Application of industrial scale genomics to discovery of therapeutic targets in heart failure.

In recent years intense activity in both academic and industrial sectors has provided a wealth of information on the human genome with an associated impressive increase in the number of novel gene sequences deposited in sequence data repositories and patent applications. This genomic industrial revolution has transformed the way in which drug target discovery is now approached. In this article we discuss how various differential gene expression (DGE) technologies are being utilized for cardiovascular disease (CVD) drug target discovery. Other approaches such as sequencing cDNA from cardiovascular derived tissues and cells coupled with bioinformatic sequence analysis are used with the aim of identifying novel gene sequences that may be exploited towards target discovery. Additional leverage from gene sequence information is obtained through identification of polymorphisms that may confer disease susceptibility and/or affect drug responsiveness. Pharmacogenomic studies are described wherein gene expression-based techniques are used to evaluate drug response and/or efficacy. Industrial-scale genomics supports and addresses not only novel target gene discovery but also the burgeoning issues in pharmaceutical and clinical cardiovascular medicine relative to polymorphic gene responses.

Gene expression profiling in an in vitro model of angiogenesis.

In the present study we have used a novel, comprehensive mRNA profiling technique (GeneCalling) for determining differential gene expression profiles of human endothelial cells undergoing differentiation into tubelike structures. One hundred fifteen cDNA fragments were identified and shown to represent 90 distinct genes. Although some of the genes identified have previously been implicated in angiogenesis, potential roles for many new genes, including OX-40, white protein homolog, KIAA0188, a homolog of angiopoietin-2, ADAMTS-4 (aggrecanase-1), and stanniocalcin were revealed. Support for the biological significance was confirmed by the abrogation of the changes in the expression of angiogenesis inhibitors and in situ hybridization studies. This study has significantly extends the molecular fingerprint of the changes in gene expression that occur during endothelial differentiation and provides new insights into the potential role of a number of new molecules in angiogenesis.

Gene transfer of type 1 interleukin-1 receptor extracellular-domain complementary DNA into rabbit synovial cell line HIG-82 results in cellular blockade of interleukin-1 signal transduction.

OBJECTIVE: To produce, by means of expression cloning, a soluble type 1 interleukin-1 receptor (sIL-1R), and to assess its inhibitory properties on the IL-1 pathway. METHODS: High-affinity IL-1R sites were identified in a human chondrosarcoma cell line by means of 125I-IL-1beta binding. A 1-kilobase complementary DNA (cDNA) encoding the ligand-binding domain of the type 1 IL-1R was cloned by using polymerase chain reaction, and the cDNA was inserted into a mammalian expression vector pRc/CMV. The sIL-1R expression vector was transfected into a rabbit synovial cell line (HIG-82) and a stably transfected cell population was selected. The production of sIL-1R was confirmed in the medium of transfected cells using 125I-IL-1beta binding. 35S labeling of transfected cultures, followed by immunoprecipitation and gel electrophoresis, was used to characterize the size of the recombinant sIL-1R. Stromelysin and IL-1alpha steady-state messenger RNA (mRNA) levels were assessed by Northern blotting. Prostaglandin E2 (PGE2) release was measured by enzyme-linked immunosorbent assay. RESULTS: IL-1R on the surface of HIG-82 cells bound 125I-IL-1beta with an equilibrium dissociation constant (Kd) of 67.3 +/- 7.8 pM (mean +/- SD). Transfection of the sIL-1R expression vector into a synovial cell line in vitro resulted in the appearance of an sIL-1R protein that bound 125I-IL-1beta with high affinity in the medium (Kd = 108 +/- 5 pM). Two protein bands (Mr 42 kd and 47 kd) were immunoprecipitated with an antibody against type 1 T cell-derived sIL-1R. Expression of sIL-1R was accompanied by a marked decrease in both stromelysin and IL-1alpha steady-state mRNA levels. In conjunction, there was a significant inhibition of basal and IL-1-stimulated PGE2 released by sIL-1R-producing cells. CONCLUSION: The data suggest that gene transfer of type 1 sIL-1R into the synovium may be an effective means of inhibiting IL-1-induced metalloproteinase expression and inflammatory responses.

Increased secretion and activity of matrix metalloproteinase-3 in synovial tissues and chondrocytes from experimental osteoarthritis.

