Publications

HUMAN CHONDROCYTES RESPOND DISCORDANTLY TO THE PROTEIN ENCODED BY THE OSTEOARTHRITIS SUSCEPTIBILITY GENE GDF5

By: Madhushika Ratnayake, Frank Plöger, Mauro Santibanez-Koref, John Loughlin

A genetic deficit mediated by SNP rs143383 that leads to reduced expression of GDF5 is strongly associated with large-joint osteoarthritis. We speculated that this deficit could be attenuated by the application of exogenous GDF5 protein and as a first step we have assessed what effect such application has on primary osteoarthritis chondrocyte gene expression. Chondrocytes harvested from cartilage of osteoarthritic patients who had undergone joint replacement were cultured with wildtype recombinant mouse and human GDF5 protein. We also studied variants of GDF5, one that has a higher affinity for the receptor BMPR-IA and one that is insensitive to the GDF5 antagonist noggin. As a positive control, chondrocytes were treated with TGF-b1. Chondrocytes were cultured in monolayer and micromass and the expression of genes coding for catabolic and anabolic proteins of cartilage were measured by quantitative PCR. The expression of the GDF5 receptor genes and the presence of their protein products was confirmed and the ability of GDF5 signal to translocate to the nucleus was demonstrated by the activation of a luciferase reporter construct. The capacity of GDF5 to elicit an intracellular signal in chondrocytes was demonstrated by the phosphorylation of intracellular Smads. Chondrocytes cultured with TGF-b1 demonstrated a consistent down regulation of MMP1, MMP13 and a consistent upregulation of TIMP1 and COL2A1 with both culture techniques. In contrast, chondrocytes cultured with wildtype GDF5, or its variants, did not show any consistent response, irrespective of the culture technique used. Our results show that osteoarthritis chondrocytes do not respond in a predictable manner to culture with exogenous GDF5. This may be a cause or a consequence of the osteoarthritis disease process and will need to be surmounted if treatment with exogenous GDF5 is to be advanced as a potential means to overcome the genetic deficit conferring osteoarthritis susceptibility at this gene.

A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

By: Elisa Degenkolbe, Jana König, Julia Zimmer, Maria Walther, Carsten Reißner, Joachim Nickel, Frank Plöger, Jelena Raspopovic, James Sharpe, Katarina Dathe, Jacqueline T. Hecht, Stefan Mundlos, Sandra C. Doelken, Petra Seemann

Growth and Differentiation Factor 5 (GDF5) is a secreted growth factor that belongs to the Bone Morphogenetic Protein (BMP) family and plays a pivotal role during limb development. GDF5 is a susceptibility gene for osteoarthritis (OA) and mutations in GDF5 are associated with a wide variety of skeletal malformations ranging from complex syndromes such as acromesomelic chondrodysplasias to isolated forms of brachydactylies or multiple synostoses syndrome 2 (SYNS2). Here, we report on a family with an autosomal dominant inherited combination of SYNS2 and additional brachydactyly type A1 (BDA1) caused by a single point mutation in GDF5 (p.W414R). Functional studies, including chondrogenesis assays with primary mesenchymal cells, luciferase reporter gene assays and Surface Plasmon Resonance analysis, of the GDF5W414R variant in comparison to other GDF5 mutations associated with isolated BDA1 (p.R399C) or SYNS2 (p.E491K) revealed a dual pathomechanism characterized by a gain- and loss-of-function at the same time. On the one hand insensitivity to the main GDF5 antagonist NOGGIN (NOG) leads to a GDF5 gain of function and subsequent SYNS2 phenotype. Whereas on the other hand, a reduced signaling activity, specifically via the BMP receptor type IA (BMPR1A), is likely responsible for the BDA1 phenotype. These results demonstrate that one mutation in the overlapping interface of antagonist and receptor binding site in GDF5 can lead to a GDF5 variant with pathophysiological relevance for both, BDA1 and SYNS2 development. Consequently, our study assembles another part of the molecular puzzle of how loss and gain of function mutations in GDF5 affect bone development in hands and feet resulting in specific types of brachydactyly and SYNS2. These novel insights into the biology of GDF5 might also provide further clues on the pathophysiology of OA.

ENHANCED RECONSTRUCTION OF LONG BONE ARCHITECTURE BY A GROWTH FACTOR MUTANT COMBINING POSITIVE FEATURES OF GDF-5 AND BMP-2

By: Kerstin Kleinschmidt, Frank Ploeger, Joachim Nickel, Julia Glockenmeier, Pierre Kunz, Wiltrud Richter

The latest innovation of the BIOPHARM Growth Factor Platform Technology, the proprietary growth factor BB-1, has been reviewed in the publication “Enhanced reconstruction of long bone architecture by a growth factor mutant combining positive features of GDF-5 and BMP-2”, published by Kleinschmidt et al. in Biomaterials 34 (2013). The review demonstrates that BB-1, a recently patented Growth Factor developed by BIOPHARM, shows a superior bone growth potential over currently available bone morphogenic proteins (BMP).

GENERATION OF AMYLOID-B IS REDUCED BY THE INTERACTION OF CALRETICULIN WITH AMYLOID PRECURSOR PROTEIN, PRESENILIN AND NICASTRIN

By: Nina Stemmer, Elena Strekalova, Nevena Djogo, Frank Plöger, Gabriele Loers, David Lutz, Friedrich Buck, Marek Michalak, Melitta Schachner, Ralf Kleene

Dysregulation of the proteolytic processing of amyloid precursor protein by c-secretase and the ensuing generation of amyloid-b is associated with the pathogenesis of Alzheimer’s disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the c-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the c-secretase cleavage site within amyloid precursor protein and showed that this Ca2+- and N-glycan-independent interaction is mediated by amino acids 330–344 in the C-terminal Cdomain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the c-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the c-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-b42 levels in culture supernatants, while transfection with the P-domain increases amyloid-b40 levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330–344 to amyloid precursor protein overexpressing cells result in elevated amyloid-b40 and amyloid-b42 levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the c-secretase complex regulates the proteolytic processing of amyloid precursor protein by the c-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer’s disease.

NEW INSIGHTS INTO THE MOLECULAR MECHANISM OF MULTIPLE SYNOSTOSES SYNDROME (SYNS): MUTATION WITHIN THE GDF5 KNUCKLE EPITOPE CAUSES NOGGIN-RESISTANCE.

By: Schwaerzer GK, Hiepen C, Schrewe H, Nickel J, Ploeger F, Sebald W, Mueller T, Knaus P.

Growth and differentiation factor 5 (GDF5), a member of the bone morphogenetic protein (BMP) family, is essential for cartilage, bone, and joint formation. Antagonists such as noggin counteract BMP signaling by covering the ligand's BMP type I (BMPRI) and type II (BMPRII, ActRII, ActRIIB) interaction sites. The mutation GDF5-S94N is located within the BMPRII interaction site, the so-called knuckle epitope, and was identified in patients suffering from multiple synostoses syndrome (SYNS). SYNS is characterized by progressive symphalangism, carpal/tarsal fusions, deafness and mild facial dysmorphism. Here we present a novel molecular mechanism of a GDF5 mutation affecting chondrogenesis and osteogenesis. GDF5-S94N exhibits impaired binding to BMPRII causing alleviated Smad and non-Smad signaling and reduced chondrogenic differentiation of ATDC5 cells. Surprisingly, chondrogenesis in mouse micromass cultures was strongly enhanced by GDF5-S94N. By using quantitative techniques (SPR, reporter gene assay, ALP assay, qPCR), we uncovered that this gain of function is caused by strongly reduced affinity of GDF5-S94N to the BMP/GDF antagonist noggin and the consequential lack of noggin inhibition. Thus, since noggin is upregulated during chondrogenic differentiation, GDF5-S94N exceeds the GDF5 action, which results in the phenotypic outcome of SYNS. The detailed molecular characterization of GDF5-S94N as a noggin-resistant growth factor illustrates the potential of GDF5 mutants in applications with defined therapeutical needs.

INTRANASAL DELIVERY OF GROWTH DIFFERENTIATION FACTOR 5 TO THE CENTRAL NERVOUS SYSTEM.

By: Hanson LR, Fine JM, Hoekman JD, Nguyen TM, Burns RB, Martinez PM, Pohl J, Frey WH 2nd.

CONTEXT: Growth differentiation factor 5 (GDF5), in addition to its role in bone and joint development, protects dopaminergic (DA) neurons from degeneration, and is a potential therapeutic agent for Parkinson's disease. Its large size and insolubility at physiologic pH are obstacles for drug administration to the central nervous system (CNS) in humans.
OBJECTIVE: In this study, formulations to deliver GDF5 to the brain using intranasal (IN) administration were developed.
MATERIALS AND METHODS: IN administration of GDF5 in acidic buffer, 20 mM sodium acetate (NaAc) at pH 4.25, was performed in rats. Also, a lipid microemulsion (LME) comprised of olive oil and phosphatidylserine (PS) was used to formulate GDF5 at neutral pH for IN administration. Tissue concentrations of GDF5 were determined by both gamma counting and enzyme-linked immunosorbent assay (ELISA).
RESULTS: IN administration of GDF5 in acidic buffers bypassed the blood-brain barrier (BBB), resulting in delivery to the brain with limited systemic exposure. IN administration of GDF5-LME increased drug targeting to the midbrain eightfold when compared to IN administration of GDF5 in acidic buffer.
DISCUSSION AND CONCLUSION: This study is the first to show that GDF5 can be formulated at neutral pH and can be directly delivered to the CNS via IN administration, with biologically relevant concentrations in the midbrain where it may be used to treat Parkinson's disease.

TIME KINETICS OF BONE DEFECT HEALING IN RESPONSE TO BMP-2 AND GDF-5 CHARACTERISED BY IN VIVO BIOMECHANICS

By: Madhushika Ratnayake, Frank Plöger, Mauro Santibanez-Koref, John Loughlin

This study reports that treatment of osseous defects with different growth factors initiates distinct rates of repair. We developed a new method for monitoring the progression of repair, based upon measuring the in vivo mechanical properties of healing bone. Two different members of the bone morphogenetic protein (BMP) family were chosen to initiate defect healing: BMP-2 to induce osteogenesis, and growth-and-differentiation factor (GDF)-5 to induce chondrogenesis. To evaluate bone healing, BMPs were implanted into stabilised 5 mm bone defects in rat femurs and compared to controls. During the first two weeks, in vivo biomechanical measurements showed similar values regardless of the treatment used. However, 2 weeks after surgery, the rhBMP-2 group had a substantial increase in stiffness, which was supported by the imaging modalities. Although the rhGDF-5 group showed comparable mechanical properties at 6 weeks as the rhBMP-2 group, the temporal development of regenerating tissues appeared different with rhGDF-5, resulting in a smaller callus and delayed tissue mineralisation. Moreover, histology showed the presence of cartilage in the rhGDF-5 group whereas the rhBMP-2 group had no cartilaginous tissue. Therefore, this study shows that rhBMP-2 and rhGDF-5 treated defects, under the same conditions, use distinct rates of bone healing as shown by the tissue mechanical properties. Furthermore, results showed that in vivo biomechanical method is capable of detecting differences in healing rate by means of change in callus stiffness due to tissue mineralisation.

THE EFFECT OF TWO POINT MUTATIONS IN GDF-5 ON ECTOPIC BONE FORMATION IN A BETA-TRICALCIUMPHOSPHATE SCAFFOLD.

By: Madhushika Ratnayake, Frank Plöger, Mauro Santibanez-Koref, John Loughlin

The osteoinductivity of human growth-and-differentiation factor-5 (GDF-5) is well established, but a reduced amount of ectopic bone is formed compared to other members of the bone morphogenetic protein (BMP) family like BMP-2. We hypothesized that swap of two BMP-receptor-interacting residues of GDF-5 to amino acids present in BMP-2 (methionine to valine at the sites 453 and 456) may improve the bone formation capacity of the mutant GDF-5. Heterotopic bone formation of a mutant GDF-5 coated beta-TCP carrier was compared to carriers coated with similar amounts (10 microg) of GDF-5 and BMP-2 in SCID mice. Four week explants revealed 6-fold higher ALP activity in the mutant GDF-5 versus the wild type GDF-5 group (p < 0.0001) and 1.4-fold higher levels compared to BMP-2 (p < 0.006). Bone area in histology was significantly higher in mutant GDF-5 versus all other groups at 4 weeks; however, at 8 weeks BMP-2 reached a similar neo-bone formation like mutant GDF-5. Micro-CT evaluation confirmed higher values in the mutant GDF-5 and BMP-2 groups compared to wild type GDF-5. In conclusion, the mutant GDF-5 showed superior bone formation capacity than GDF-5, and a faster induction at similar final outcome as BMP-2. Mutant GDF-5 thus represents a promising new GDF-5 variant for bone regeneration possibly acting via an increased binding affinity to the BMP-type I receptor.

ELUCIDATION OF THE DISULFIDE BRIDGE PATTERN OF THE RECOMBINANT HUMAN GROWTH AND DIFFERENTIATION FACTOR 5 DIMER AND THE INTERCHAIN CYS/ALA MUTANT MONOMER.

By: Trachsel C, Kämpfer U, Bechtold R, Schaller J, Schürch S.

Growth and differentiation factor 5 (GDF5) is involved in many developmental processes such as chondrogenesis and joint and bone formation. A recombinant monomeric human GDF5 mutant rGDF5(C84A) is in vitro as potent as the dimeric native form, and clinical investigations of rGDF5(C84A) are in progress. Native homodimeric GDF5 belongs to the transforming growth factor beta (TGF-beta) superfamily; each monomer contains a cystine knot formed by three intrachain disulfide bridges, and the monomers are connected via an interchain disulfide bridge. The disulfide bridge pattern of recombinant homodimeric rGDF5 was recently elucidated by X-ray diffraction. A combination of proteolytic degradation with thermolysin, separation of the generated fragments by reverse-phase high-performance liquid chromatography (RP-HPLC), and subsequent analyses of the disulfide-linked peptides by electrospray-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, amino acid analysis, and Edman degradation led to the unambiguous identification of the disulfide bridge pattern of the monomeric mutant rGDF5(C84A) and of the homodimeric rGDF5 in solution. The cystine knot of homodimeric rGDF5 exhibits the pattern Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7 (three intrachain disulfide bonds), and the monomers are connected by a single interchain disulfide bridge (Cys4-Cys4) in accordance with other members of the TGF-beta superfamily. The monomeric mutant rGDF5(C84A) exhibits the same cystine knot pattern as homodimeric rGDF5.

