Michael D. Barker

Three-Dimensional Tissue Engineering of Hyaline Cartilage: Comparison of Adult Nasal and Articular Chondrocytes

Adult chondrocytes are less chondrogenic than immature cells, yet it is likely that autologous cells from adult patients will be used clinically for cartilage engineering. The aim of this study was to compare the postexpansion chondrogenic potential of adult nasal and articular chondrocytes. Bovine or human chondrocytes were expanded in monolayer culture, seeded onto polyglycolic acid (PGA) scaffolds, and cultured for 40 days. Engineered cartilage constructs were processed for histological and quantitative analysis of the extracellular matrix and mRNA. Some engineered constructs were implanted in athymic mice for up to six additional weeks before analysis. Using adult bovine tissues as a cell source, nasal chondrocytes generated a matrix with significantly higher fractions of collagen type II and glycosaminoglycans as compared with articular chondrocytes. Human adult nasal chondrocytes proliferated approximately four times faster than human articular chondrocytes in monolayer culture, and had a markedly higher chondrogenic capacity, as assessed by the mRNA and protein analysis of in vitro-engineered constructs. Cartilage engineered from human nasal cells survived and grew during 6 weeks of implantation in vivo whereas articular cartilage constructs failed to survive. In conclusion, for adult patients nasal septum chondrocytes are a better cell source than articular chondrocytes for the in vitro engineering of autologous cartilage grafts. It remains to be established whether cartilage engineered from nasal cells can function effectively when implanted at an articular site.

Activity of matrix metalloproteinase-9 against native collagen types I and III

Interstitial collagen types I, II and III are highly resistant to proteolytic attack, due to their triple helical structure, but can be cleaved by matrix metalloproteinase (MMP) collagenases at a specific site, approximately three‐quarters of the length from the N‐terminus of each chain. MMP‐2 and ‐9 are closely related at the structural level, but MMP‐2, and not MMP‐9, has been previously described as a collagenase. This report investigates the ability of purified recombinant human MMP‐9 produced in insect cells to degrade native collagen types I and III. Purified MMP‐9 was able to cleave the soluble, monomeric forms of native collagen types I and III at 37 °C and 25 °C, respectively. Activity against collagens I and III was abolished by metalloproteinase inhibitors and was not present in the concentrated crude medium of mock‐transfected cells, demonstrating that it was MMP‐9‐derived. Mutated, collagenase‐resistant type I collagen was not digested by MMP‐9, indicating that the three‐quarters/one‐quarter locus was the site of initial attack. Digestion of type III collagen generated a three‐quarter fragment, as shown by comparison with MMP‐1‐mediated cleavage. These data demonstrate that MMP‐9, like MMP‐2, is able to cleave collagens I and III in their native form and in a manner that is characteristic of the unique collagenolytic activity of MMP collagenases.

Expression of ADAMTS metalloproteinases in the retinal pigment epithelium derived cell line ARPE-19: Transcriptional regulation by TNFα

ADAMTS (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type I motifs) are multidomain proteins with demonstrated metalloproteinase functionality and have potential roles in embryonic development, angiogenesis and cartilage degradation. We present here investigations of ADAMTS expression in an ocular cell type, ARPE-19, with a view to implicating them in retinal matrix turnover. Expression analysis was undertaken using a combination of reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting experiments, which together detected the expression of mRNAs for several ADAMTS proteins, all of which have active site motifs characteristic of matrix metalloproteases (MMPs). These included ADAMTS1, ADAMTS2, ADAMTS3, ADAMTS5, ADAMTS6, ADAMTS7 and ADAMTS9. The expression of mRNA isoforms for ADAMTS7 and ADAMTS9 were also detected. Following stimulation with TNFalpha, ADAMTS1, ADAMTS6 and both ADAMTS9 transcripts expressed in ARPE-19 cells showed a potent upregulation. The expression of ADAMTS genes in ARPE-19 cells and the transcriptional stimulation of some family members by TNFalpha may implicate them in inflammatory eye disease and the compromise of retinal matrix structure, which is evident in age-related macular degeneration (ARMD) and other retinal pathologies.

