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Osteoarthritis (OA) is a common, debilitating, chronic disease with no disease-modifying drug approved to date. We discovered LNA043—a derivative of angiopoietin-like 3 (ANGPTL3)—as a potent chondrogenesis inducer using a phenotypic screen with human mesenchymal stem cells. We show that LNA043 promotes chondrogenesis and cartilage matrix synthesis in vitro and regenerates hyaline articular cartilage in preclinical OA and cartilage injury models in vivo. LNA043 exerts at least part of these effects through binding to the fibronectin receptor, integrin α5β1 on mesenchymal stem cells and chondrocytes. In a first-in-human (phase 1), randomized, double-blinded, placebo-controlled, single ascending dose, single-center trial (NCT02491281; sponsored by Novartis Pharmaceuticals), 28 patients with knee OA were injected intra-articularly with LNA043 or placebo (3:1 ratio) either 2 h, 7 d or 21 d before total knee replacement. LNA043 met its primary safety endpoint and showed short serum pharmacokinetics, cartilage penetration and a lack of immunogenicity (secondary endpoints). Post-hoc transcriptomics profiling of cartilage revealed that a single LNA043 injection reverses the OA transcriptome signature over at least 21 d, inducing the expression of hyaline cartilage matrix components and anabolic signaling pathways, while suppressing mediators of OA progression. LNA043 is a novel disease-modifying OA drug candidate that is currently in a phase 2b trial (NCT04864392) in patients with knee OA.

The validation of a diffusion chamber comprising a donor and a receptor side separated by a cartilage membrane was undertaken according to the basic principles described by Peng et al. (1998). The study had three targets: first to evaluate the chamber as in vitro system by the examination of the diffusibility of compound through bovine cartilage samples; second the analysis of the affinity of compound (RS-130830) to cartilage; third to test the influence of two pre-incubation periods (one or three nights) of the cartilage samples. The validation of the chamber as in vitro system for the analysis of compound diffusibility and affinity to cartilage was performed using membrane slices of fresh bovine cartilage and a hydroxamic acid derivative (RS-130830) known as matrix metalloproteinase inhibitor (MMPI). The influence of the pre-incubation of cartilage was also examined. Compound concentrations in donor, receptor and membrane were determined by high performance liquid chromatography–mass spectrometry (HPLC–MS). Diffusion could be demonstrated after 6 h and finally 24 h incubation: the compound concentration in the receptor increased from 0 to 35 μM (mean) while it decreased in the donor from 200 to 144 μM (mean). We also found compound in the cartilage membrane (approximately 1.2 nmol (mean)). Pre-incubation of cartilage samples in culture buffer is suitable as a storage procedure, since the results on the donor side only were influenced significantly but not for the receptor and the cartilage affinity. Thus, the system could clearly reflect relevant properties of the tested compound with regard to its diffusibility and affinity to cartilage tissue.

Endothelial intercellular adhesion molecule 1 (ICAM-1) and ICAM-2 are both involved in lymphocyte extravasation during immunosurveillance and inflammation. To define their exact role during T-cell extravasation, we used mouse T cells and ICAM-1-/-ICAM-2-/- brain endothelioma cells. ICAM-1-/-ICAM-2-/- brain endothelioma cells did not support transendothelial migration (TEM) of T cells in vitro. Re-expression of different ICAM-1 mutants in the ICAM-1-/-ICAM-2-/- endothelioma line bEndI1/2.1 or in the ICAM-1-/- endothelioma line bEndI1.1 demonstrated that the extracellular domain of ICAM-1 suffices to support T-cell adhesion while the presence of the cytoplasmic tail was strictly required for TEM. Surprisingly, tyrosine phosphorylation of endothelial ICAM-1 was not necessary for TEM of T cells or for Rho guanosine triphosphatase (RhoGTPase) activation. Furthermore, cytoplasmic deletion mutants of ICAM-1 were unable to mediate RhoGTPase activation. Thus, our data demonstrate that the cytoplasmic tail of endothelial ICAM-1—independently from tyrosine phosphorylation—is essential for supporting TEM of T lymphocytes, while Rho signaling is involved in endothelial cells. (Blood. 2003;102:3675-3683)