PERIODONTAL WOUND HEALING/REGENERATION FOLLOWING IMPLANTATION OF RECOMBINANT HUMAN GROWTH/DIFFERENTIATION FACTOR-5 (RHGDF-5) IN AN ABSORBABLE COLLAGEN SPONGE CARRIER INTO ONE-WALL INTRABONY DEFECTS IN DOGS: A DOSE-RANGE STUDY.

By: Kim TG, Wikesjö UM, Cho KS, Chai JK, Pippig SD, Siedler M, Kim CK.

AIM:
Recombinant human growth/differentiation factor-5 (rhGDF-5) is being evaluated as a candidate therapy in support of periodontal regeneration. The objective of this study was to evaluate cementum and alveolar bone formation, and aberrant healing events following surgical implantation of rhGDF-5 in an absorbable collagen sponge (ACS) carrier using an established periodontal defect model.
MATERIALS AND METHODS:
Bilateral 4 x 5 mm (width x depth), one-wall, critical-size, intrabony periodontal defects were surgically created at the mandibular second and fourth pre-molar teeth in 15 Beagle dogs. Five animals received 1 microg/defect and five animals 20 microg/defect rhGDF-5 in unilateral defect sites. Contralateral sites received treatments reported elsewhere. Five animals received rhGDF-5/ACS with 0 (buffer control) and 100 microg/defect rhGDF-5 in contralateral defect sites. The animals were euthanized at 8 weeks post-surgery for histologic and histometric evaluation.
RESULTS:
Surgical implantation of rhGDF-5 stimulated significant periodontal regeneration. Cementum formation was significantly enhanced in sites implanted with rhGDF-5 (1 and 100 microg) compared with control (p<0.05). Similarly, bone formation height was significantly greater in sites receiving rhGDF-5 (1 and 100 microg) compared with control (p<0.05). There were no significant or remarkable differences in bone and cementum formation within the selected dose interval (1, 20 and 100 microg rhGDF-5). None of the control or the rhGDF-5 sites exhibited root resorption, ankylosis, or other aberrant tissue reactions.
CONCLUSION:
Surgical implantation of rhGDF-5/ACS may be used safely to support periodontal wound healing/regeneration in intrabony periodontal defects without complications.

A PRELIMINARY REPORT ON THE EFFECT OF DIMERIC RHGDF-5 AND ITS MONOMERIC FORM RHGDF-5C465A ON BONE HEALING OF RAT CRANIAL DEFECTS.

By: Dupoirieux L, Pohl J, Hanke M, Pourquier D.

INTRODUCTION:
The purpose of the study was to compare the efficacy on rat skull defects of two bone growth factors derived from the GDF-5 family.
MATERIAL AND METHODS:
The study was conducted on 17 adult Wistar rats. On each animal, two symmetrical 6-mm wide, full-thickness, skull defects were carried out in the parietal regions. In 15 out of 17 animals, both experimental defects were filled by the implants. In the group I (n=2), both defects were left empty for control. The 15 other rats were divided into 3 groups: In group II (n=5), a collagen sponge was implanted. In group III (n=5), a collagen sponge impregnated with rhGDF-5 (the genuine dimeric form) was implanted. In group IV (n=5), a collagen sponge impregnated with rhGDF-5C465A (a monomeric form of GDF-5) was implanted. All animals were sacrificed at 8 weeks. The harvested specimens were processed for contact radiography and standard histological examination. The quantitative results were assessed with a semi-quantitative histological scoring system.
RESULTS:
One animal in the group II was excluded because it died of unknown reasons. In group I, no bone healing was observed in the defects. In group II, no bone healing was observed in 4 out of 10 defects, and partial bone healing was observed in 5 out of 10 defects. In group III, partial bone healing was also observed in 3 out of 8 defects and complete bone healing in 4 out of 8 defects. In group IV, partial bone healing was observed in 8 out of 10 defects and complete bone healing in 2 out of 10 defects.
CONCLUSION:
Bone healing was improved in all treated groups. Further studies are necessary to determine the optimal formulation of these composite implants.

MUTATIONS IN GDF5 REVEAL A KEY RESIDUE MEDIATING BMP INHIBITION BY NOGGIN.

By: Seemann P, Brehm A, König J, Reissner C, Stricker S, Kuss P, Haupt J, Renninger S, Nickel J, Sebald W, Groppe JC, Plöger F, Pohl J, Schmidt-von Kegler M, Walther M, Gassner I, Rusu C, Janecke AR, Dathe K, Mundlos S.

Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP-related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP-inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.

GDF5 AND BMP2 INHIBIT APOPTOSIS VIA ACTIVATION OF BMPR2 AND SUBSEQUENT STABILIZATION OF XIAP.

By: Liu Z, Shen J, Pu K, Katus HA, Plöger F, Tiefenbacher CP, Chen X, Braun T.

GDF5 and BMP2, members of the TGF-beta superfamily of growth factors, are known to regulate apoptosis in different cell types either positively or negatively. We wanted to investigate the effects of GDF5 and BMP2 on vascular smooth muscle cells and mouse embryonic fibroblasts and disclose the mechanism by which GDF5 and BMP2 might exert anti-apoptotic effects. The effect of GDF5 and BMP2 on proliferation and/or programmed cells death was assessed in isolated human vascular smooth muscle cells and mouse embryonic fibroblasts. We demonstrate that GDF5 and BMP2 prevent apoptosis induced by serum starvation in mouse embryonic fibroblasts but not in smooth muscle cells via the BMP receptor 2 (BMPR2), which is often mutated in hereditary cases of primary pulmonary hypertension. GDF5 and BMP2 stimulate the interaction of BMPR-2 with XIAP thereby reducing the ubiquitination of XIAP, which results in enhanced protein stability. The increased concentration of XIAP counteracts apoptosis by binding and inactivating activated caspases. We conclude that the inhibition of apoptosis in mouse embryonic fibroblasts by BMP2 and GDF5 does not depend on more complex signal transduction pathways such as smad and MAPK signaling but on direct stabilization of XIAP by BMPR2.

EMERGING NEUROMODULATORY MOLECULES FOR THE TREATMENT OF NEUROGENIC ERECTILE DYSFUNCTION CAUSED BY CAVERNOUS NERVE INJURY.

By: Bella AJ, Lin G, Cagiannos I, Lue TF.

Advances in the neurobiology of growth factors, neural development, and prevention of cell death have resulted in a heightened clinical interest for the development of protective and regenerative neuromodulatory strategies for the cavernous nerves (CNs), as therapies for prostate cancer and other pelvic malignancies often result in neuronal damage and debilitating loss of sexual function. Nitric oxide released from the axonal end plates of these nerves within the corpora cavernosa causes relaxation of smooth muscle, initiating the haemodynamic changes of penile erection as well as contributing to maintained tumescence; the loss of CN function is primarily responsible for the development of erectile dysfunction (ED) after pelvic surgery and serves as the primary target for potential neuroprotective or regenerative strategies. Evidence from pre-clinical studies for select neuromodulatory approaches is reviewed, including neurotrophins, glial cell line-derived neurotrophic factors (GDNF), bone morphogenic proteins, immunophilin ligands, erythropoetin (EPO), and stem cells

SINUS FLOOR AUGMENTATION WITH RECOMBINANT HUMAN GROWTH AND DIFFERENTIATION FACTOR-5 (RHGDF-5): A HISTOLOGICAL AND HISTOMORPHOMETRIC STUDY IN THE GOETTINGEN MINIATURE PIG.

By: Gruber RM, Ludwig A, Merten HA, Achilles M, Poehling S, Schliephake H.

AIM:
The aim of this study was to test the hypothesis that recombinant human growth and differentiation factor-5 (rhGDF-5) enhances bone formation in sinus floor augmentations in miniature pigs.
MATERIAL AND METHODS:
The maxillary sinus floors in 12 adult female Goettingen minipigs were augmented with beta-tricalcium phosphate (beta-TCP) on one side. The contralateral test side was augmented using two concentrations of rhGDF-5 (400 microg rhGDF-5/g beta-TCP; 800 microg rhGDF-5/g beta-TCP) delivered on beta-TCP (six animals each). One dental implant was inserted into each sinus floor augmentation. After 4 and 12 weeks, histological and histomorphometric assessment of non-decalcified histological specimens was performed.
RESULTS:
The results showed significantly higher mean values of volume density (VD) of newly formed bone using the concentration of 400 microg/g beta-TCP (22.8%) compared with the respective control (8%) after 4 weeks (P=0.05). The bone-to-implant contact rates were also significantly enhanced after 4 weeks between test sites (400 microg: 41.9%; 800 microg: 40.6%) and control sites (400 microg: 7.8%; 800 microg: 16.4%) (400 microg: P=0.024; 800 microg: P=0.048).
CONCLUSION:
It is concluded that rhGDF-5 delivered on beta-TCP significantly enhanced early bone formation compared with beta-TCP alone in sinus lift procedures in miniature pigs.

DEVELOPMENT OF AN INJECTABLE COMPOSITE AS A CARRIER FOR GROWTH FACTOR-ENHANCED PERIODONTAL REGENERATION.

By: Herberg S, Siedler M, Pippig S, Schuetz A, Dony C, Kim CK, Wikesjö UM.

AIM:
Biomaterials are often applied in periodontal therapy; however, not always well adapted for tissue regeneration. The objective of this study was to evaluate the physico-chemical properties and biocompatibility of an injectable, in situ setting composite for growth factor-enhanced periodontal regeneration.
MATERIAL AND METHODS:
The composite constitutes bioresorbable poly(lactic-co-glycolic acid) (PLGA) and additives forming in situ a matrix designed as a carrier for recombinant human growth/differentiation factor-5 (rhGDF-5). In vitro characterization included the porosity, biointeraction, biodegradation, injectability, and biological activity of released rhGDF-5. Biocompatibility was compared with granular beta-tricalcium phosphate and an absorbable collagen sponge using a canine periodontal defect model.
RESULTS:
The PLGA composite showed a highly porous (500-1000 mum) space-providing structure. It effectively induced coagulation exhibiting an intimate interaction with the fibrin clot. The biphasic biodegradation was complete within 4 weeks. The composite was conveniently injectable (90.4+/-3.6 N) for ease of use. It exhibited a sustained rhGDF-5 release over 4 weeks (40.8%) after initial burst (3.4%) detected by ALP activity. Sites receiving the composite showed limited, if any, residuals and had no appreciable negative effect on periodontal wound healing. There were no noteworthy inflammatory lesions in sites receiving the PLGA composite.
CONCLUSION:
Characteristics of the PLGA composite makes it an attractive matrix to support native wound healing and rhGDF-5-enhanced periodontal regeneration.

INTRACAVERNOUS GROWTH DIFFERENTIATION FACTOR-5 THERAPY ENHANCES THE RECOVERY OF ERECTILE FUNCTION IN A RAT MODEL OF CAVERNOUS NERVE INJURY.

By: Fandel TM, Bella AJ, Lin G, Tantiwongse K, Lin CS, Pohl J, Lue TF.

INTRODUCTION:
Neurogenic erectile dysfunction remains a serious complication in the postprostatectomy population. Effective protective and regenerative neuromodulatory strategies are needed.
AIM:
To determine the effect of growth differentiation factor-5 (GDF-5) on erectile function and its mechanism in a rat model of cavernous nerve (CN) injury.
MAIN OUTCOME MEASURES:
Erectile function was assessed by CN electrostimulation at 4 weeks. Penile tissues were examined by real-time polymerase chain reaction (PCR) and immunohistochemical analyses.
METHODS:
Forty-eight male Sprague-Dawley rats were randomly divided into six equal groups: one group underwent sham operation (uninjured controls), while five groups underwent bilateral CN crush. Crush-injury groups were treated at the time of injury with intracavernous injection of a slow-release suspension of liquid microparticles containing no GDF-5 (vehicle), 0.4 microg (low concentration), 2 microg (intermediate concentration), or 10 microg GDF-5 (high concentration). One untreated group served as injured controls.
RESULTS:
GDF-5 enhanced erectile recovery and significantly increased intracavernous pressure in the low and intermediate-concentration groups vs. injured controls. Low-concentration GDF-5 demonstrated the best functional preservation, as the intracavernous pressure increase in this group did not differ significantly from uninjured controls. A dose-response relationship was confirmed for the effects of GDF-5 in penile tissue. Low-concentration GDF-5 showed better preservation of the penile dorsal nerves and antiapoptotic effects in the corpus cavernosum (P < 0.05 vs. injured controls). Although high concentration GDF-5 did not confer meaningful erectile recovery, this dose was more effective at decreasing transforming growth factor-beta than low-concentration GDF-5.
CONCLUSIONS:
Intracavernous injection of low (0.4 microg) or intermediate-concentration GDF-5 (2 microg) was effective in preserving erectile function in a rat model of neurogenic erectile dysfunction. The underlying mechanism appears to involve neuron preservation and antiapoptosis.

BRACHYDACTYLY TYPE A2 ASSOCIATED WITH A DEFECT IN PROGDF5 PROCESSING.

By: Plöger F, Seemann P, Schmidt-von Kegler M, Lehmann K, Seidel J, Kjaer KW, Pohl J, Mundlos S.

We investigated a family with a brachydactyly type A2 and identified a heterozygous arginine to glutamine (R380Q) substitution in the growth/differentiation factor 5 (GDF5) in all affected individuals. The observed mutation is located at the processing site of the protein, at which the GDF5 precursor is thought to be cleaved releasing the mature molecule from the prodomain. In order to test the effect of the mutation, we generated the GDF5-R380Q mutant and a cleavage-resistant proGDF5 mutant (R380A/R381A) in vitro. Both mutants were secreted from chicken micromass cultures, but showed diminished biological activity. Western blot analyses showed that wt GDF5 was processed by the chicken micromass cells, whereas the mutants were not, indicating that the mutations interfere with processing and that this leads to a strong reduction of biological activity. To test the requirements for GDF5 processing in vitro we produced recombinant human (rh) proGDF5 wild-type protein in Escherichia coli. The results show that unprocessed (rh) proGDF5 is virtually inactive but can be proteolytically activated by different enzymes such as trypsin, furin, and MMP3. (rh) proGDF5 could thus be used as a locally administered depot form with retarded release of activity. In contrast to mature rhGDF5, (rh) proGDF5 shows a high solubility at physiological pH, a characteristic that might be useful for therapeutic applications.