Alanine scanning mutagenesis of CCR3 reveals that the three intracellular loops are essential for functional receptor expression

Intracellular signaling mediated by the eotaxin receptor, CCR3, has been implicated in allergic diseases involving the recruitment and activation of eosinophils. To investigate the structural requirements of the three intracellular loops (ICL) of CCR3, a panel of 15 alanine triplet mutants were generated and their effects on function assessed by assays of cell surface expression and chemotactic responsiveness. While the majority of constructs were efficiently expressed when compared with their wild‐type counterpart, their abilities to migrate in response to eotaxin were relatively poor, suggesting that all three intracellular loops of CCR3 are involved to some degree in coupling to G proteins. – Another panel of 7 point mutants were then constructed to examine the DRY motif which resides in ICL2 and is highly conserved throughout the chemokine receptors identified to date. The conservative mutants D130E and R131K were well tolerated and gave chemotactic responses approaching 35 % of wild‐type CCR3, but the less conserved substitutions D130A, D130N and R131L were non‐functioning. Tyrosine 131 was particularly sensitive to mutation as both Y131F and Y131S mutants were poorly expressed and were chemotactically inactive. Together, this data suggest that the acidic / basic / polar nature of the DRY motif is a prerequisite for CCR3 function.

Clinical features of a novel TIMP-3 mutation causing Sorsby's fundus dystrophy: implications for disease mechanism

AIMS To describe the phenotype in three family members affected by a novel mutation in the gene coding for the enzyme tissue inhibitor of metalloproteinase-3 (TIMP-3). METHODS Three members of the same family were seen with a history of nyctalopia and visual loss due to maculopathy. Clinical features were consistent with Sorsbys fundus dystrophy. Exon 5 of the gene coding for TIMP-3 was amplified by the polymerase chain reaction, single strand conformation polymorphism analysis undertaken and exon 5 amplicons were directly sequenced. RESULTS Onset of symptoms was in the third to fourth decade. Five of six eyes had geographic macular atrophy rather than neovascularisation as a cause for central visual loss. Peripheral retinal pigmentary disturbances were present. Scotopic ERGs were abnormal in all three. Mutation analysis showed a G→T transversion in all three resulting in a premature termination codon, E139X, deleting most of the carboxy terminal domain of TIMP-3. CONCLUSIONS The patients described had a form of Sorsbys fundus dystrophy which fell at the severe end of the spectrum of this disease. Postulated disease mechanisms include deposition of dimerised TIMP-3 protein.

Expression of mutant and wild-type TIMP3 in primary gingival fibroblasts from Sorsby's fundus dystrophy patients

Gingival fibroblast cell lines were derived from Sorsbys fundus dystrophy (SFD) patients carrying the S181C TIMP3 and the E139X TIMP3 mutations. These cell lines were grown in culture to study expression of the wild-type and mutant tissue inhibitor of metalloproteinase 3 (TIMP3) alleles from a normal diploid cell type. Firstly, patient cells were found to co-express the wild-type and mutant TIMP3 alleles, S181C TIMP3 or E139X TIMP3, at the mRNA level using restriction fragment length polymorphism (RFLP) analysis. A SpeI RFLP for E139X TIMP3 is described. Low levels of endogenous TIMP3 protein expression were elevated using the natural polysaccharide calcium pentosan polysulfate (CaPPs) in combination with the cytokine IL-1alpha. Immunoblotting detected protein expression from both wild-type and mutant alleles, S181C TIMP3 or E139X TIMP3. S181C TIMP3 from these cells was found to dimerise and retain MMP2 inhibitory activity. To facilitate studies of the E139X TIMP3 protein, the allele was expressed using HighFive insect cells. In this cell type, the E139X TIMP3 was synthesised as a mixture of monomer and dimer. Both monomeric and dimeric E139X TIMP3 protein retained MMP2 inhibitory activity in gelatin zymography. Expression of mutant E139X or S181C TIMP3 protein from a normal diploid patient-derived fibroblast cell had no effect on either MMP2 or MMP9 expression or activation whilst transcribed from their normal promoter context.