We report the cloning of the full-length cDNA of a new murine protein kinase, mPKL12. The sequence reveals a 305-amino-acid protein that contains the characteristic subdomains of the kinase superfamily and particular homology indicating a ser/thr specificity. We have also identified its human homologue gene (94% identical) and the putative homologue proteins from Saccharomyces cerevisiae and Arabidoposis thaliana. These four sequences appear to form a new subfamily of protein kinases, close in size to the theoretical minimal catalytic domain, therefore suggesting that they could be the catalytic unit of a more complex holoenzyme. Using Escherichia coli-purified protein, we have demonstrated that the mPKL12 enzyme possesses an intrinsic kinase activity, capable of phosphorylating enolase and also of promoting autophosphorylation, with a ser/thr specificity. Tissue expression analysis of mPKL12 showed that it is ubiquitously expressed, although at very low levels. RT-PCR analysis of several cell lines also supports this view, therefore suggesting that PKL12 may play a role in a very general cellular function, probably related with the secretory pathway.

CD44 is expressed in various isoforms on numerous cell types and tissues during embryogenesis and in the mature organism. CD44 may also be involved in tumor growth. To study the multiple roles of CD44, we abolished expression of all known isoforms of CD44 in mice by targeting exons encoding the invariant N-terminus region of the molecule. Surprisingly, mice were born in Mendelian ratio without any obvious developmental or neurological deficits. Hematological impairment was evidenced by altered tissue distribution of myeloid progenitors with increased levels of colony-forming unit–granulocyte-macrophage (CFU-GM) in bone marrow and reduced numbers of CFU-GM in spleen. Fetal liver colony-forming unit–spleen and granulocyte colony-stimulating factor mobilization assays, together with reduced CFU-GM in peripheral blood, suggested that progenitor egress from bone marrow was defective. In what was either a compensatory response to CD44 deficiency or an immunoregulatory defect, mice also developed exaggerated granuloma responses to Cryotosporidium parvum infection. Finally, tumor studies showed that SV40-transformed CD44-deficient fibroblasts were highly tumorigenic in nude mice, whereas reintroduction of CD44s expression into these fibroblasts resulted in a dramatic inhibition of tumor growth.

A 730-base pair element regulates expression of the Drosophila gap gene Krüppel (Kr) in response to the fly anterior morphogen bicoid (bcd). Two hormone receptor-like proteins, encoded by the genes knirps (kni) and tailless (tll), bind specifically to the element. In vitro, kni protein competes with the homeodomain-containing bcd protein in binding to a 16-base pair target sequence. In vivo experiments suggest that both kni and tll act as competitive repressors of bcd-mediated activation of Kr. These results suggest a mechanism by which developmental genes can be regulated in response to an activating morphogen gradient antagonized by repressors.

The transcription factor Hey1, a known Notch target gene of the HES family, has recently been described as a target gene of bone morphogenetic protein-2 (BMP-2) during osteoblastic differentiation in vitro. As the role of Hey1 in skeletal physiology is unknown, we analyzed bones of mice ubiquitously lacking or overexpressing Hey1. This strategy enabled us to evaluate whether Hey1 modulation in the whole organism could serve as a drug or antibody target for therapy of diseases associated with bone loss. Hey1 deficiency resulted in modest osteopenia in vivo and increased number and activity of osteoclasts generated ex vivo. Hey1 overexpression resulted in distinct progressive osteopenia and inhibition of osteoblasts ex vivo, an effect apparently dominant to a mild inhibition of osteoclasts. In both Hey1 deficient and overexpressing mice, males were less affected than females and skeleton was not affected during development. Bone histomorphometry did not reveal major changes in animals at 20 weeks, suggesting that modulation had occurred before. Adult Hey1 transgenics also displayed increased type X collagen expression and an enlarged hypertrophic zone in the growth plate. Taken together, our data suggest that ubiquitous in vivo Hey1 regulation affects osteoblasts, osteoclasts and chondrocytes. Due to the complex role of Hey1 in bone, inhibition of Hey1 does not appear to be a straightforward therapeutic strategy to increase the bone mass.