BONE REGENERATION.

By: Holy, C., Volenec, FJ., Geesin, J., and Bruder, SP.

MODULATION OF VEGF EXPRESSION IN RAT BONE MARROW STROMAL CELLS BY GDF-5.

By: Sena K, Sumner DR, Virdi AS.

Angiogenesis is essential for bone formation and several bone morphogenetic proteins (BMPs) have been shown to induce angiogenesis through osteoblast-derived vascular endothelial growth factor (VEGF)-A. Growth differentiation factor-5 (GDF-5) is a member of the BMP family expressed in bone and known to induce angiogenesis in vivo. In this study, the effects of GDF-5 on osteogenic differentiation and expression of VEGF-related genes were determined using rat bone marrow stromal cells. GDF-5 stimulated osteogenic differentiation. It also upregulated the expression of VEGF-A after 3 hr, accompanied by a trend of decrease in its receptor VEGFR-2 at 6 and 24 hr. VEGF-D and its receptor VEGFR-3 showed peak expression at later time points. This regulation may be further controlled by neuropilin 2 that exhibited a parallel profile to VEGF-D. These observations indicate that GDF-5 stimulates osteogenic differentiation and has a potential to induce angiogenesis through osteoblast-derived VEGF-A in bone.

THE EFFECT OF GROWTH DIFFERENTIATION FACTOR-5-COATED SUTURES ON TENDON REPAIR IN A RAT MODEL.

By: Dines JS, Weber L, Razzano P, Prajapati R, Timmer M, Bowman S, Bonasser L, Dines DM, Grande DP.

Tendon ruptures are common injuries that are often treated surgically. Growth Differentiation Factor-5 (GDF-5) has been shown to accelerate tendon healing with varying degrees of success. We used a novel technique to apply recombinant human GDF-5 (rhGDF-5) to suture and hypothesized that controlled, local delivery of rhGDF-5 can be used to enhance tendon repair. Tendons of 92 rats were transected and repaired with sutures. All researchers were blinded to the following treatment groups (24 rats in each group): 0 rhGDF (control), 24 ng/cm rhGDF, 55 ng/cm rhGDF, and 556 ng/cm rhGDF. Rats were euthanized at 3 weeks (n = 48) and at 6 weeks (n = 48). Sutures were coated with rhGDF-5 using a novel dip-coat technique. Enzyme-linked immunosorbent assay confirmed consistent and reproducible delivery of rhGDF-5. Within each group, 8 were tested biomechanically, and 4 were assessed histologically. Histologic grading at 3 weeks showed improved healing in tendons repaired with coated suture versus controls. By 6 weeks, there were no significant differences. At 3 weeks, minimal isolated cartilage formation was observed; 6-week samples showed more extensive presence, typically surrounding suture fibers. At 3 weeks, tendons repaired with rhGDF-5-coated sutures resulted in significantly higher ultimate tensile load and stiffness compared with control sutures (P < .05) At 6 weeks, there were no significant differences in the mechanical properties of repaired tendons. At 3 weeks, rhGDF-5 induced significant tendon hypertrophy that was more pronounced than at 6 weeks. In addition, tendons repaired with rhGDF-5 showed an increased rate of healing versus control repairs at 3 weeks. This study showed that a novel dip-coating technique can be used to deliver growth factors in varying concentrations to local repair sites to accelerate tendon healing.

DIFFERENTIAL EFFECTS OF GDF5 ON THE MEDIAL AND LATERAL RAT VENTRAL MESENCEPHALON.

By: Clayton KB, Sullivan AM.

Growth/differentiation factor 5 (GDF5) is a member of the transforming growth factor-beta (TGF-beta) superfamily that has potent neurotrophic and protective effects on dopaminergic neurones and is expressed in the developing rat substantia nigra (the ventral mesencephalon; VM). GDF5 has the potential to be used in the treatment of Parkinson's disease (PD), a neurodegenerative disorder characterised by the selective degeneration of nigrostriatal dopaminergic neurones. One therapy being explored for PD involves transplantation of fetal VM tissue into the striatum in order to replace lost dopaminergic neurones. The majority of transplantation studies have used transplants incorporating the whole VM. The principal location of dopaminergic neurones in the E14 rat VM is in the medial VM. In the present study, the effects of GDF5 on cultures prepared from medial, lateral and whole E14 rat VM tissue were compared. GDF5 treatment increased the number of dopaminergic neurones in whole and lateral, but not in medial, VM cultures, whereas it increased total cell number in medial, but not in whole or lateral, VM cultures. RT-PCR studies showed that the receptors for GDF5 were differentially expressed in E14 VM; the expression of BMPR-IB and Ror2 was low in medial but high in lateral VM tissue. This study suggests that GDF5 increases the number of dopaminergic neurones in whole VM cultures by acting on BMPR-IB and Ror2-expressing cells in the lateral VM.

EFFECT OF GDF-5 ON LIGAMENT HEALING.

By: Tashiro T, Hiraoka H, Ikeda Y, Ohnuki T, Suzuki R, Ochi T, Nakamura K, Fukui N.

The effects of growth and differentiation factor-5 (GDF-5) on ligament healing were studied using a gap injury model of the medial collateral ligament in rat knee joints. The administration of GDF-5 once at the time of surgery significantly improved the mechanical properties of the femur-ligament-tibia complex. At 3 weeks after surgery, 30 microg of GDF-5 improved the ultimate tensile strength of the complex by 41%, and the stiffness by 60%, compared with the vehicle control (p < 0.05 for both; Fisher's PLSD test). The observation with a transmission electron microscopy revealed that GDF-5 increased the diameter of collagen fibrils in the repair tissue, which was considered to be a possible mechanism for the positive result in the biomechanical testing. Quantitative PCR and in situ hybridization revealed enhanced type I procollagen expression by GDF-5, and the PCR analysis also revealed that the GDF-5 treatment reduced the expression of type III procollagen relative to type I procollagen. The PCR analysis further showed that the expression of decorin and fibromodulin was relatively reduced against type I procollagen by the growth factor, which was considered to be responsible for the increase of collagen fibril diameter in the repair tissue. No adverse effects were observed, and the use of GDF-5 was considered a promising approach to facilitate ligament healing.

GDF5 IS A SECOND LOCUS FOR MULTIPLE-SYNOSTOSIS SYNDROME.

By: Dawson K, Seeman P, Sebald E, King L, Edwards M, Williams J 3rd, Mundlos S, Krakow D.

Multiple-synostosis syndrome is an autosomal dominant disorder characterized by progressive symphalangism, carpal/tarsal fusions, deafness, and mild facial dysmorphism. Heterozygosity for functional null mutations in the NOGGIN gene has been shown to be responsible for the disorder. However, in a cohort of six probands with multiple-synostosis syndrome, only one was found to be heterozygous for a NOGGIN mutation (W205X). Linkage studies involving the four-generation family of one of the mutation-negative patients excluded the NOGGIN locus, providing genetic evidence of locus heterogeneity. In this family, polymorphic markers flanking the GDF5 locus were found to cosegregate with the disease, and sequence analysis demonstrated that affected individuals in the family were heterozygous for a novel missense mutation that predicts an R438L substitution in the GDF5 protein. Unlike mutations that lead to haploinsufficiency for GDF5 and produce brachydactyly C, the protein encoded by the multiple-synostosis-syndrome allele was secreted as a mature GDF5 dimer. These data establish locus heterogeneity in multiple-synostosis syndrome and demonstrate that the disorder can result from mutations in either the NOGGIN or the GDF5 gene.

LOCAL APPLICATION OF A COLLAGEN TYPE I/HYALURONATE MATRIX AND GROWTH AND DIFFERENTIATION FACTOR 5 INFLUENCES THE CLOSURE OF OSTEOCHONDRAL DEFECTS IN A MINIPIG MODEL BY ENCHONDRAL OSSIFICATION.

By: Jung M, Tuischer JS, Sergi C, Gotterbarm T, Pohl J, Richter W, Simank HG.

This pilot study evaluated the effect of growth and differentiation factor-5 (rhGDF-5) combined with a collagen type I/hyaluronate matrix (c/h) on osteochondral defect repair in a minipig model. Defects created in both medial femoral condyles of 20 minipigs were treated with c/h (n = 10), c/h + rhGDF-5 (n = 10) or were left empty. After 3 and 12 months, five animals of each group were sacrificed. Evaluation included macroscopic and histological scoring and quantitative histomorphometry of synthesized bone. C/h and c/h + rhGDF-5 treatment increased trabecular bone formation in the upper third of the defect compared to empty controls, showing significance for c/h + rhGDF-5 (p = 0.05) but not between c/h and c/h + rhGDF-5 treatment. Cartilage regeneration and macroscopic outcome were not improved by c/h or c/h + rhGDF-5 treatment. Since c/h remnants were seen even one year postoperatively in the defect, possibly inhibiting further bone and cartilage healing, other matrices in combination with rhGDF-5 may provide further improvement in osteochondral defect treatment.

GROWTH AND DIFFERENTIATION FACTOR-5: NEW FRONTIERS TO BONE REPAIR AND SPINAL FUSION.

By: Holy C, Volenec FJ, Pohl J, Bechtold R und Bruder SP.

Recombinant human growth/differentiation factor-5 is a novel bioactive currently in clinical trials. This growth factor was discovered in 1992 and underwent significant preclinical evaluation, as described herein. It was implanted, lyophilized on a mineralized collagen strip- in spinal and long-bone defects, in animal ranging from rats to nonhuman primates. Bone formation resulting from the use of this growth factor was found in all cases to be at least equivalent to that observed with autograft. In addition, saline solutions of this bioactive successfully restored disc height in rabbit models. This new molecule may therefore open new therapeutic avenues for spine regeneration.

EFFECTS OF GROWTH DIFFERENTIATION FACTOR-5 ON THE INTERVERTEBRAL DISC--IN VITRO BOVINE STUDY AND IN VIVO RABBIT DISC DEGENERATION MODEL STUDY.

By: Chujo T, An HS, Akeda K, Miyamoto K, Muehleman C, Attawia M, Andersson G, Masuda K.

STUDY DESIGN:
In vitro studies on the effects of recombinant human growth and differentiation factor-5 (rhGDF-5) on matrix metabolism of bovine intervertebral disc cells and an in vivo study on the effect of rhGDF-5 in the rabbit anular puncture model.
OBJECTIVE:
To determine the reparative capacity of rhGDF-5 on the intervertebral disc.
SUMMARY OF BACKGROUND DATA:
The in vitro and in vivo effects of rhGDF-5, a crucial protein in the developing musculoskeletal system, on repair of the degenerated intervertebral disc remain unidentified.
METHODS:
In vitro, bovine nucleus pulposus and anulus fibrosus cells were cultured with or without rhGDF-5 (100 or 200 ng/mL). On days 7, 14, and 21, the contents of deoxyribonucleic acid and proteoglycan, and the synthesis of proteoglycan and collagen were assessed. In vivo, 16 adolescent New Zealand white rabbits received anular punctures in 2 lumbar discs. Four weeks later, phosphate buffered saline or rhGDF-5 (10 ng, 1 and 100 mug) was injected into the nucleus pulposus. The rabbits were followed up for 16 weeks for disc height, magnetic resonance imaging, and histologic grading.
RESULTS:
In vitro, rhGDF-5 increased the deoxyribonucleic acid and proteoglycan contents of both cell types significantly after day 14. rhGDF-5 at 200 ng/mL significantly stimulated proteoglycan synthesis (nucleus pulposus: +138%, anulus fibrosus: +24%) and collagen synthesis (nucleus pulposus: +95%, anulus fibrosus: +23%) at day 21. In vivo, the injection of rhGDF-5 resulted in a restoration of disc height, improvement of magnetic resonance imaging scores, and histologic grading scores with statistical significance (P < 0.05-0.001).
CONCLUSION:
A single injection of rhGDF-5 has a reparative capacity on intervertebral discs, presumably based on its effects to enhance extracellular matrix production in vitro.

SUPERIOR EFFECT OF MD05, BETA-TRICALCIUM PHOSPHATE COATED WITH RECOMBINANT HUMAN GROWTH/DIFFERENTIATION FACTOR-5, COMPARED TO CONVENTIONAL BONE SUBSTITUTES IN THE RAT CALVARIAL DEFECT MODEL.

By: Poehling S, Pippig SD, Hellerbrand K, Siedler M, Schütz A, Dony C.

BACKGROUND:
MD05 consists of beta-tricalcium phosphate (beta-TCP) coated with recombinant human growth/differentiation factor-5 (rhGDF-5) and is under evaluation as an osteoinductive and osteoconductive bone graft material for use in dental and maxillofacial applications. The objective of this study was to compare the bone regenerative properties of MD05 with those of conventional commercially available bone substitutes.
METHODS:
Full-thickness, 6-mm diameter, calvarial critical-size defects (two per animal) were created in adult Sprague-Dawley rats. Groups of rats were implanted with the following: 1) MD05; 2) bovine bone mineral; 3) bovine bone mineral with collagen; 4) bovine bone mineral with synthetic peptide, 5) beta-TCP (from two different manufacturers); or 6) no filling material (sham controls). Blinded macroscopic analysis, histopathologic analysis, and histomorphometric analysis were carried out 6 weeks after implantation.
RESULTS:
New bone formation assessed histomorphometrically was about five times greater with MD05 than with the other bone substitutes tested, and bone repair was well advanced in MD05-filled defects after 6 weeks. The extent of fibrous tissue and residual implant were significantly lower in the MD05 group. In contrast to the other materials, the use of MD05 was associated with the complete osseous bridging of the defect and with the presence of normal bone marrow. The osteoinductive effect of rhGDF-5 was apparent from the more pronounced bone ingrowth observed with MD05 compared to the beta-TCP carrier alone. All implants showed good biocompatibility.
CONCLUSION:
MD05 achieved superior bone regeneration compared to conventional materials and is a promising new bone substitute for dental and maxillofacial applications.

HEALOS/RECOMBINANT HUMAN GROWTH AND DIFFERENTIATION FACTOR-5 INDUCES POSTEROLATERAL LUMBAR FUSION IN A NEW ZEALAND WHITE RABBIT MODEL.

By: Magit DP, Maak T, Trioano N, Raphael B, Hamouria Q, Polzhofer G, Drespe I, Albert TJ, Grauer JN.

