4, although its rate of endocytosis is slower than that of ADAMTS-5. Domain deletion mutagenesis of ADAMTS-4 identified that the cysteine-rich and spacer domains are responsible for binding to LRP1, whereas the thrombospondin 1 and spacer domains are responsible in ADAMTS-5. The estimated t½ value of ADAMTS-4 endocytosis was about 220 min, whereas that of ADAMTS-5 was 100 min. The difference in half-lives between the two enzymes is explained by the 13-fold lower affinity of ADAMTS-4 for LRP1 compared with that of ADAMTS-5. Studies using soluble ligand binding clusters of LRP1 showed that ADAMTS-4 binds to clusters II and IV with similar KD,app values of 98 and 73 nm, respectively, whereas ADAMTS-5 binds to cluster II, III, and IV with KD,app values of 3.5, 41, and 9 nm, respectively. Thus, ADAMTS-5 competitively inhibits ADAMTS-4 endocytosis but not vice versa. This study highlights that the affinity between a ligand and LRP1 dictates the rate of internalization and suggests that LRP1 is a major traffic controller of the two aggrecanases, especially under inflammatory conditions, where the protein levels of ADAMTS-4 increase, but those of ADAMTS-5 do not.

CCBE1 enhances lymphangiogenesis via A disintegrin and metalloprotease with thrombospondin motifs-3-mediated vascular endothelial growth factor-C activation.

BACKGROUND: Hennekam lymphangiectasia-lymphedema syndrome (Online Mendelian Inheritance in Man 235510) is a rare autosomal recessive disease, which is associated with mutations in the CCBE1 gene. Because of the striking phenotypic similarity of embryos lacking either the Ccbe1 gene or the lymphangiogenic growth factor Vegfc gene, we searched for collagen- and calcium-binding epidermal growth factor domains 1 (CCBE1) interactions with the vascular endothelial growth factor-C (VEGF-C) growth factor signaling pathway, which is critical in embryonic and adult lymphangiogenesis. METHODS AND RESULTS: By analyzing VEGF-C produced by CCBE1-transfected cells, we found that, whereas CCBE1 itself does not process VEGF-C, it promotes proteolytic cleavage of the otherwise poorly active 29/31-kDa form of VEGF-C by the A disintegrin and metalloprotease with thrombospondin motifs-3 protease, resulting in the mature 21/23-kDa form of VEGF-C, which induces increased VEGF-C receptor signaling. Adeno-associated viral vector-mediated transduction of CCBE1 into mouse skeletal muscle enhanced lymphangiogenesis and angiogenesis induced by adeno-associated viral vector-VEGF-C. CONCLUSIONS: These results identify A disintegrin and metalloprotease with thrombospondin motifs-3 as a VEGF-C-activating protease and reveal a novel type of regulation of a vascular growth factor by a protein that enhances its proteolytic cleavage and activation. The results suggest that CCBE1 is a potential therapeutic tool for the modulation of lymphangiogenesis and angiogenesis in a variety of diseases that involve the lymphatic system, such as lymphedema or lymphatic metastasis.

Relationship between gene expression and lung function in Idiopathic Interstitial Pneumonias.

BACKGROUND: Idiopathic interstitial pneumonias (IIPs) are a group of heterogeneous, somewhat unpredictable diseases characterized by progressive scarring of the interstitium. Since lung function is a key determinant of survival, we reasoned that the transcriptional profile in IIP lung tissue would be associated with measures of lung function, and could enhance prognostic approaches to IIPs. RESULTS: Using gene expression profiling of 167 lung tissue specimens with IIP diagnosis and 50 control lungs, we identified genes whose expression is associated with changes in lung function (% predicted FVC and % predicted DLCO) modeled as categorical (severe vs mild disease) or continuous variables while adjusting for smoking status and IIP subtype; false discovery rate (FDR) approach was used to correct for multiple comparisons. This analysis identified 58 transcripts that are associated with mild vs severe disease (categorical analysis), including those with established role in fibrosis (ADAMTS4, ADAMTS9, AGER, HIF-1α, SERPINA3, SERPINE2, and SELE) as well as novel IIP candidate genes such as rhotekin 2 (RTKN2) and peptidase inhibitor 15 (PI15). Protein-protein interactome analysis of 553 genes whose expression is significantly associated with lung function when modeled as continuous variables demonstrates that more severe presentation of IIPs is characterized by an increase in cell cycle progression and apoptosis, increased hypoxia, and dampened innate immune response. Our findings were validated in an independent cohort of 131 IIPs and 40 controls at the mRNA level and for one gene (RTKN2) at the protein level by immunohistochemistry in a subset of samples. CONCLUSIONS: We identified commonalities and differences in gene expression among different subtypes of IIPs. Disease progression, as characterized by lower measures of FVC and DLCO, results in marked changes in expression of novel and established genes and pathways involved in IIPs. These genes and pathways represent strong candidates for biomarker studies and potential therapeutic targets for IIP severity.