ICAM-2 has been implicated in leukocyte transmigration in vitro, but there is little in vivo evidence to support this. To address this, neutrophil migration was investigated in ICAM-2–deficient mice (KO) and in wild-type (WT) mice treated with an anti–ICAM-2 blocking monoclonal antibody (mAb) (3C4). In a peritonitis model, IL-1β–induced accumulation of neutrophils was significantly reduced in mice treated with 3C4 (51% inhibition) and in KO mice (41% inhibition). In contrast, TNF-α– or thioglycolate-induced responses were not suppressed in KO mice. Analysis of IL-1β–induced leukocyte responses in cremasteric venules of KO animals by intravital microscopy indicated a defect in transmigration (44% inhibition) but not rolling or adhesion. As found before, TNF-α–induced leukocyte transmigration was unaltered in the KO mice. WT mice treated with the anti–ICAM-2 mAb also exhibited a selective reduction in leukocyte transmigration in response to IL-1β while an anti–ICAM-1 mAb inhibited both leukocyte adhesion and transmigration. Interestingly, mAb 3C4 significantly suppressed IL-1β–induced neutrophil transmigration in PE-CAM-1 KO animals in the peritonitis model but not in the cremaster muscle. The findings provide direct evidence for the involvement of ICAM-2 in neutrophil transmigration in vivo, though this role appears to be stimulus specific. Furthermore, ICAM-2 appears capable of mediating PECAM-1–independent leukocyte transmigration.

Osteoarthritis (OA) is a primarily non-inflammatory, degenerative joint disease characterized by progressive loss of articular cartilage, subchondral bone sclerosis, osteophyte formation, changes in the synovial membrane, and an increased volume of synovial fluid with reduced viscosity and hence changed lubrication properties. As OA is the most common type of arthritis and a leading cause of disability, there is a largely unmet medical need for disease-modifying and symptomatic treatment. Due to the localized nature of the disease, intraarticular (IA) drug injection is an attractive treatment approach for OA. The various glucocorticoid and hyaluronic acid (HA) formulations, which are currently available on the market for IA treatment, provide only short-term pain relief or/and often do not provide adequate pain relief. The available oral drugs for symptomatic treatment also have shortcomings, most notably side effects. Therefore, there is still a large unmet need for novel OA drugs, which provide effective long-term pain relief and/or have disease-modifying properties. To achieve long-term drug exposure, different established formulations such as suspensions and hydrogels, and also novel approaches such as lipid based formulations and nano- or microparticles are currently in development. The development of novel drugs in combination with new formulations for IA treatment of OA, represents a promising approach in this challenging area of research.

Endothelial junctions maintain endothelial integrity and vascular homeostasis. They modulate cell trafficking into tissues, mediate cell-cell contact and regulate endothelial survival and apoptosis. Junctional adhesion molecules such as vascular endothelial (VE)–cadherin and CD31/platelet endothelial cell adhesion molecule (PECAM) mediate contact between adjacent endothelial cells and regulate leukocyte transmigration and angiogenesis. The leukocyte adhesion molecule intercellular adhesion molecule 2 (ICAM-2) is expressed at the endothelial junctions. In this study we demonstrate that endothelial ICAM-2 also mediates angiogenesis. Using ICAM-2–deficient mice and ICAM-2–deficient endothelial cells, we show that the lack of ICAM-2 expression results in impaired angiogenesis both in vitro and in vivo. We show that ICAM-2 supports homophilic interaction, and that this may be involved in tube formation. ICAM-2–deficient cells show defective in vitro migration, as well as increased apoptosis in response to serum deprivation, anti-Fas antibody, or staurosporine. ICAM-2 signaling in human umbilical vein endothelial cells (HUVECs) was found to activate the small guanosine triphosphatase (GTPase) Rac, which is required for endothelial tube formation and migration. These data indicate that ICAM-2 may regulate angiogenesis via several mechanisms including survival, cell migration, and Rac activation. Our findings identify a novel pathway regulating angiogenesis through ICAM-2 and a novel mechanism for Rac activation during angiogenesis.