STUDY DESIGN:
Posterolateral lumbar spine fusions in New Zealand white rabbits.
OBJECTIVE:
To evaluate the efficacy of recombinant human growth and differentiation factor-5 (rhGDF-5) lyophilized to a Healos carrier (cross-linked type I collagen with hydroxyapatite coating; DePuy Spine, Inc., Raynham, MA) in inducing fusion.
SUMMARY OF BACKGROUND DATA:
Bone graft substitutes have become an area of considerable interest. rhGDF-5 is one such product. Limited lumbar preclinical studies have been performed with this product.
METHODS:
Single-level, intertransverse process fusions were performed in 67 rabbits using iliac crest autograft (n = 13), Healos alone (n = 13), or 0.5, 1.0, or 1.5 mg/cc rhGDF-5 lyophilized to Healos (n = 13 per group). At 8 weeks, the rabbits were euthanized. Fusion masses were assessed.
RESULTS:
There were 2 animals (3%) lost to complication. Manual palpation revealed fusion rates for autograft of 38% (5/13), Healos alone of 0% (0/13), and each of the Healos/rhGDF-5 groups of 100% (13/13). Histologic analyses were 95% sensitive and 95% specific for confirming fusion. Histologic differences were found among the treatment groups.
CONCLUSIONS:
In this rabbit fusion model, Healos/rhGDF-5 induced fusion in 100% of the rabbits studied. This rate was significantly higher than the fusion rate induced by autograft (38%). Overall, these results support continued research of Healos/rhGDF-5 as a potential bone graft alternative.

UPREGULATION OF ID PROTEIN BY GROWTH AND DIFFERENTIATION FACTOR 5 (GDF5) THROUGH A SMAD-DEPENDENT AND MAPK-INDEPENDENT PATHWAY IN HUVSMC.

By: Chen X, Zankl A, Niroomand F, Liu Z, Katus HA, Jahn L, Tiefenbacher C.

GDF5 (growth and differentiation factor five), a member of the TGF-beta superfamily, binds specifically to BMPR1b, BMPR2 and ACTR2a receptors forming a heterodimeric complex, thereby inducing phosphorylation of smad1, 5, 8 and translocation to the nucleus. ID1 (inhibitor of differentiation or DNA binding) is essential for G1 to S phase transition inhibiting DNA binding thereby playing an important role in the control of differentiation, proliferation and angiogenesis. The objective of this study was, therefore, to characterize the signal transduction pathway of GDF5, especially the involvement of ID1, in human umbilical vein smooth muscle cells (HUVSMC). We observed the expression of BMPR1a, BMPR1b, BMPR2, ACTR2a, smad1, smad 5, ID1, ID2 and ID3 in HUVSMC. Application of GDF5 upregulated ID1 and ID3 expression by involvement of the smad signaling pathway. GDF5 caused phorsphorylation of smad1 followed by upregulation of ID1 and ID3. Co-incubation with anti-GDF5 prevented these effects. GDF5 significantly inhibited phosphorylation of p38 MAPK and induced phosphorylation of ERK. The specific inhibitor of p38 MAPK or ERK, SB203580 or U0126 did not induce ID protein expression. Smad1 siRNA transfection inhibited the upregulation of ID protein. GDF5 had chemotactic activity in HUVSMC; this effect was partly blocked by transfection of smad1 or ID1 siRNA. Our results indicate that GDF5 induces ID1 and ID3 in HUVSMC by a smad-dependent, MAPK-independent pathway. GDF5 binds to specific receptors, thereby inducing phosphorylation and translocation of smad1 to the nucleus where it is involved in the regulation of transcription. Since ID1 has been shown to be crucial for cell cycle control, we propose that GDF5 could be involved in the process of angiogenesis.

THE EFFECT OF INTRACAVERNOSAL GROWTH DIFFERENTIATION FACTOR-5 THERAPY IN A RAT MODEL OF CAVERNOSAL NERVE INJURY.

By: Fandel TM, Bella AJ, Tantiwongse K, Garcia M, Nunes L, Thüroff JW, Tanagho EA, Pohl J, Lue TF.

OBJECTIVE:
To determine whether the intracavernosal application of growth differentiation factor-5 (GDF-5) influences nerve regeneration and erectile function after cavernosal nerve injury in a rat model.
MATERIALS AND METHODS:
Thirty-two male Sprague-Dawley rats were randomly divided into four equal groups: eight had a sham operation (uninjured controls), while 24 had bilateral cavernosal nerve crush. The crush-injury groups were treated at the time of injury with an impregnated collagen sponge implanted into the right corpus cavernosum. The sponge contained no GDF-5 (injured controls), 2 microg (low concentration), or 20 microg GDF-5 (high concentration). Erectile function was assessed by cavernosal nerve electrostimulation at 8 weeks. Midshaft penile tissue samples were histochemically evaluated for neuronal nitric oxide synthase (nNOS)-containing fibres in the dorsal penile nerve.
RESULTS:
There was no erectile dysfunction in the uninjured control group, as shown by a mean (sem) maximal increase in intracavernosal pressure (ICP) of 149.5 (17.0) cmH(2)O on stimulation. By comparison, the ICP decreased in the injured control group, by 21.3 (6.7) cmH(2)O. After cavernosal nerve injury, the recovery of erectile function was greatest in the low-concentration GDF-5 group; the maximum ICP increase was 40.8 (13.3) cmH(2)O, vs 24.3 (5.9) cmH(2)O for 20 microg GDF-5. Histologically, the low-concentration group had significantly more nNOS-containing nerve fibres, at 163 (24.7), than the high-concentration group, at 76 (17.3), or injured controls, at 67 (23.8). By contrast, the uninjured controls had a mean of 538 (40.6) nerve fibres in the dorsal nerve.
CONCLUSION:
Bilateral cavernosal nerve crush resulted in erectile dysfunction with accompanying neurological changes in the rat. The intracavernosal application of GDF-5 enhanced the recovery of erectile function and n-NOS nerve preservation, with a 2-microg dose giving the most promising results.

MONOMERIC AND DIMERIC GDF-5 SHOW EQUAL TYPE I RECEPTOR BINDING AND OLIGOMERIZATION CAPABILITY AND HAVE THE SAME BIOLOGICAL ACTIVITY.

By: Sieber C, Plöger F, Schwappacher R, Bechtold R, Hanke M, Kawai S, Muraki Y, Katsuura M, Kimura M, Rechtman MM, Henis YI, Pohl J, Knaus P.

Growth and differentiation factor 5 (GDF-5) is a homodimeric protein stabilized by a single disulfide bridge between cysteine 465 in the respective monomers, as well as by three intramolecular cysteine bridges within each subunit. A mature recombinant human GDF-5 variant with cysteine 465 replaced by alanine (rhGDF-5 C465A) was expressed in E. coli, purified to homogeneity, and chemically renatured. Biochemical analysis showed that this procedure eliminated the sole interchain disulfide bond. Surprisingly, the monomeric variant of rhGDF-5 is as potent in vitro as the dimeric form. This could be confirmed by alkaline phosphatase assays and Smad reporter gene activation. Furthermore, dimeric and monomeric rhGDF-5 show comparable binding to their specific type I receptor, BRIb. Studies on living cells showed that both the dimeric and monomeric rhGDF-5 induce homomeric BRIb and heteromeric BRIb/BRII oligomers. Our results suggest that rhGDF-5 C465A has the same biological activity as rhGDF-5 with respect to binding to, oligomerization of and signaling through the BMP receptor type Ib.

THE INFLUENCE OF SURFACE COATINGS OF DICALCIUM PHOSPHATE (DCPD) AND GROWTH AND DIFFERENTIATION FACTOR-5 (GDF-5) ON THE STABILITY OF TITANIUM IMPLANTS IN VIVO.

By: Simank HG, Stuber M, Frahm R, Helbig L, van Lenthe H, Müller R.

Mechanical stability of implants is usually tested by pull out or push out tests which destroy the interface between the implant and bone. Pull out tests do not ideally reflect the clinical situation. In contrast, applying submaximal load leads to more physiologic micro-displacement between implant and bone. The aim of this study was to evaluate a new non-destructive mechanical testing device on different modifications of titanium implants. In 18 rabbits we investigated the influence of a dicalcium phosphate (DCPD) coating, or of a growth and differentiation factor-5 (GDF-5) coating, or a combination of both on the stability of titanium implants. The stability of implant was assessed by a non-destructive micro-measurement. In the same specimens the interface was investigated by micro-CT and histological evaluation. Surface modifications had a positive effect on the implant stability regarding displacement (p=0.001). Mechanical stability correlated with the quality of peri-implant tissue. Micro-displacement correlated negatively with the bone formation around the implants in histomorphometric evaluation (p=0.02). Amount of peri-prosthetic soft tissue showed a positive correlation with micro-displacement (p=0.01). Our findings indicate the positive effect of DCPD and GDF-5 coatings on stability of titanium implants. Results demonstrate the non-destructive testing to be an effective method to evaluate mechanical stability of implants.

COMBINATION OF ENGINEERED NEURAL CELL ADHESION MOLECULES AND GDF-5 FOR IMPROVED NEURITE EXTENSION IN NERVE GUIDE CONCEPTS.

By: Niere M, Braun B, Gass R, Sturany S, Volkmer H.

Current therapeutical approaches for the treatment of severe lesions in the peripheral nervous system rely on the use of autologous tissue or the body's own Schwann cells. However, these approaches are limited and alternative strategies for peripheral nerve regeneration are required. Here we evaluate combinations of a variety of neuronal regeneration factors including engineered cell adhesion molecules and growth factors in embryonic model neurons to test the possible improvement of artificial nerve guides by cooperative mechanisms. Cell adhesion molecules L1 and neurofascin synergistically promote neurite elongation. The outgrowth promoting properties of both proteins can be combined and further increased within one chimeric protein. Addition of growth and differentiation factor 5 (GDF-5) further enhances neurite outgrowth in a substrate-independent manner. This effect is not due to a protective mode of action of GDF-5 against pro-apoptotic stimuli. Consequently, the study supports the idea that different modes of action of pro-regenerative factors may contribute synergistically to neurite outgrowth and emphasizes the applicability of combinations of proteins specifically involved in development of the nervous system for therapeutical approaches.

RECOMBINANT HUMAN GROWTH/DIFFERENTIATION FACTOR-5 (RHGDF-5) INDUCED BONE FORMATION IN MURINE CALVARIAE.

By: Yoshimoto T, Yamamoto M, Kadomatsu H, Sakoda K, Yonamine Y, Izumi Y.

OBJECTIVES:
Growth/differentiation factor-5 (GDF-5), a member of the transforming growth factor-beta superfamily, shows a close structural relationship to bone morphogenetic proteins and plays crucial roles in skeletal morphogenesis. Recombinant human (rh) GDF-5 was reported as a suitable factor for enhancing healing in bone defect and inducing ectopic bone formation. The purpose of the present study was to investigate the mechanism of bone formation induced by rhGDF-5 in murine calvariae by radiological, histological and immunohistochemical methods. Cell proliferation was also examined in vitro.
MATERIAL AND METHODS:
Cells including primary osteoblasts, periosteum cells and connective tissue fibroblasts were isolated enzymatically from neonatal murine calvariae or head skin. In the presence or absence of rhGDF-5, cell proliferation was estimated by tetrazolium reduction assay. To examine the mechanism of osteoinduction, rhGDF-5/atelocollagen (AC) composite or 0.01 N HCl/AC composite were injected into murine calvariae subcutaneously. Tissue was examined radiologically, histologically and immunohistochemically.
RESULTS:
In the presence of rhGDF-5, proliferation of primary osteoblasts, periosteum cells, and connective tissue fibroblasts was increased significantly in culture. Immunohistochemical observations showed cells at the site injected with rhGDF-5/AC displayed immunoreactivity for proliferating cell nuclear antigen (PCNA). Newly formed bone- and cartilage-like tissue contained chondrocyte osteocyte and osteoclastic cells, and were immunoreactive for both type I and II collagen.
CONCLUSION:
Exposure to GDF-5 promotes proliferation and differentiation of calvarial cells, which give rise to ectopic bone formation.

ADENOVIRUS-MEDIATED GENE TRANSFER OF GROWTH AND DIFFERENTIATION FACTOR-5 INTO TENOCYTES AND THE HEALING RAT ACHILLES TENDON.

By: Rickert M, Wang H, Wieloch P, Lorenz H, Steck E, Sabo D, Richter W.

Growth and differentiation factor-5 (GDF-5) is known to induce tendon tissue and stimulate tendon healing. The hypothesis was that adenoviral GDF-5 transfer leads to transitory transgene expression and improves Achilles tendon healing. In vitro experiments were first performed with rat tenocytes. Transgene expression was evaluated by RT-PCR, Western blotting and GDF-5-ELISA. In vivo virus dosage and transgene expression were examined by a marker gene transfer (LacZ and luciferase). In the main experiment in 131 rats, adenovirus particles (3 x 10(10)) were injected into transected Achilles tendons. The time course of GDF-5 mRNA expression was assessed by real-time RT-PCR. Histology and biomechanical testing were used to evaluate tendon healing and tensile strength. In vitro GDF-5 was secreted with a maximum after 2 weeks (330 ng GDF-5/10(6) cells per 24 hr). In vivo GDF-5 transgene expression showed a maximum at 4 weeks. At 8 weeks, GDF-5 specimens were thicker (p<0.05) with a trend to higher strength (p=0,064). Histology showed greater cartilage formation in type II collagen stains than in controls. Injection of adenovirus particles successfully can deliver the GDF-5 gene in healing tendons and leads to thicker tendon regenerates after 8 weeks. This technique might become a new approach for nonsurgical treatment of tendon injuries.

ACTIVATING AND DEACTIVATING MUTATIONS IN THE RECEPTOR INTERACTION SITE OF GDF5 CAUSE SYMPHALANGISM OR BRACHYDACTYLY TYPE A2.

By: Seemann P, Schwappacher R, Kjaer KW, Krakow D, Lehmann K, Dawson K, Stricker S, Pohl J, Plöger F, Staub E, Nickel J, Sebald W, Knaus P, Mundlos S.