OBJECTIVE: The aim of this study was to define the relative regulation of matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinases-1 (TIMP-1), in chondrocytes and synovium in experimental osteoarthritis (EOA). METHODS: Partial-meniscectomized (PM) rabbits, surgical sham controls (SH), and normal non-surgical controls (N) were killed at times corresponding to early degenerative lesions (4 weeks) and increasingly progressive stages of EOA at 8 and 12 weeks post-PM. MMP-3 activity was measured in conditioned media from chondrocytes and synovium using a peptide cleavage assay with substance P (SP) as the substrate. TIMP-1 was quantitated using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Early degenerative lesions (4 weeks post-PM) were characterized by inflammatory responses in the synovium accompanied by a significant rise of MMP-3 activity in synovial cultures (P < 0.05). At 8 weeks there was no discernible inflammation, and MMP-3 activity in EOA synovial cultures was comparable to that in the controls; this was followed by a second increase in MMP-3 activity in EOA samples at 12 weeks. MMP-3 activity was significantly elevated in EOA chondrocyte cultures at 8 weeks post-PM relative to N controls, corresponding to the most destructive phase of EOA, but not in the early phase (4 weeks) or 'late' degenerative phase (12 weeks). Medium derived from chondrocytes contained little or no TIMP-1. Synovia secreted relatively higher amounts of TIMP-1, and this was elevated at 8 weeks post-PM relative to the SH controls. The majority (approximately 90%) of MMP-3 activity could be inhibited using recombinant TIMP-1 or a hydroxamate MMP inhibitor. Complete inhibition was achieved with EDTA or 1,10 phenanthroline. CONCLUSION: Together, these data indicate that in EOA, MMP-3 is initially upregulated in the synovium which may play a pivotal role in the pathogenesis of cartilage lesions. In contrast, chondrocyte-derived MMP-3 is upregulated in the later phases of EOA, contributing further to progression of cartilage lesions.

Temporal pattern of cysteine endopeptidase (cathepsin B) expression in cartilage and synovium from rabbit knees with experimental osteoarthritis: gene expression in chondrocytes in response to interleukin-1 and matrix depletion.

OBJECTIVE: To determine the temporal pattern of expression of cathepsin-B in chondrocytes and synovium in experimental osteoarthritis, and to determine possible mechanisms for upregulation and secretion of cathepsin-B from chondrocytes. METHODS: Experimental osteoarthritis was induced with partial medial meniscectomy (PM); sham operated (SH) and normal (N) rabbits were used as controls. Cathepsin-B mRNA expression was assessed with northern blotting with a 32P labelled cDNA probe. Cathepsin-B was measured in conditioned media or cell extracts using a fluorogenic substrate Z-Arg-Arg-AMC. Chondrocyte monolayers were used to determine cathepsin-B expression in response to interleukin-1 beta (IL-1 beta). Cartilage explants were used to test the effect of matrix depletion on cathepsin-B release. RESULTS: Chondrocytes obtained from experimental osteoarthritis knees did not show cathepsin-B mRNA upregulation. However, isolated chondrocytes secreted cathepsin-B into the culture medium. Enzyme release was significantly higher at 8 weeks relative to controls, but not at 12 weeks or 4 weeks. Enzyme released from synovium was significantly higher in PM group compared with SH group at 4 and 8 weeks. IL-1 beta was ineffective in upregulating steady state cathepsin-B mRNA in chondrocytes; however, it upregulated the intracellular enzyme, and this was blocked with cycloheximide. Enzymatic depletion of cartilage matrix after exposure of explants to IL-1 resulted in release of significantly higher amounts of cathepsin-B into the medium by matrix depleted chondrocytes compared with intact explants. CONCLUSIONS: In experimental osteoarthritis, cathepsin-B is upregulated in synovial tissue during the early degenerative phase. Progression of experimental osteoarthritis is accompanied by upregulation of cathepsin-B in cartilage. Cartilage and synovial cathepsin-B levels decline as experimental osteoarthritis advances to more degenerative states. IL-1 upregulates intracellular cathepsin-B by increasing cathepsin-B protein synthesis; it is not an effective stimulus for enzyme secretion. Depletion of cartilage matrix during progression of experimental osteoarthritis may contribute to secretion of cathepsin-B and perpetuation of cartilage destruction.

Prostromelysin and procollagenase genes are differentially up-regulated in chondrocytes from the knees of rabbits with experimental osteoarthritis.