Osteoarthritic (OA) cartilage degeneration and cartilage destruction in rheumatoid arthritis depends significantly on enzymatic degradation of cartilage proteoglycan aggrecan. A member of the ADAMTS family of proteases, ADAMTS-1 was described to have "aggrecanase" activity. We investigated the quantitative expression and distribution of ADAMTS-1 in healthy and OA cartilage and in cultured articular chondrocytes with and without stimulation by interleukin 1beta (IL-1beta) and insulin-like growth factor-I (IGF-I).Conventional and online polymerase chain reaction (PCR) technology was used to determine ADAMTS-1 mRNA expression levels of ADAMTS-1. Protein was localized using immunostaining with different polyclonal antibodies.Conventional and online semiquantitative PCR showed significant levels of ADAMTS-1 mRNA expression in normal and OA chondrocytes in vivo and in vitro. Only a slight increase was observed in OA cartilage. After stimulation with IL-1beta a downregulation of ADAMTS-1 was observed, whereas IGF did not appear to change mRNA expression levels in vitro. The in vivo mRNA expression results were confirmed by the presence of significant protein staining with antibodies for ADAMTS-1 in normal and OA chondrocytes as well as Western blotting analysis. Whereas a significantly stronger stain was seen in normal articular cartilage in the upper zones, in OA cartilage as well the middle zone showed enhanced staining.Our results confirm the expression and presence of ADAMTS-1 in articular cartilage. However, they also point out that ADAMTS-1 appears to be constitutively expressed by adult articular chondrocytes and overall is not strongly upregulated in OA. Thus our data suggest that ADAMTS-1 is the first matrix-degrading enzyme downregulated by the catabolic factor IL-1beta in vitro.

Animal models for osteoarthritis (OA) face the difficulty to display the key pathological events of a naturally slowly progressing disease in a protracted time frame. To serve as pharmacological models, in addition, these changes need to be meaningful and synchronous enough to allow predictions regarding the potential effects of new therapies. Therefore, high expectations have been placed in models where the OA-like pathology develops spontaneously, without any defined stimulus potentially limiting the true disease relevance of the tissue reactions thus induced.

Control of growth and differentiation during mammalian embryogenesis is regulated by growth factors from embryonic and/or maternal sources. Cytokines are polypeptide growth factors that are released by a variety of activated immune and nonimmune cells. To identify novel members of the cytokine family that could be involved in the growth and differentiation of the preimplantation embryo, we studied the expression pattern of several genes encoding cytokines and their receptors during mouse preimplantation developmentin vitroWe found that poly(A)+mRNAs for IL-1, IL-3, IL-6, IL-7, and TNFαare differentially expressed at several stages of mouse preimplantation development, including unfertilized oocytes. Immunostaining of preimplantation embryos using monoclonal antibodies specific for several cytokines and their receptors revealed that at least some of these mRNAs are translated into mature proteins during preimplantation development (IL-1, IL-6, and TNFα). Positive staining for IL-1 and IL-6 receptors was also detected at these stages of development. The controlled expression of these “inflammatory-type” cytokines and their receptors suggests a role for these growth factors during the early phases of mouse ontogeny.

The Drosophila gap gene knirps (kni) is required for abdominal segmentation. It encodes a steroid/thyroid orphan receptor-type transcription factor which is distributed in a broad band of nuclei in the posterior region of the blastoderm. To identify essential domains of the kni protein (KNI), we cloned and sequenced the DNA encompassing the coding region of nine kni mutant alleles of different strength and kni-homologous genes of related insect species. We also examined in vitro-modified versions of KNI in various assay systems both in vitro and in tissue culture. The results show that KNI contains several functional domains which are arranged in a modular fashion. The N-terminal 185-amino-acid region which includes the DNA-binding domain and a functional nuclear location signal fails to provide kni activity to the embryo. However, a truncated KNI protein that contains additional 47 amino acids exerts rather strong kni activity which is functionally defined by a weak kni mutant phenotype of the embryo. The additional 47-amino-acid stretch includes a transcriptional repressor domain which acts in the context of a heterologous DNA-binding domain of the yeast transcriptional activator GAL4. The different domains of KNI as defined by functional studies are conserved during insect evolution.