Here we describe 2 mutations in growth and differentiation factor 5 (GDF5) that alter receptor-binding affinities. They cause brachydactyly type A2 (L441P) and symphalangism (R438L), conditions previously associated with mutations in the GDF5 receptor bone morphogenetic protein receptor type 1b (BMPR1B) and the BMP antagonist NOGGIN, respectively. We expressed the mutant proteins in limb bud micromass culture and treated ATDC5 and C2C12 cells with recombinant GDF5. Our results indicated that the L441P mutant is almost inactive. The R438L mutant, in contrast, showed increased biological activity when compared with WT GDF5. Biosensor interaction analyses revealed loss of binding to BMPR1A and BMPR1B ectodomains for the L441P mutant, whereas the R438L mutant showed normal binding to BMPR1B but increased binding to BMPR1A, the receptor normally activated by BMP2. The binding to NOGGIN was normal for both mutants. Thus, the brachydactyly type A2 phenotype (L441P) is caused by inhibition of the ligand-receptor interaction, whereas the symphalangism phenotype (R438L) is caused by a loss of receptor-binding specificity, resulting in a gain of function by the acquisition of BMP2-like properties. The presented experiments have identified some of the main determinants of GDF5 receptor-binding specificity in vivo and open new prospects for generating antagonists and superagonists of GDF5.

DIFFERENTIAL EFFECTS OF GROWTH/DIFFERENTIATION FACTOR 5 AND GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR ON DOPAMINERGIC NEURONS AND ASTROGLIA IN CULTURES OF EMBRYONIC RAT MIDBRAIN.

By: Wood TK, McDermott KW, Sullivan AM.

Parkinson's disease is characterized by the progressive degeneration of midbrain dopaminergic neurons. Several studies have examined the effects of the dopaminergic neurotrophins growth/differentiation factor 5 (GDF5) and glial cell line-derived neurotrophic factor (GDNF) on these neurons in vitro. However, there is little information regarding their effects on astroglial cells. Here, the effects of GDF5 and GDNF on dopaminergic neuronal and astroglial survival and differentiation in embryonic rat midbrain cultures were examined. Both GDF5 and GDNF enhanced the survival and differentiation of dopaminergic neurons. GDF5 significantly increased the survival of astroglial cells, whereas GDNF had no significant effect on these cells. The possible involvement of astroglia in the dopaminergic neurotrophic effect induced by GDF5 was investigated by examining the effect of GDF5 on the survival of dopaminergic neurons in glia-depleted midbrain cultures. There was no significant difference between the survival of dopaminergic neurons in glia-depleted cultures treated with GDF5 and that in mixed cell cultures treated with GDF5, suggesting that GDF5 acts directly on dopaminergic neurons in exerting its neurotrophic effect. GDF5 and GDNF have been established as potent neurotrophic factors for dopaminergic neurons. However, the effects of adding a combination of these neurotrophins to midbrain cultures have not been previously examined. The present study found that combined treatment with GDF5 and GDNF significantly increased the survival of dopaminergic neurons in cultures compared with that in cultures treated with either neurotrophin alone. This was an additive effect, indicating that these neurotrophins act on separate subpopulations of dopaminergic neurons.

THE ROLE OF GROWTH/DIFFERENTIATION FACTOR 5 (GDF5) IN THE INDUCTION AND SURVIVAL OF MIDBRAIN DOPAMINERGIC NEURONES: RELEVANCE TO PARKINSON'S DISEASE TREATMENT.

By: Sullivan AM, O'Keeffe GW.

Growth/differentiation factor-5 (GDF5) is a member of the transforming growth factor-beta superfamily which has potent effects on dopaminergic neurones in vitro and in vivo. GDF5 is under investigation as a potential therapeutic agent for Parkinson's disease (PD), which is caused by the progressive degeneration of dopaminergic neurones projecting from the substantia nigra (SN) to the striatum. In the rat ventral mesencephalon (VM; the developing SN), GDF5 expression peaks at embryonic day 14, the time at which dopaminergic neurones undergo terminal differentiation. Addition of GDF5 protein to cultures of embryonic rat VM increases the survival and improves the morphology of dopaminergic neurones in these cultures. GDF5 treatment also increases the number of cells which adopt a dopaminergic phenotype in cultures of VM progenitor cells. Intracerebral administration of GDF5 has potent neuroprotective and restorative effects on the nigrostriatal pathway in animal models of PD. Furthermore, addition of GDF5 protein to embryonic rat dopaminergic neuronal transplants improves their survival and function in a rat model of PD. Thus, GDF5 has potential applications to PD therapy as a dopaminergic neuroprotective agent and as a factor that may induce a dopaminergic neuronal fate in unrestricted progenitor cells.

CRYSTAL STRUCTURE OF RECOMBINANT HUMAN GROWTH AND DIFFERENTIATION FACTOR 5: EVIDENCE FOR INTERACTION OF THE TYPE I AND TYPE II RECEPTOR-BINDING SITES.

By: Schreuder H, Liesum A, Pohl J, Kruse M, Koyama M.

The crystal structure of human growth differentiation factor 5 (GDF5) was solved at 2.4A resolution. The structure is very similar to the structure of bone morphogenetic factor 7 (BMP7) and consists of two banana-shaped monomers, linked via a disulfide bridge. The crystal packing of GDF5 is the same as the crystal packing of BMP7. This is highly unusual since only 25-30% of the crystal contacts involve identical residues. Analysis of the crystal packing revealed that residues of the type I receptor epitope are binding to residues of the type II receptor-binding epitope. The fact that for both BMP family members the type I and type II receptor-binding sites interact suggests that the complementary sites on the receptors may interact as well, suggesting a way how preformed receptor heterodimers may form, similar to the preformed receptors observed for the erythropoietin receptor and the BMP2 receptors.

EFFECTS OF GROWTH/DIFFERENTIATION FACTOR 5 ON THE SURVIVAL AND MORPHOLOGY OF EMBRYONIC RAT MIDBRAIN DOPAMINERGIC NEURONES IN VITRO.

By: O'Keeffe GW, Dockery P, Sullivan AM.

Growth/differentiation factor 5 (GDF5) is a member of the transforming growth factor-beta superfamily that is expressed in the developing CNS, including the ventral mesencephalon (VM). GDF5 has been shown to increase the survival of dopaminergic neurones in animal models of Parkinson's disease. This study was aimed at characterising the effects of GDF5 on dopaminergic neurones in vitro. Treatment with GDF5 induced a three-fold increase in the number of dopaminergic neurones in embryonic day 14 rat VM cultures after six days in vitro. A significant increase was also observed in the numbers of astrocytes in GDF5-treated cultures. GDF5 treatment also had significant effects on the morphology of dopaminergic neurones in these cultures; total neurite length, number of branch points and somal area were all significantly increased after six days in vitro. Analysis of neurite length and numbers of branch points at each level of the neuritic field revealed that the most pronounced effects of GDF5 were on the secondary and tertiary levels of the neuritic field. The specific type I receptor for GDF5, bone morphogenetic protein receptor (BMPR)-Ib, was found to be strongly expressed in freshly-dissected E14 VM tissue, but its expression was lost with increasing time in culture. Accordingly, treatment with GDF5 for 24 h from the time of plating induced increases in the numbers of dopaminergic neurones, while treatment with GDF5 for 24 h after six days in vitro did not. This study shows that GDF5 can promote both the survival and morphological differentiation of VM dopaminergic neurones in vitro, lending support to its potential as a candidate dopaminergic neurotrophin for use in the treatment of Parkinson's disease.

NEUROPROTECTIVE EFFECTS OF DELAYED ADMINISTRATION OF GROWTH/DIFFERENTIATION FACTOR-5 IN THE PARTIAL LESION MODEL OF PARKINSON'S DISEASE.

By: Hurley FM, Costello DJ, Sullivan AM.

Neurotrophic factors have the potential for therapeutic use in Parkinson's disease (PD) to support the remaining dopaminergic neurons and protect them against the ongoing disease process. We have examined the effects of the neurotrophin growth and differentiation factor-5 (GDF-5) in a rat model of Parkinson's disease, the intrastriatal 6-hydroxydopamine (6-OHDA) lesion. GDF-5 (25 microg) was injected into either the striatum or substantia nigra (SN) of adult rats at 1 or 2 weeks after 6-hydroxydopamine administration. The behavioral effects of GDF-5 treatment were examined in vivo by amphetamine-induced rotational testing. Injection of GDF-5 into the nigra at either 1 or 2 weeks, or into the striatum at 1 week, after the lesion induced significant decreases in rotations. Post-mortem immunocytochemistry after 6 weeks showed that GDF-5 administration into either site protected dopaminergic cell bodies of the nigra when injected at 1 but not 2 weeks after 6-hydroxydopamine. However, no significant protection of striatal dopaminergic fiber density was observed after GDF-5 treatment. This study shows that the delayed administration of a single dose of GDF-5 has significant protective effects on the damaged adult rat nigrostriatal pathway, reinforcing its therapeutic potential for Parkinson's disease.

ENDOSCOPIC INSTRUMENTED POSTEROLATERAL LUMBAR FUSION WITH HEALOS AND RECOMBINANT HUMAN GROWTH/DIFFERENTIATION FACTOR-5.

By: Jahng TA, Fu TS, Cunningham BW, Dmitriev AE, Kim DH.

OBJECTIVE:
The goal of this study was to use a minimally invasive endoscopic surgical technique in a sheep model to evaluate the efficacy of an osteoinductive growth factor, recombinant human growth/differentiation factor-5 (also designated MP52), and an osteoconductive matrix formulation (Healos; DePuy AcroMed, Inc., Mountain View, CA) for inducing and facilitating bone formation.
METHODS:
Twelve mature sheep underwent bilateral posterolateral lumbar fusion and pedicle screw fixation via a posterior endoscopic approach. Each sheep received two different types of graft material, autogenous iliac crest bone, or a bone graft substitute (MP52 with Healos), inserted into the right and left sides of the spine in an alternating fashion. Groups of four sheep were killed at 2, 4, and 6 months postoperatively for manual, radiographic, and histological evaluation.
RESULTS:
No neurological impairments, infections, or other complications were observed. After 2 months, partial fusion on both sides was observed, but radiographic evaluation showed greater bone growth on the side that received the bone graft substitute. Solid posterolateral fusion was observed in both autograft and bone graft substitute sites at 4 and 6 months, and autograft and Healos MP52 fusion sites were essentially the same at histological examination. There was no abnormal overgrowth of new bone from either of these two materials.
CONCLUSION:
Endoscopic posterolateral lumbar arthrodesis and instrumentation is feasible, safe, and effective in a sheep model. Healos is a useful bonding agent that mimics natural bone in posterolateral intertransverse fusion sites. Combined with MP52, it produced fusion comparable to that of autogenous bone graft. Minimally invasive techniques and bone graft substitutes could eliminate morbidity and increase the likelihood of successful fusion.

EXPRESSION OF GROWTH DIFFERENTIATION FACTOR-5 IN THE DEVELOPING AND ADULT RAT BRAIN.

By: O'Keeffe GW, Hanke M, Pohl J, Sullivan AM.

Expression of the dopaminergic neurotrophin GDF-5 in developing rat ventral mesencephalon (VM) was found to begin at embryonic day (E) 12 and peak on E14, when dopaminergic neurones undergo terminal differentiation. In the adult rat, GDF-5 was found to be restricted to heart and brain, being expressed in many areas of the brain, including striatum and midbrain. This indicates a role for GDF-5 in the development and maintenance of dopaminergic neurones.

EFFECTS OF LOCAL APPLICATION OF GROWTH AND DIFFERENTIATION FACTOR-5 (GDF-5) IN A FULL-THICKNESS CARTILAGE DEFECT MODEL.

By: Simank HG, Sergi C, Jung M, Adolf S, Eckhardt C, Ehemann V, Ries R, Lill C, Richter W.

Our purpose in this study was to investigate the effects of growth and differentiation factor-5 (GDF-5) on cartilage and subchondral bone in a rabbit model harboring an osteochondral defect for a period of 6 months. Absorbable composites were implanted in adult rabbits (18 controls, 18 animals with collagen-I matrix, and 18 animals with matrix plus GDF-5). After 4, 8, or 24 weeks the specimens were studied by histology, microcomputed tomography (microCT) and flow-cytometric analysis (FACS). Implantation of GDF-5 resulted in an improved histological appearance. This was the result of significantly improved defect filling at 4 and 8 weeks. At 24 weeks, however, there was no difference between the groups. The histological examination disclosed a predominance of fibrocartilage, and remodeled subchondral bone was also observed. MicroCT documented normal bone density in all groups, excluding subchondral sclerosis. At 24 weeks, FACS analysis revealed high apoptotic activity in the GDF-5-treated group. As far as we are aware, this is the first report of the effects of GDF-5 in a full-thickness cartilage defect model. We assume that recombinant GDF-5 contained on the carrier is probably able to accelerate the regeneration of the osteochondral defect owing to its availability. However, control of the protein delivery may require further investigation.

RELEASE OF ACTIVE AND DEPOT GDF-5 AFTER ADENOVIRUS-MEDIATED OVEREXPRESSION STIMULATES RABBIT AND HUMAN INTERVERTEBRAL DISC CELLS.

By: Wang H, Kroeber M, Hanke M, Ries R, Schmid C, Poller W, Richter W.

To develop new therapeutic options for the treatment of disc degeneration we tested the possibility of overexpression of active growth and differentiation factor (GDF) 5 and of transforming growth factor (TGF) beta(1) by adenoviral gene transfer and characterized its effect on cell proliferation and matrix synthesis of cultured rabbit and human intervertebral disc cells. Recombinant adenovirus encoding for GDF-5 or TGF-beta(1) was developed and transgene expression characterized by RT-PCR, western blot and ELISA. Growth and matrix synthesis of transduced cells was measured by [(3)H]thymidine or [(35)S]sulfate incorporation. Disc cells expressed the receptors BMPR1A, BMPR1B, and BMPR2, which are relevant for GDF-5 action. Adenovirus efficiently transferred the GDF-5 gene or the TGF-beta(1) gene to rabbit and human intervertebral disc cells. About 50 ng GDF-5 protein/10(6 )cells per 24 h or 7 ng TGF-beta(1) protein/10(6 )cells per 24 h was produced. According to western blotting, two GDF-5 forms, with molecular weights consistent with the activated GDF-5 dimer and the proform, were secreted over the 3 weeks following gene transfer. Overexpressed GDF-5 and TGF-beta(1) were bioactive and promoted growth of rabbit disc cells in monolayer culture. Our results suggest that ex vivo gene delivery of GDF-5 and TGF-beta(1) is an attractive approach for the release of mature and pre-GDF-5 in surrounding tissue. This leads us to hope that it will prove possible to improve the treatment of degenerative disc disease by means of ex vivo gene transfer of single or multiple growth factors.