OBJECTIVE: To determine the relative expressions of matrix metalloprotease (MMP) genes pro-MMP1 and pro-MMP3 in the cartilage of rabbits with experimentally induced osteoarthritis (OA), and to assess the role of the chondrocyte in this process. METHODS: OA was induced in rabbits after partial medial meniscectomy. Rabbits were killed at 4 weeks or 8 weeks, and total cellular RNA was prepared from cartilage and probed by Northern blotting with pro-MMP 32P-labeled complementary DNA. Monolayer chondrocytes were used to assess MMP-inducing activity of chondrocyte factor(s). RESULTS: Pro-MMP messenger RNAs (mRNAs) were up-regulated in experimental OA cartilage; pro-MMP3 mRNA expression exceeded that of pro-MMP1. Conditioned medium from OA-derived chondrocytes up-regulated pro-MMP mRNAs in normal chondrocytes. CONCLUSION: Up-regulation of MMP genes in this OA model may contribute to cartilage degradation. Chondrocytes up-regulate MMP genes via an autocrine pathway.

The proteoglycan synthesis repertoire of rabbit chondrocytes maintained in type II collagen gels.

Repair of experimental articular cartilage lesions employing cultured rabbit articular chondrocytes requires a detailed knowledge of the phenotypic stability of these cells. A suitable matrix vehicle for use in chondrocyte transplantation is a much sought-after component of any transplantation paradigm. We studied the proteoglycan synthesis repertoire of young immature rabbit articular chondrocytes maintained in chick type II collagen gels or collagen gels supplemented with recombinant human transforming growth factor-beta 1 (rhTGF beta 1). Maintenance of chondrocytes in type II collagen gels increased the percentage 35SO4-labeled proteoglycans reaching equilibrium in the A1D1 or D1 fraction of CsCl density gradient when compared to chondrocytes maintained in polystyrene microwell cultures. Although rhTGF beta 1 supplementation increased the percentage of A1D1/D1 proteoglycan by chondrocytes grown on polystyrene, rhTGF beta 1 did not augment this percentage increase in A1D1/D1 when added to collagen II gels. Rabbit chondrocytes synthesized two core proteins derived from the high-density aggregatable proteoglycans. LI and LII have apparent molecular sizes of 480 kDa and 390 kDa, respectively. Both core protein forms were found in the medium fraction, but the predominant core protein form associated with the cell fraction was LI. Maintenance of chondrocytes in collagen II gels increased synthesis of both core proteins. In addition to the large core proteins, three other core proteins with properties on SDS PAGE characteristic of the small dermatan sulfate proteoglycans, biglycan and decorin, were identified. Synthesis of these core proteins was stimulated by maintenance in collagen gels. Furthermore, they were preferentially retained in the gel matrix. Chondrocytes maintained on glass or in type II collagen gels stained with monoclonal antibodies specific for chondroitin-6-sulfate, chondroitin-4-sulfate and keratan sulfate. However, while chondrocytes grown on glass slides failed to stain with monoclonal antibody 3B3 in the absence of chondroitinase ABC digestion, chondrocytes grown in collagen II gels stained intensely in the absence of enzyme pretreatment. These results were confirmed by Western blots.

Immunological analysis of proteoglycan structural changes in the early stage of experimental osteoarthritic canine cartilage lesions.

Specific modifications of the proteoglycan (PG) structure of osteoarthritic (OA) dog cartilage in relation to endogenous metalloprotease activity were examined using murine anti-proteoglycan monoclonal antibodies (MoAbs). OA lesions were induced over a period of 8 weeks in crossbred dogs (Pond-Nuki model). The articular cartilage was removed and homogenized in a Tris buffer, pH 7.5, and then divided into four groups: direct PG extraction, no addition, presence of 1 mM p-aminophenyl mercuric acetate (APMA), and presence of 1 mM APMA and 10 mM o-phenanthroline, incubated for 42 h at 37 degrees C followed by PG extraction. MoAbs reactive with PG protein and carbohydrate epitopes included 1C6, 3B3, 5D4, D1B2, and m4D6. The results showed marked alterations induced by APMA activation of the endogenous metalloproteases. PG changes were apparent at at least three sites: one was either in the hyaluronic acid-binding region or between the hyaluronic-binding region and the G2 globular domain, another was between the keratan-sulfate-rich domain and the chondroitin sulfate-attachment domain, and a third was in the chondroitin sulfate-attachment domain. Constitutive metalloprotease activity resulted in less marked PG alterations with preservation of functional PG aggregation to hyaluronan.

Serum keratan sulfate. Quantitative and qualitative comparisons in inflammatory versus noninflammatory arthritides.