Summary The segmented body pattern along the longitudinal axis of the Drosophila embryo is established by a cascade of specific transcription factor activities. This cascade is initiated by maternal gene products that are localized at the polar regions of the egg. The initial long-range positional information of the maternal factors, which are transcription factors (or are factors which activate or localize transcription factors), is transferred through the activity of the zygotic segmentation genes. The gap genes act at the top of this regulatory hierarchy. Expression of the gap genes occurs in discrete domains along the longitudinal axis of the preblastoderm and defines specific, overlapping sets of segment primordia. Their protein products, which are DNA-binding transcription factors mostly of the zinc finger type, form broad and overlapping concentration gradients which are controlled by maternal factors and by mutual interactions between the gap genes themselves. Once established, these overlapping gap protein gradients provide spatial cues which generate the repeated pattern of the subordinate pair-rule gene expression, thereby blue-printing the pattern of segmental units in the blastoderm embryo. Our results show different strategies by which maternal gene products, in combination with various gap gene proteins, provide position-dependent sets of transcriptional activator/repressor systems which regulate the spatial pattern of specific gap gene expression. Region-specific combinations of different transcription factors that derive from localized gap gene expression eventually generate the periodic pattern of pair-rule gene expression by the direct interaction with individual cis-acting “stripe elements” of particular pair-rule gene promotors. Thus, the developmental fate of blastoderm cells is programmed according to their position within the anterior-posterior axis of the embryo: maternal transcription factors regulate the region-specific expression of first zygotic transcription factors which, by their specific and unique combinations, control subordinate zygotic transcription factors, thereby subdividing the embryo into increasingly smaller units later seen in the larva.

The Drosophila embryo is composed of serially repeating units, or segments, which differentiate into particular structures and patterns according to their position along the longitudinal axis of the embryo. The generation and diversification of the segments depend on two distinct but integrated processes during early embryogenesis: the subdivision of the embryo into metameric units, and the specification of their identities. Both processes occur simultaneously at the blastoderm stage of embryogenesis which is reached after about 2 h postfertilization. At this stage, the embryo is a single-layered sheet of cells which develops in a characteristic way: nuclei generated by the initial syncitial cleavage divisions migrate to the periphery (egg cortex), and undergo three more divisions before membranes grow down from the cortex to separate each nucleus, thereby forming the cellular blastoderm (for details see Campos-Ortega and Hartenstein 1985). By the completion of this process, the fate of each newly formed cell is already established with regard to its role in the segmentation process. The purpose of this review is to survey the recent progress towards understanding the molecular mechanism of how blastoderm cells are programmed for specific developmental pathways. We first summarize the different components of the genetic circuitry that establish the segment pattern along the longitudinal axis of the Drosophila embryo. We then focus on the activities of the different maternal and zygotic segmentation genes, most of which encode DNA-binding proteins that bind cis-regulatory elements of developmental target genes in a complex network of interactions.

Drosophila proved an excellent system to study molecular processes in establishing the body pattern of an embryo. Genes which are active during oogenesis provide localized cues which regulate a cascade of zygotic genes that determines the developmental fate of the blastoderm cells along the longitudinal axis of the embryo.

ICAM-2-deficient mice exhibit prolonged accumulation of eosinophils in lung interstitium concomitant with a delayed increase in eosinophil numbers in the airway lumen during the development of allergic lung inflammation. The ICAM-2-dependent increased and prolonged accumulation of eosinophils in lung interstitium results in prolonged, heightened airway hyperresponsiveness. These findings reveal an essential role for ICAM-2 in the development of the inflammatory and respiratory components of allergic lung disease. This phenotype is caused by the lack of ICAM-2 expression on non-hematopoietic cells. ICAM-2 deficiency on endothelial cells causes reduced eosinophil transmigration in vitro. ICAM-2 is not essential for lymphocyte homing or the development of leukocytes, with the exception of megakaryocyte progenitors, which are significantly reduced.