HEPARIN-BINDING EPIDERMAL GROWTH FACTOR-LIKE GROWTH FACTOR: A COMPONENT IN CHROMAFFIN GRANULES WHICH PROMOTES THE SURVIVAL OF NIGROSTRIATAL DOPAMINERGIC NEURONES IN VITRO AND IN VIVO.

By: Hanke M, Farkas LM, Jakob M, Ries R, Pohl J, Sullivan AM.

Chromaffin cells can restore function to the damaged nigrostriatal dopaminergic system in animal models of Parkinson's disease. It has been reported that a protein which is released from chromaffin granules can promote the survival of dopaminergic neurones in vitro and protect them against N-methylpyridinium ion toxicity. This neurotrophic effect has been found to be mediated by astroglial cells and blocked by inhibitors of the epidermal growth factor (EGF) receptor signal transduction pathway. Here we report the identification of bovine heparin-binding EGF-like growth factor (HB-EGF) in chromaffin granules and the cloning of the respective cDNA from bovine-derived adrenal gland. Protein extracts from bovine chromaffin granules were found to promote the survival of embryonic dopaminergic neurones in culture, to the same extent as recombinant human HB-EGF. Furthermore, the neurotrophic action of the chromaffin granule extract could be abolished by antiserum to recombinant human HB-EGF. We also show that intracerebral injection of recombinant human HB-EGF protected the nigrostriatal dopaminergic system in an in vivo adult rat model of Parkinson's disease. Intracerebral administration of this protein at the same time as a 6-hydroxydopamine lesion of the medial forebrain bundle was found to spare dopamine levels in the striatum and tyrosine hydroxylase-immunopositive neurones in the midbrain. This study has found that the main component in chromaffin granules responsible for their neurotrophic effect on dopaminergic neurones is HB-EGF. Furthermore, HB-EGF has significant protective effects on nigrostriatal dopaminergic neurones in vivo, making it a potential candidate for use in the treatment of Parkinson's disease.

EFFECTS OF GROWTH/DIFFERENTIATION FACTOR-5 ON HUMAN PERIODONTAL LIGAMENT CELLS.

By: Nakamura T, Yamamoto M, Tamura M, Izumi Y.

OBJECTIVES:
Growth/differentiation factor-5 (GDF-5), a member of the transforming growth factor-beta superfamily, shows a close structural relationship to bone morphogenetic proteins and plays crucial roles in skeletal, tendon, and ligament morphogenesis. The mRNA encoding GDF-5 is also expressed during odontogenesis, especially in dental follicle tissue. While this suggests that GDF-5 participates in the formation of alveolar bone and the periodontal ligament, cementum, and dental root, the physiologic role of GDF-5 in these tissues in adulthood remains unclear. We therefore investigated GDF-5 effects upon cultures of human periodontal ligament (HPDL) cells.
MATERIAL AND METHODS:
HPDL cells were obtained from healthy periodontal ligaments of individuals. Tetrazolium reduction assay was carried out for cell proliferation assay. Alkaline phosphatase (ALP) activity was estimated by measuring light absorbance at 405 nm. Reverse transcription-polymerase chain reaction (RT-PCR) and northern analysis were performed for gene expression in cultured HPDL cells. Sulfated glycosaminoglycan (sGAG) synthesis was evaluated by histochemical staining and a quantitative dye-binding method.
RESULTS:
Expression of GDF-5 and its receptor was demonstrated in HPDL cells by RT-PCR. ALP activity in HPDL cells was significantly decreased by addition of rhGDF-5 at 10-1000 ng/ml (p < 0.05). Although northern analysis showed little change in gene expression for collagen alpha2(I) in rhGDF-5-stimulated HPDL cells, rhGDF-5 dose-dependently enhanced cell proliferation. This proliferative effect persisted for 16 d. Alcian blue staining and dye-binding assays indicated that sGAG synthesis was enhanced by rhGDF-5.
CONCLUSION:
rhGDF-5 may provide an environment fostering periodontal healing or regeneration by affecting extracellular matrix metabolism.

EXPRESSION OF ACTIVINS C AND E INDUCES APOPTOSIS IN HUMAN AND RAT HEPATOMA CELLS.

By: Vejda S, Erlach N, Peter B, Drucker C, Rossmanith W, Pohl J, Schulte-Hermann R, Grusch M.

Activins C and E (homodimers of the betaC and betaE subunits), which are almost exclusively expressed in the liver, are members of the transforming growth factor beta (TGFbeta) superfamily of growth factors. We examined their expression in three different hepatoma cell lines and found that, compared with normal liver or primary hepatocytes, human hepatoblastoma (HepG2), human hepatocellular carcinoma (Hep3B) and rat hepatoma (H4IIEC3) cells have either completely lost or drastically reduced the expression of activins C and E. In order to elucidate the biological function of these proteins we transiently transfected HepG2, Hep3B and H4IIEC3 cell lines with rat activin betaC or betaE cDNA to study the consequences of restoring activin expression in hepatoma cells. Transfection with activin betaA, a known inhibitor of hepatic DNA synthesis and inducer of apoptosis, served as a positive control. We found that transfection of the three cell lines with activin betaC or betaE, as well as with activin betaA, reduced the increase in cell number by up to 40% compared with cells transfected with a control plasmid. Co-culture with a CHO cell clone secreting activin C also inhibited HepG2 cell multiplication. Furthermore, the three hepatoma cell lines studied showed an enhanced rate of apoptosis and elevated levels of active caspases in response to activin transfection. These results indicate that activins C and E share the potential to induce apoptosis in liver derived cell lines with activin A and TGFbeta1.

BONE AUGMENTATION USING RHGDF-5-COLLAGEN COMPOSITE.

By: Kuniyasu H, Hirose Y, Ochi M, Yajima A, Sakaguchi K, Murata M, Pohl J.

The aim of this study was to evaluate the effectiveness of local application of growth differentiation factor-5 (GDF-5)-collagen composite on bone augmentation on the rat calvaria. GDF-5-collagen composite is made from recombinant human GDF-5 (rhGDF-5) and purified bovine type I atelocollagen. The GDF-5 solution was mixed with 0.3% atelocollagen acid solution, and the mixture was lyophilized. The spongy lyophilized material was pressed into the shape of a minidisk to make the GDF-5-collagen composite. The GDF-5-collagen composite contained 1, 10, or 100-microg rhGDF-5. The control collagen composite contained 0-microg rhGDF-5. The GDF-5-collagen composite or control collagen composite was inserted beneath the calvarial periosteum of 4-week-old rats. At 3 weeks after implantation, the implants containing 1-microg rhGDF-5 had mostly induced new bone formation on the cranial side. In the implants containing 10- microg rhGDF-5, bone formation had proceeded to the center of the GDF-5-collagen composite from the periosteal and the cranial sides, and bone marrow was seen focally. The augmented bone showed a connected trabecular structure with abundant vascularization. The implants containing 100-microg rhGDF-5 were nearly entirely replaced by new bone with bone marrow, and the augmented bone was firmly connected with the original bone. Neither cartilage nor bone formation was found in the control collagen composite. Thus, we conclude that the GDF-5-collagen composite may be a superior biomaterial for bone augmentation and this composite could be useful as a local osteoinductive device.

THE BOOROOLA MUTATION IN SHEEP IS ASSOCIATED WITH AN ALTERATION OF THE BONE MORPHOGENETIC PROTEIN RECEPTOR-IB FUNCTIONALITY.

By: Fabre S, Pierre A, Pisselet C, Mulsant P, Lecerf F, Pohl J, Monget P, Monniaux D.

The hyperprolificacy phenotype of Booroola ewes is due to the presence of the FecB(B) allele at the FecB locus, recently identified as a single amino acid substitution (Q249R) in the bone morphogenetic protein (BMP) type-IB receptor (BMPR1B), and is associated with a more precocious differentiation of ovarian granulosa cells (GCs). To evaluate the consequences of the Booroola mutation on BMPR1B functions, the action of ligands of the transforming growth factor-beta (TGFbeta)/BMP family that act through (growth and differentiation factor-5, BMP-4) or independently of (activin A, TGFbeta-1) BMPR1B were studied on primary cultures of GCs from homozygous FecB(+) and FecB(B) ewes. All the tested TGFbeta/BMP family ligands inhibited progesterone secretion by FecB(+) GCs. Those inhibitory effects were lower for GCs from preovulatory (5-7 mm diameter) than from small antral follicles (1-3 mm diameter). The presence of the Booroola mutation was associated with a 3- to 4-fold (P<0.001) decreased responsiveness of GCs from FecB(B) compared with FecB(+) small follicles to the action of BMPR1B ligands. In contrast, TGFbeta-1 and activin A had similar inhibitory effects on progesterone secretion by GCs from FecB(+) and FecB(B) small follicles. No difference between genotypes was observed with GCs from preovulatory follicles. In transfection experiments with HEK-293 cells, co-expression of FecB(+) BMPR1B and BMPR2 resulted in a 2.6-fold (P<0.01) induction of the activity of a BMP-specific luciferase reporter construct by BMP-4. Interestingly, no response to BMP-4 was observed when cells were transfected with the FecB(B) form of the BMPR1B receptor. Overall, these data strongly suggest that the Q249R mutation is associated with a specific alteration of BMPR1B signaling in hyperprolific Booroola ewes.

SPINAL FUSION WITH RECOMBINANT HUMAN GROWTH AND DIFFERENTIATION FACTOR-5 COMBINED WITH A MINERALIZED COLLAGEN MATRIX.

By: Spiro RC, Thompson AY, Poser JW.

The availability of recombinant osteoinductive growth factors and new osteoconductive matrices offers an alternative to the use of autogenous bone (autograft) for grafting indications. This study evaluates the bone-forming activity of a mineralized collagen matrix combined with recombinant human growth and differentiation factor-5 in a rabbit posterolateral spinal fusion model. The activity of three distinct matrix-growth factor formulations is assessed by radiographic, histologic, and mechanical strength methods. Results show that the radiographic density, histologic quality, and mechanical strength of fusion at 12 weeks post-treatment rank consistently within the treatment groups. Optimal formulations are shown to perform similar to autograft in both the rate and strength of fusion. Fusion rates as high as 80% are observed within specific matrix/growth factor formulations. The average biomechanical strength of treated motion segments in the most efficacious formulation is 82% higher than that obtained with autograft, although this difference is not statistically significant. The fusion mass formed in response to matrix/growth factor formulations is composed of normal trabecular bone with a thin outer cortical plate and modest hematopoietic bone marrow. These results demonstrate that the combination of a mineralized collagen matrix with recombinant human growth and differentiation factor-5 maximizes the inherent conductive and inductive properties of each component, respectively, to provide an effective alternative to autograft for bone grafting procedures.

A GROWTH AND DIFFERENTIATION FACTOR-5 (GDF-5)-COATED SUTURE STIMULATES TENDON HEALING IN AN ACHILLES TENDON MODEL IN RATS.

By: Rickert M, Jung M, Adiyaman M, Richter W, Simank HG.

Growth and differentiation factor-5 (GDF-5) is essential for normal skeletal development and induces tendon-and ligament-like structures at ectopic sites. Therefore, we investigated the influence of a GDF-5-coated suture on the healing Achilles tendon in rats. The right Achilles tendon in 80 rats was transected and sutured with an absorbable polyglactin suture. Animals were randomized to an uncoated-suture control and a GDF-5-coated suture group. At 1, 2, 4 and 8 weeks after surgery the repair tissue was evaluated biomechanically and histologically. Biomechanical testing revealed significantly thicker tendons, which were stiffer at 1, 2, and 4 weeks, in the experimental group than in the control group. The maximum tensile strength was significantly increased at 2 weeks after surgery. Histologically we found cartilage-like cell nests 4 weeks after tendon repair, which were positive for type II collagen. In conclusion, local growth factor delivery by a coated suture material showed a promising beneficial effect on tendon repair. The appearance of cartilage-like structures may demonstrate the chondroinductive capacity of GDF-5, which in these circumstances, however, might be overcome by modifications of the GDF-5 dose and/or the suture material.

GROWTH FACTOR THERAPY FOR EXP. NECROSIS OF THE FEMORAL HEAD.

By: Simank H-G, Manggold J, Sebald W, Ries Rainer, Richter W, Ewerbeck V, Consolato S.

Intro:
Osteotropic growth factors enhance bone repair, but their efficacy in an area of necrotic bone is not known. The purpose of this study was to investigate the effects and potential side effects of an intraosseous application of absorbable Bone Morphogenetic Protein-2 (BMP-2) and Growth and Differentiation Factor-5 (GDF-5) composites in a sheep model for partial necrosis of the femoral head.
Material and Methods:
The direct injection of ethanol under fluoroscopy into the superior centre of the right femoral head produced histologically documented necrosis of the central region of the head in ten sheep. Another 27 sheep constituted the sample to study the effects of BMP-2 and GDF-5. Necrosis was produced in the same fashion. Four weeks later nine sheep received 300 µg recombinant BMP-2 and nine sheep (control group) the carrier alone. The animals were sacrificed at three, six and twelve weeks after implantation.
Results:
Bone density analysis and microscopic examination indicated that bone formation was induced as early as 3 weeks postoperatively in the growth factor composites, documented by histological scoring and histomorphometric analysis. No severe local side effects were observed.
Discussion:
The application of an absorbable growth factor composite in combination with established surgical techniques is a promising approach that may enhance the healing of devitalised bone defects.

SPINAL FUSION WITH RHGDF-5/MP52 IN COMBINATION WITH COLLAGEN MATRIX.

By: Spiro RC, Liu LS, Heidaran MA, Thompson AY, Ng CK, Pohl J, Poser JW.