CD11c(+) macrophages and levels of TNF-α and MMP-3 are increased in synovial and adipose tissues of osteoarthritic mice with hyperlipidaemia.

To understand more clearly the link between osteoarthritis and hyperlipidaemia, we investigated the inflammatory macrophage subsets and macrophage-regulated matrix metalloprotease-3 (MMP-3) and A disintegrin and metalloprotease with thrombospondin motifs-4 (ADAMTS4) in synovial (ST) and adipose tissues (AT) of osteoarthritic mice with hyperlipidaemia (STR/Ort). CD11c(+) F4/80(+) CD11b(+) macrophage populations in the ST and AT of 9-month-old STR/Ort and C57BL/6J mice were characterized and compared by flow cytometry and real-time polymerase chain reaction (PCR) analyses. Expression of tumour necrosis factor (TNF)-α, MMP-3 and ADAMTS4, and the response of these factors to anionic liposomal clodronate induced-macrophage depletion were also evaluated by real-time PCR. Expression of TNF-α in CD11c(+) cells, which were isolated by magnetic beads, was compared to CD11c(-) cells. In addition, the effect of TNF-α on cultured synovial fibroblasts and adipocytes was investigated. CD11c(+) F4/80(+) CD11b(+) macrophages were increased in ST and AT of STR/Ort mice. The CD11c(+) cell fraction highly expressed TNF-α. Expression of TNF-α and MMP3 was increased in ST and AT, and was decreased upon macrophage depletion. TNF-α treatment of cultured synovial fibroblasts and adipocytes markedly up-regulated MMP-3. CD11c(+) F4/80(+) CD11b(+) macrophages were identified as a common inflammatory subset in the AT and ST of STR/Ort mice with hyperlipidaemia. The induction of inflammation in AT and ST may be part of a common mechanism that regulates MMP3 expression through TNF-α. Our findings suggest that increased numbers of CD11c(+) macrophages and elevated levels of TNF-α and MMP-3 in AT and ST may explain the relationship between hyperlipidaemia and OA.

ADAMTS-4 activity in synovial fluid as a biomarker of inflammation and effusion.

OBJECTIVE: To evaluate the potential of ADAMTS-4 (aggrecanase -1) activity in synovial fluid (SF) as a biomarker of knee injury and joint disease. DESIGN: We have measured ADAMTS-4 activity in the synovial fluid of 170 orthopaedic patients with different degrees of joint pathology, using a commercial ADAMTS-4 fluorescence resonance energy transfer (FRET) substrate assay. Patients were classified at arthroscopy as (i) macroscopically normal, (ii) with an injury of the meniscus, anterior cruciate ligament or chondral/osteochondral defects or (iii) with osteoarthritis, and the influence of independent factors (age, patient group, effusion and synovial inflammation) on ADAMTS-4 activity levels was assessed. RESULTS: In most patients (106/170) ADAMTS-4 activity was undetectable; ADAMTS-4 ranged from 0 to 2.8 ng/mL in synovial fluid from patients with an injury, 0-4.1 ng/mL in osteoarthritic patients and 4.0-12.3 ng/mL in patients with large effusions. Four independent variables each significantly influenced ADAMTS-4 activity in synovial fluid (all P < 0.001): age (concordance = 0.69), presence of osteoarthritis (OA) (concordance = 0.66), level of effusion (concordance = 0.78) and inflammation (concordance = 0.68). Not only did effusion influence the amount of ADAMTS-4 activity most strongly, but it also did this in an ordered manner (P < 0.001). CONCLUSIONS: The main finding of this study is that ADAMTS-4 levels in synovial fluid are most strongly correlated with inflammation and severity of effusion in the knee. Further study is required to determine if it could provide a useful tool to aid clinical diagnoses, indicate treatment, to monitor progression of joint degeneration or OA or alternatively the success of treatment.