Multiple signalling pathways in the C5a-induced expression of adhesion receptor Mac-1

The CD11/CD18 family of leukocyte glycoproteins is essential in the process of adherence to endothelial and other cells that occurs during the acute inflammatory response. The cell surface expression of one member of this family, CD11b/CD18, or Mac-1, is increased on monocytes, neutrophils and other cell types by a number of agents, including chemotactic peptides and lipid mediators. The intracellular signalling mechanisms which control Mac-1 expression are not fully understood. In this report we have investigated the role of G proteins and extracellular Ca2+ in the stimulation of Mac-1 upregulation by the chemoattractant C5a in the human monocyte-like cell line, U937. Two signal transduction pathways are apparently involved and can be distinguished by their sensitivity to pertussis toxin, which inhibits activation of the Gi class of G proteins. The results indicate that a pertussis toxin-insensitive influx of extracellular Ca2+ may be one part of a network of signals leading to Mac-1 upregulation on U937 cells. This is in contrast to the stimulation of this process in neutrophils by chemotactic peptide, which is reported to be entirely dependent on pertussis toxin sensitive G proteins and independent of extracellular Ca2+.

Lentiviral shRNA knock-down of ADAMTS-5 and -9 restores matrix deposition in 3D chondrocyte culture

Aggrecan is one of the two major constituents of articular cartilage, and during diseases such as osteoarthritis (OA) it is subject to degradation by proteolytic enzymes. The primary proteases responsible for aggrecan cleavage are the aggrecanases, identified as members of the ADAMTS family of proteases, which are upregulated in response to inflammatory stimuli. It is uncertain which of the six aggrecanases (ADAMTS‐1, ‐4, ‐5, ‐8, ‐9 and ‐15) are primarily responsible for the degradation of aggrecan in human cartilage. Here we show that four of the six aggrecanases are expressed in immortalized chondrocyte cell‐lines and can be upregulated in response to inflammatory cytokines. Using RNA interference, we demonstrate robust knock‐down of ADAMTS‐5 and ‐9 expression in these cells and, by culturing them on three‐dimensional (3D) scaffolds, show that reduction in expression of ADAMTS‐5 enzyme results in an increase in matrix deposition. These data suggest that the quality of tissue‐engineered cartilage matrix might be improved by targeted depletion of aggrecanase expression. Moreover, this work also provides further evidence that ADAMTS‐5 may be a therapeutic target in the treatment of arthritic disease. Copyright

A peptide derived from TIMP-3 inhibits multiple angiogenic growth factor receptors and tumour growth and inflammatory arthritis in mice

Abstract The binding of vascular endothelial growth factor (VEGF) to VEGF receptor-2 (VEGFR-2) on the surface of vascular endothelial cells stimulates many steps in the angiogenic pathway. Inhibition of this interaction is proving of value in moderating the neovascularization accompanying age-related macular degeneration and in the treatment of cancer. Tissue inhibitor of metalloproteinases-3 (TIMP-3) has been shown to be a natural VEGFR-2 specific antagonist—an activity that is independent of its ability to inhibit metalloproteinases. In this investigation we localize this activity to the C-terminal domain of the TIMP-3 molecule and characterize a short peptide, corresponding to part of this domain, that not only inhibits all three VEGF-family receptors, but also fibroblast growth factor and platelet-derived growth factor receptors. This multiple-receptor inhibition may explain why the peptide was also seen to be a powerful inhibitor of tumour growth and also a partial inhibitor of arthritic joint inflammation in vivo.

Site directed mutagenesis of the complement C5a receptor—Examination of a model for its interaction with the ligand C5a

C5a is a 74 amino acid peptide cleaved from the fifth component of the complement system after activation of either the alternative or classical pathways. It is a potent chemoattractant for neutrophils and monocytes binding to identical receptors on the cell surface. Following the cloning of the cDNA encoding for the human complement C5a receptor, revealing it to be a member of the rhodopsin superfamily of G-protein coupled receptors, a model for the interaction of the C5a receptor with its ligand was proposed, the structure for the receptor being modelled on that of the well defined receptor bacteriorhodopsin. In this model two key residues of the receptor, aspartate82 and either glutamate179 or glutamate 180 were proposed to make up part of the binding site for C5a, acting as counter ions for arginine74 and arginine40, respectively of the C5a molecule. Replacement of aspartate82, glutamate179 and glutamate180 of the C5a receptor with asparagine and glutamine, respectively was shown to have little effect on the dissociation constant of the receptor as detected by Scatchard analysis and competitive binding assays. Hence this modus operandi for the interaction of C5a with its receptor can be rejected.