Growth and differentiation factor-5 (GDF-5) is a divergent member of the transforming growth factor-ß/bone morphogenetic protein (BMP) superfamily that is required for proper skeletal patterning and development in the vertebrate limb. Based on the homology of GDF-5 with other bone-inducing BMP family members, the inductive activity of a recombinant form of human GDF-5 (rhGDF-5/MP52) was evaluated in a series of in vitro assays and in vivo bone-formation models. The in vitro response to rhGDF-5/MP52 resulted in the formation of chondrogenic nodules in fetal rat calvarial cells cultured in the context of collagen or collagen/hyaluronate extracellular matrices.Matrices loaded with rhGDF-5 induced ectopic cartilaginous and osseous tissue when implanted in subcutaneous or intramuscular sites. In non-human primate long-bone-defect and spinal-fusion models, rhGDF-5/MP52 combined with a mineralized collagen matrix induced bone formation in a manner equivalent to autogenous bone. These results highlight the unique potential of rhGDF-5/MP52 in a wide variety of orthopaedic applications.

LARGE-SCALE REFOLDING OF THERAPEUTIC PROTEINS

By: Honda J., Andou H., Mannen T., Sugimoto S.

DIRECT REFOLDING OF RECOMBINANT HUMAN GROWTH DIFFERENTIATION FACTOR 5 FOR LARGE-SCALE PRODUCTION PROCESS.

By: Honda J, Andou H, Mannen T, Sugimoto S.

An efficient downstream process for the production of recombinant human growth differentiation factor 5 (rhGDF5) has been developed for industrial application utilizing a novel "direct refolding" method. In this method, the starting material is an inclusion body produced in Escherichia coli, and the critical step is the direct refolding step that follows directly after solubilization of the inclusion body. rhGDF5 can be refolded at a markedly high concentration of 2.4 mg.ml(-1), which is 24 times that hitherto achieved by the proteins of the TGF-beta superfamily. The refolding yield is 63%, and after purification by diafiltration, isoelectric precipitation and reverse-phase chromatography, the final purification yield is 20% with purity higher than 99%. The yield is more than twice that of a conventionally established process having three chromatography steps and the purity is equivalent. The first pilot-scale trial shows a refolding yield of 51% and a final yield of 11%. The final yield is 1.4 times that of the conventional process, and further optimization at pilot-scale is expected to bring this figure up to or above that of laboratory-scale. As a result, the calculated production cost of rhGDF5 has been reduced dramatically. This type of efficient and simple process is beneficial particularly in the large-scale production of recombinant proteins in which high yield and quality are required.

LOCALIZATION OF ACTIVIN BETA(A)-, BETA(B)-, AND BETA(C)-SUBUNITS IN HUMANPROSTATE AND EVIDENCE FOR FORMATION OF NEW ACTIVIN HETERODIMERS OF BETA(C)-SUBUNIT.

By: Mellor SL, Cranfield M, Ries R, Pedersen J, Cancilla B, de Kretser D, Groome NP, Mason AJ, Risbridger GP.

Activin ligands are formed by dimerization of activin ss(A)- and/or ss(B)-subunits to produce activins A, AB, or B. These ligands are members of the transforming growth factor-ss superfamily and act as growth and differentiation factors in many cells and tissues. New additions to this family include activin ss(C)-, ss(D)-, and ss(E)-subunits. The aim of this investigation was to examine the localization of and dimerization among activin subunits; the results demonstrate that activin ss(C) can form dimers with activin ss(A) and ss(B) in vitro, but not with the inhibin alpha-subunit. Using a specific antibody, activin ss(C) protein was localized to human liver and prostate and colocalized with ss(A)- and ss(B)-subunits to specific cell types in benign and malignant prostate tissues. Activin C did not alter DNA synthesis of the prostate tumor cell line, LNCaP, or the liver tumor cell line, HepG2, in vitro when added alone or with activin A. Therefore, the capacity to form novel activin heterodimers (but not inhibin C) resides in the human liver and prostate. Activin A, AB, and B have diverse actions in many tissues, including liver and prostate, but there is no known biological activity for activin C. Thus, the evidence of formation of activin AC or BC heterodimers may have significant implications in the regulation of levels and/or biological activity of other activins in these tissues.

EXTRACELLULAR MATRIX INFLUENCES CELL DIFFERENTIATION THROUGH RHGDF-5.

By: Heidaran MA, Daverman R, Thompson A, Ng CK, Pohl J, Poser JW, Spiro RC.

Activin ligands are formed by dimerization of activin ss(A)- and/or ss(B)-subunits to produce activins A, AB, or B. These ligands are members of the transforming growth factor-ss superfamily and act as growth and differentiation factors in many cells and tissues. New additions to this family include activin ss(C)-, ss(D)-, and ss(E)-subunits. The aim of this investigation was to examine the localization of and dimerization among activin subunits; the results demonstrate that activin ss(C) can form dimers with activin ss(A) and ss(B) in vitro, but not with the inhibin alpha-subunit. Using a specific antibody, activin ss(C) protein was localized to human liver and prostate and colocalized with ss(A)- and ss(B)-subunits to specific cell types in benign and malignant prostate tissues. Activin C did not alter DNA synthesis of the prostate tumor cell line, LNCaP, or the liver tumor cell line, HepG2, in vitro when added alone or with activin A. Therefore, the capacity to form novel activin heterodimers (but not inhibin C) resides in the human liver and prostate. Activin A, AB, and B have diverse actions in many tissues, including liver and prostate, but there is no known biological activity for activin C. Thus, the evidence of formation of activin AC or BC heterodimers may have significant implications in the regulation of levels and/or biological activity of other activins in these tissues.

CHARACTERIZATION OF BONE MORPHOGENETIC PROTEIN FAMILY MEMBERS AS NEUROTROPHIC FACTORS FOR CULTURED SENSORY NEURONS.

By: Farkas LM, Jászai J, Unsicker K, Krieglstein K.

The bone morphogenetic proteins have been implicated in several inductive processes throughout vertebrate development including nervous system patterning. Recently, these proteins have also emerged as candidates for regulating survival of mesencephalic dopaminergic and sympathetic neurons. Interestingly, we have found that several bone morphogenetic proteins can be detected in developing embryonic day 14 rat dorsal root ganglia by means of reverse transcription-polymerase chain reaction and immunocytochemistry. To further elucidate their potential role during the period of ontogenetic neuron death, serum-free cultures of dorsal root sensory neurons from developing chick and rat embryos were treated with distinct bone morphogenetic proteins with or without simultaneous addition of other "established" neurotrophic factors. Our results show that bone morphogenetic proteins exert survival promoting effects on their own, and that they can positively modulate the effects of neurotrophins on sensory neurons. In particular, growth/differentiation factor-5, bone morphogenetic protein-2, -4, -7 and -12 significantly increased the survival promoting effects of neurotrophin-3 and nerve growth factor on cultured dorsal root ganglion neurons. These results fit well into the current concept that neurotrophic factors may act synergistically in ensuring neuronal survival. Moreover, these data suggest potential instructive interactions of bone morphogentic proteins and neurotrophins during sensory neuron development. Finally, the documented neurotrophic capacity of bone morphogenetic protein family members may have potential relevance for the treatment of peripheral neuropathies.

P38 MITOGEN-ACTIVATED PROTEIN KINASE FUNCTIONALLY CONTRIBUTES TO CHONDROGENESIS INDUCED BY GROWTH/DIFFERENTIATION FACTOR-5 IN ATDC5 CELLS.

By: Nakamura K, Shirai T, Morishita S, Uchida S, Saeki-Miura K, Makishima F.

Recent studies of intracellular signal transduction mechanisms for the transforming growth factor-beta (TGF-beta) superfamily have focused on Smad proteins, but have paid little attention to mitogen-activated protein (MAP) kinase cascades. Here we demonstrate that growth/differentiation factor-5 (GDF-5), but neither bone morphogenetic protein-2 (BMP-2) nor TGF-beta1, fully promotes the early phase of the chondrogenic response by inducing cellular condensation followed by cartilage nodule formation in a mouse chondrogenic cell line, ATDC5. We investigated which, if any, of the three major types of MAP kinase plays a functional role in the promotion of chondrogenesis induced by GDF-5. GDF-5 induced phosphorylation of p38 MAP kinase and extracellular signal-regulated kinase (ERK) but not that of c-Jun N-terminal kinase (JNK). The phosphorylation of p38 MAP kinase was also induced by BMP-2 and TGF-beta1. An inhibitor of p38 and p38 beta MAP kinase, SB202190, showed complete inhibition of cartilage nodule formation but failed to affect alkaline phosphatase (ALP) activity induced by GDF-5. Expression of the type II collagen gene, a hallmark of chondrogenesis in vertebrates, was also induced by GDF-5 treatment and strongly suppressed by SB202190. On the other hand, although an inhibitor of MAP/ERK kinase, PD98059, inhibited the rapid phosphorylation of ERK by GDF-5, it inhibited neither ALP activity nor cartilage nodule formation induced by GDF-5. These results strongly suggest that the p38 MAP kinase cascade is involved in GDF-5 signaling pathways and that a role of the p38 MAP kinase pathway is necessary over a longer period to promote chondrogenesis in ATDC5 cells.

MIDBRAIN DOPAMINERGIC NEURONS ARE PROTECTED FROM RADICAL INDUCED DAMAGE BY GDF-5 APPLICATION. SHORT COMMUNICATION.

By: Lingor P, Unsicker K, Krieglstein K.

Embryonic day 14 rat midbrain cultures were kept for 7 days in vitro and then intoxicated with radical donors iron and sodium-nitroprusside for 24 h. Tyrosine-hydroxylase positive neurons in cultures which were additionally treated with growth/differentiation factor-5 (GDF-5) survived to a significantly higher percentage as compared to sister cultures without factor supplementation. Since the degeneration of TH positive cells is a key feature in Parkinson's disease, GDF-5 might be a putative therapeutical agent for this disorder.

MINIMAL PROMOTER COMPONENTS OF THE HUMAN GROWTH/DIFFERENTIATION FACTOR-5 GENE.

By: Sugiura T, Hötten G, Kawai S.

Growth/differentiation factor-5 (GDF-5) is a new member of the BMP family supposed to be involved in chondrogenesis. We cloned the human GDF-5 gene from lambda phage library and sequenced its 3.5-kb 5'-flanking region. The transcription start site was mapped by 5'-RACE to the sequence that coincides with the initiator element. Electrophoresis mobility shift assays (EMSA) demonstrated a zinc finger transcription factor, YY1, to bind to the sequence surrounding the transcription start site. To localize positive and negative regulatory elements in the GDF-5 5'-upstream region, we constructed a series of progressively deleted promoter-reporter plasmids. The transient transfection assay with human osteoblastic Hos cells indicated that a minimal enhancer element resides within -117 to -27 relative to the transcription initiation site. Since the GDF-5 promoter was active even in fibroblastic L cells, a mechanism governing its chondrocyte-restrictive expression needs to be explored.

BMPS AND GDFS IN THE HUMAN CORNEA.

By: You L, Kruse FE, Pohl J, Völcker HE.

Purpose:
To investigate transcription of members of the transforming growth factor (TGF)-ß superfamily and corresponding receptors in human corneal epithelium and stroma.
Methods:
Transcription of bone morphogenetic proteins (BMP)-2, BMP-3, BMP-4, BMP-5, and BMP-7; growth-differentiation factor (GDF-5), and BMP receptors (BMPR) types I (BMPR-IA, BMPR-IB) and II (BMPR-II) was investigated by reverse transcription-polymerase chain reaction (RT-PCR) in ex vivo and cultured cells. For verification, PCR fragments were cloned and sequenced. DNA dot blot analysis was performed to estimate the level of transcription. RNA dot blots were performed to determine expression of GDF-5. Expression of BMP receptor proteins was investigated by immunohistochemistry. Single cell clonal growth proliferation assays were performed using recombinant human GDF-5 and TGF-ß1.
Results:
The level of transcription was higher in cultured stroma for all factors, but the level for the receptors was higher in cultured epithelium. In contrast GDF-5 was transcribed only in stromal cells, suggesting that this cytokine may be an important mediator between keratocytes and epithelial cells. Furthermore, GDF-5 inhibited proliferation of corneal epithelial and stromal cells.
Conclusions:
Given the importance of the TGF-ß family during embryonic development, the results suggest that its members may be components of the corneal cytokine network and may participate in the regulation of cellular proliferation and differentiation.

NEUROPROTECTIVE EFFECTS OF GDF-5 IN VARIOUS REGIONS OF THE BRAIN.

By: Sullivan AM, Opacka-Juffry J, Pohl J, Blunt SB.

Growth/differentiation factor 5 (GDF-5) is a neurotrophin which protects the rat nigrostriatal dopaminergic pathway from 6-hydroxydopamine-induced damage. Here we used amphetamine-induced rotational testing, high-performance liquid chromatography and immunocytochemistry to investigate the minimum effective dose of GDF-5. We also compared the effectiveness of injecting GDF-5 into either the substantia nigra pars compacta (SNpc), the lateral ventricle (LV) or the striatum (or combinations of these sites).

EXPRESSION OF HUMAN ACTIVIN C PROTEIN IN INSECT LARVAE INFECTED WITH A RECOMBINANT BACULOVIRUS.

By: Kron R, Schneider C, Hötten G, Bechtold R, Pohl J.

In order to generate dimeric recombinant transforming growth factor-beta (TGF-beta) proteins, expensive eucaryotic cell systems, such as CHO cells, are usually used. An alternative represents the expression of such proteins in insects using a baculovirus expression system. In this study, recombinant human activin C protein was expressed in Noctuidae larvae. On SDS-PAGE, the expressed protein has a size of about 15 kD under reducing conditions and of about 20 kD under non-reducing conditions. This suggests that activin C is expressed as a dimer and disulfide bridges can be formed. Compared with expression in eucaryotic cell culture systems, expression in insect larvae presents a rapid and low cost method, without the need for expensive tissue culture scale-ups or special equipment.

GROWTH/DIFFERENTIATION FACTOR 5 AND GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR ENHANCE SURVIVAL AND FUNCTION OF DOPAMINERGIC GRAFTS IN A RAT MODEL OF PARKINSON'S DISEASE.

By: Sullivan AM, Pohl J, Blunt SB.

Growth/differentiation factor 5 is a member of the transforming growth factor beta superfamily, which has neurotrophic and neuroprotective effects on dopaminergic neurons both in vitro and in vivo. Here we investigate the effects of growth/differentiation factor 5 on foetal mesencephalic grafts transplanted into a rat model of Parkinson's disease, and compare them with those of glial cell line-derived neurotrophic factor. Mesencephalic tissue was suspended in solutions containing either growth/differentiation factor 5 or glial cell line-derived neurotrophic factor prior to transplantation into the left striatum of rats with 6-hydroxydopamine lesions of the left medial forebrain bundle. Both proteins enhanced graft-induced compensation of amphetamine-stimulated rotations. Positron emission tomography studies showed that both neurotrophins increased graft-induced recovery of striatal binding of [11C]RTI-121, a marker for dopaminergic nerve terminals. Post mortem analysis at 8 weeks after transplantation showed that both neurotrophins significantly increased the survival of grafted dopaminergic neurons. This study shows that growth/differentiation factor 5 is at least as effective as glial cell line-derived neurotrophic factor in enhancing the survival and functional activity of mesencephalic grafts, and thus is an important candidate for use in the treatment of Parkinson's disease.

EFFECTS OF GROWTH FACTORS ON THE PROLIFERATION OF HUMAN KERATINOCYTES AND FIBROBLASTS IN VITRO.

By: Kim DS, Korting HC, Schäfer-Korting M.

Growth/differentiation factor-5 (GDF-5) is a new member of the transforming growth factor-beta (TGF-beta) superfamily of multifunctional peptide growth factors that appear to mediate many key events in cell growth and development. The effects of GDF-5 and other growth factors (epidermal growth factor, EGF; TGF-beta 1) on the proliferation of human keratinocytes and fibroblasts compared with desoximetasone and calcipotriol have been investigated. The proliferation rate was determined by a hemocytometer, MTT assay and the incorporation of [3H]-thymidine. Moreover, cell cycle analyses were performed and the influence on interleukin-1 alpha (IL-1 alpha) production in keratinocytes was measured by enzyme-linked immunosorbent assay (ELISA) because of its pronounced proinflammatory effect. In keratinocytes, GDF-5 stimulated cell proliferation to a minor extent. The drug already proved to be effective at very low concentrations (0.1 ng/ml). Growth stimulatory effects with EGF have been observed only in keratinocyte basal medium (KBM), but not in keratinocyte growth medium (KGM). TGF-beta 1 markedly inhibited the proliferation of keratinocytes at concentrations > 1 ng/ml. Calcipotriol and desoximetasone also showed a dose-dependent cell growth inhibition in epidermal cell cultures. IL-1 alpha synthesis was greatly suppressed by calcipotriol 10(-8)-10(-6) M. EGF at 10 ng/ml, in contrast, strongly stimulated IL-1 alpha production. Neither GDF-5 nor TGF-beta 1 had a significant effect on IL-1 alpha production in keratinocyte monolayer cultures. In fibroblasts, GDF-5 induced very weak antiproliferative effects. Calcipotriol and desoximetasone also inhibited cell growth in fibroblast cultures whereas proliferation and DNA synthesis were strongly stimulated by 1 ng/ml EGF. There was, however, a contradiction between TGF-beta 1 results on fibroblasts. Whereas TGF-beta 1 increased proliferation in cell number determination and in the thymidine incorporation assay, MTT assays showed slight antiproliferative effects. Due to these controversial results, in addition cell cycle analysis was employed. TGF-beta 1 led to an increased S phase, which indicates a stimulation of cell division. The different results obtained with the MTT test suggest that TGF-beta 1 may stimulate cell division of fibroblasts not only by increasing the S phase, but also by shortening the G1 phase of the cell cycle.

CHARACTERIZATION OF GROWTH/DIFFERENTIATION FACTOR 5 (GDF-5) AS A NEUROTROPHIC FACTOR FOR CULTURED NEURONS FROM CHICKEN DORSAL ROOT GANGLIA.

By: Farkas LM, Scheuermann S, Pohl J, Unsicker K, Krieglstein K.

Growth/differentiation factor-5 (GDF-5), a morphogenetic protein, has previously been shown to act as a neurotrophic factor for midbrain dopaminergic neurons. To further elucidate the neurotrophic potential of GDF-5, serum free cultures of dorsal root ganglionic (DRG) neurons from developing chick embryos were treated with GDF-5 with or without the simultaneous addition of other trophic factors. Our results show that GDF-5 has a minor promoting effect on its own, but it can enhance the survival promoting effect of neurotrophin-3 (NT-3) and nerve growth factor (NGF) on cultured DRG neurons. Our finding fits well into the concept that neurotrophic factors may act synergistically in ensuring survival of different neuronal populations. The capacity of GDF-5 to reduce the requirement of a subpopulation of sensory neurons for NT-3 may have implications for the treatment of peripheral neuropathies.

GROWTH/DIFFERENTIATION FACTOR 5 PROTECTS NIGROSTRIATAL DOPAMINERGIC NEURONES IN A RAT MODEL OF PARKINSON'S DISEASE.

By: Sullivan AM, Opacka-Juffry J, Hötten G, Pohl J, Blunt SB.

Growth/differentiation factor 5 (GDF5), a novel member of the transforming growth factor beta superfamily, promotes the survival of dopaminergic neurones in vitro. We present here the first evidence for a neuroprotective action of GDF5 in vivo. We investigated the effects of intracerebral administration of GDF5 on a rat model of Parkinson's disease. GDF5 was administered just above the substantia nigra and into the lateral ventricle immediately before ipsilateral injection of 6-hydroxydopamine into the medial forebrain bundle. GDF5 prevented the development of amphetamine-induced rotations and preserved the integrity of striatal dopaminergic nerve terminals, as measured by positron emission tomography. Post-mortem studies showed that GDF5 spared dopamine levels in the striatum and tyrosine hydroxylase positive neurones in the midbrain. This study suggests that GDF5 has potential for the treatment of Parkinson's disease.

VIRAL EXPRESSION SYSTEMS FOR RECOMBINANT PROTEINS.

By: Pohl J, Krack W, Scheffner B, Kron R.

The biological activity of several secreted recombinant proteins depends on complex folding and dimerization with other proteins. Since bacterial expressed proteins do not fulfill these requirements, in those cases one has to use eucaryotic expression systems. Usually, stably transformed cells are selected for high productivity. Prior to a decision for a cell line, it is wise to test the expression level, the correct folding and the glycosylation pattern of the desired protein. These tests can be carried out using viral expression systems, which induce high expression level without longlasting selection procedures. Cells found in these tests to be optimal, can be used for the establishment of a production cell line.

RECOMBINANT HUMAN GROWTH/DIFFERENTIATION FACTOR 5 STIMULATES MESENCHYME AGGREGATION AND CHONDROGENESIS RESPONSIBLE FOR THE SKELETAL DEVELOPMENT OF LIMBS.

By: Hötten GC, Matsumoto T, Kimura M, Bechtold RF, Kron R, Ohara T, Tanaka H, Satoh Y, Okazaki M, Shirai T, Pan H, Kawai S, Pohl JS, Kudo A.

We have expressed and biologically characterized recombinant human growth/differentiation factor 5 (huGDF5). This protein is composed of a mature homodimer consisting of 15 kD subunits. Using recombinant expressed protein, we have demonstrated that huGDF5 in vitro stimulated mesenchyme aggregation and chondrogenesis in rat limb bud cells. In vivo, partially purified huGDF5 induced cartilage and bone formation in muscular tissues of rodents. However, in contrast to the effects of other BMPs, as for example BMP-2, the osteoblastic MC3T3-E1 cells did not respond to huGDF5 as measured by alkaline phosphatase activity. These results suggest that the action of GDF5 may be relatively specific for chondrogenesis during the entire process of the endochondral bone formation. GDF5 may control the morphogenesis of cartilaginous tissue, including joints, in the skeletal development of limbs.

IDENTIFICATION OF TYPE I AND TYPE II SERINE/THREONINE KINASE RECEPTORS FOR GROWTH/DIFFERENTIATION FACTOR-5.

By: Nishitoh H, Ichijo H, Kimura M, Matsumoto T, Makishima F, Yamaguchi A, Yamashita H, Enomoto S, Miyazono K.

Growth/differentiation factor-5 (GDF-5) is a member of the bone morphogenetic protein (BMP) family, which plays an important role in bone development in vivo. Mutations in the GDF-5 gene result in brachypodism in mice and Hunter-Thompson type chondrodysplasia in human. BMPs transduce their effects through binding to two different types of serine/threonine kinase receptors, type I and type II. However, binding abilities appear to be different among the members of the BMP family. BMP-4 binds to two different type I receptors, BMP receptors type IA (BMPR-IA) and type IB (BMPR-IB), and a type II receptor, BMP receptor type II (BMPR-II). In addition to these receptors, osteogenic protein-1 (OP-1, also known as BMP-7) binds to activin type I receptor (ActR-I) as well as activin type II receptors (ActR-II and ActR-IIB). Here we investigate the binding and signaling properties of GDF-5 through type I and type II receptors. GDF-5 induced alkaline phosphatase activity in a rat osteoprogenitor-like cell line, ROB-C26. 125I-GDF-5 bound to BMPR-IB and BMPR-II but not to BMPR-IA in ROB-C26 cells and other nontransfected cell lines. Analysis using COS-1 cells transfected with the receptor cDNAs revealed that GDF-5 bound to BMPR-IB but not to the other type I receptors when expressed alone. When COS-1 cells were transfected with type II receptor cDNAs, GDF-5 bound to ActR-II, ActR-IIB, and BMPR-II but not to transforming growth factor-beta type II receptor. In the presence of type II receptors, GDF-5 bound to different sets of type I receptors, but the binding was most efficient to BMPR-IB compared with the other type I receptors. Moreover, a transcriptional activation signal was efficiently transduced by BMPR-IB in the presence of BMPR-II or ActR-II after stimulation by GDF-5. These results suggest that BMPR-IB mediates certain signals for GDF-5 after forming the heteromeric complex with BMPR-II or ActR-II.

STRUCTURE, CHROMOSOMAL LOCALIZATION, AND EXPRESSION ANALYSIS OF THE MOUSE INHIBIN/ACTIVIN BETA C (INHBC) GENE.

By: Schmitt J, Hötten G, Jenkins NA, Gilbert DJ, Copeland NG, Pohl J, Schrewe H.

The mouse inhibin/activin beta C gene (Inhbc), a member of the transforming growth factor-beta (TGF-beta) superfamily, was cloned, mapped, and characterized. The gene spans approximately 14 kb, is composed of two exons, and maps to the distal region of mouse chromosome 10, which is syntenic to chromosome 12q13.1, where the human inhibin/activin beta C gene (INHBC) maps. The primary translation product is a preproprotein of 352 amino acids. The mature C-terminal domain of 116 amino acids shares 94% identity with its human homolog. Primer extension analysis shows that transcription starts approximately 130 bp upstream of the translation initiation site, and no TATA box was found in the promoter. Ribonuclease protection analyses reveal that mouse Inhbc is predominantly expressed in adult liver. Embryonic expression is detected beginning from Day 14.5 of gestation.

CLONING OF A NEW MEMBER OF THE TGF-BETA FAMILY: A PUTATIVE NEW ACTIVIN BETA C CHAIN.

By: Hötten G, Neidhardt H, Schneider C, Pohl J.

The complete amino acid sequence of a new member of the TGF-beta superfamily has been determined through initial degenerate PCR, subsequent cloning and nucleotide sequencing of the human cDNA. Amino acid homology comparison among members of this superfamily suggests it to be a new member of the activin/inhibin subgroup. The proposed mature part of the precursor shows 53% amino acid identity to the activin beta B and 51% identity to the activin beta A chain whereas beta A and beta B show an identity of 64% to each other.

TROPHIC AND PROTECTIVE EFFECTS OF GROWTH/DIFFERENTIATION FACTOR 5, A MEMBER OF THE TRANSFORMING GROWTH FACTOR-BETA SUPERFAMILY, ON MIDBRAIN DOPAMINERGIC NEURONS.

By: Krieglstein K, Suter-Crazzolara C, Hötten G, Pohl J, Unsicker K.

Growth/differentiation factor 5 (GDF5) is a novel member of the transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. We show here that GDF5 is expresed in the developing CNS including the mesencephalon and acts as a neurotrophic, survival promoting molecule for rat dopaminergic midbrain neurons, which degenerate in Parkinson's disease. Recombinant human GDF5 supports dopaminergic neurons, dissected at embryonic day (E) 14 and cultured for 8 days under serum-free conditions, to almost the same extent as TGF-beta 3, and is as effective as glial cell line-derived neurotrophic factor (GDNF), two established trophic factors for midbrain dopaminergic neurons. In contrast to TGF-beta and GDNF, GDF5 augments numbers of astroglial cells in the cultures, suggesting that it may act indirectly and through pathways different from those triggered by TGF-beta and GDNF. GDF5 also protects dopaminergic neurons against the toxicity of N-methylpyridinium ion (MPP+), which selectively damages dopaminergic neurons through mechanisms currently debated in the etiology of Parkinson's disease (PD). GDF5 may therefore now be tested in animal models of PD and might become useful in the treatment of PD.

CLONING AND EXPRESSION OF RECOMBINANT HUMAN GROWTH/DIFFERENTIATION FACTOR 5.

By: Hötten G, Neidhardt H, Jacobowsky B, Pohl J.

The complete amino acid sequence of human Growth/differentiation factor 5 (huGdf5), a new member of the TGF-beta superfamily, has been determined through initial degenerate PCR and subsequent cloning and nucleotide sequencing of genomic DNA and cDNA encoding the precursor and flanking regions. The huGdf5 gene consists of only two coding exons. The protein is highly homologous to its murine equivalent, particularly the mature part which differs only by one amino acid. Expression in HuTK- cells using recombinant vaccinia virus revealed the expected processed dimeric mature protein. Antibodies against huGdf5 were raised in chicken.

QUANTITATIVE PCR DNA TRACE ANALYSIS IN RECOMBINANT PROTEINS

By: Merz B, Jacobowsky B, Hötten G, Neidhardt H

The Polymerase Chain Reaction which enters into the 10th year of application in molecular biology represents a powerful tool in research and diagnostics. It can as well be used in the quality control of medicinal products to quantify residual burden of cell line-DNA due to the fermentation process of recombinant biologicals. Different authorities published recommendations concerning the limits of DNA contamination in biologicals. These limits which are situated in the range of 10-100 picograms require a sensible quantification method which can be used in the analysis of pharmaceuticals. In the development of a quantitatively operating PCR method the primer design and the construction of an internal standard molecule is essential. Chinese hamster ovary cell (CHO) DNA was used as a potential contaminant of the protein erythropoetin (EPO) in the examinations. For the mentioned system a sensibility of less than 10 pg was achieved with a quantitative PCR method.