The concentration of keratan sulfate (KS) epitope was measured in the serum of patients with osteoarthritis (OA) or rheumatoid arthritis (RA) by enzyme-linked immunosorbent assay and compared with that in the serum of patients with primary fibromyalgia syndrome (PFS) and of controls who had no joint disease. By Student's tau-test, the mean serum KS concentrations in OA and RA patients measured with monoclonal antibodies (MAb) 5-D-4 and 2-D-3 were significantly increased over those in the PFS and normal groups; similar findings were observed using a nonparametric test, except that levels in RA patients showed no difference from those in PFS patients and normal subjects. There was no significant correlation between joint scores or disease duration and KS levels in OA or RA patients. Gel filtration of sera revealed mainly large, polydisperse KS-bearing fragments which eluted in a broad profile. KS purified from sera by immunoaffinity chromatography consisted mainly of high-density proteoglycans. Electrophoresis of pooled high-density KS fractions in polyacrylamide-agarose gels followed by Western blotting with MAb 5-D-4 showed diffuse bands with relative mobilities corresponding to large proteoglycans. These findings are consistent with attachment of KS to protein core fragments of various sizes; KS in patient sera is comparable in size with that in normal sera. Elevations of serum KS levels occur in the presence of cartilage degradation, but do not quantitatively define the extent or duration of articular involvement.

Serum keratan sulfate levels in rabbits with experimentally induced osteoarthritis.

Serum keratan sulfate (KS) levels were measured with an enzyme-linked immunosorbent assay using monoclonal antibody (MAb) 5-D-4 (anti-KS) in a rabbit model of osteoarthritis (OA) induced by partial meniscectomy. The partial medial meniscectomy produced pathologic changes of OA in the joints of the rabbits, which were seen when the animals were killed at 3, 6, 9, or 12 weeks postsurgery. Tibial or femoral osteophytes were seen in up to 90% of the operated joints; pitting and ulceration of medial femoral condyles were also frequently noted (77% of cases). Rabbits that underwent sham surgery, back-skin-operated rabbits, or nonoperated normal rabbits served as controls; the joints of these animals were normal at the time of killing. A rise in the level of serum KS was recorded in 50% of rabbits following partial meniscectomy, but this was matched by similar changes in the control groups. The mean serum KS level of the OA animals at serial intervals (3, 6, 9, or 12 weeks) following surgery was not significantly different from that in the control groups. When measured with a second MAb, 2-D-3, KS levels showed similar trends as with MAb 5-D-4, although lower assay values were obtained. These findings indicate that experimentally induced OA in rabbits is not associated with a significant rise in serum KS levels. KS levels did not differentiate OA from non-OA animals, nor did they parallel disease progression.

Monoclonal and polyclonal antibodies against dendrotoxin: Their effects on its convulsive activity and interaction with neuronal acceptors.

Three stable hybrid cell lines have been established that secrete monoclonal antibodies of G(1) sub-class to dendrotoxin, a convulsant polypeptide (M(r) = 7000). Using [(125)I]labelled dendrotoxin the resultant ascitic fluids were found to show no cross-reactivity with homologous toxins (toxins 1, B and E from Dendroaspis polylepis, toxin Dv-14 from Dendroaspis viridis and ?-bungarotoxin from Bungarus multicinctus). In contrast, polyclonal antibodies raised against dendrotoxin cross-reacted to varying degrees with its congeners; most importantly, the rank order of cross-reactivities was in accordance with their potencies in eliciting convulsions when injected intracerebroventricularly into rat brain. All the antibodies prevented significantly the binding of dendrotoxin to its protein acceptor in brain synaptic membranes. Moreover, when they were injected into rat brain together with lethal doses of dendrotoxin they delayed, or in some cases prevented, the onset of convulsive symptoms. Ultracentrifugation of the complexes formed by [(125)I]labelled dendrotoxin and one or more of the monoclonal antibodies showed only a single peak of radioactivity with an S(20.w) of 7S, indicating that all these mono-specific antibodies are directed to the same or overlapping epitope(s). Conversely, polyclonal antisera produced larger complexes with the antigen, revealing the presence of at least two determinants on this molecule. Such antibodies are proving helpful in identifying regions of the toxin responsible for the neurotoxicity and associated interaction with its acceptor, a putative constituent of A-current K(+)-channels.

Identification by cross-linking of a neuronal acceptor protein for dendrotoxin, a convulsant polypeptide.

Dendrotoxin, a lijow molecular weight protein from the venom of Dendroaspis angusticeps, is known to be a potent convulsant that attenuates one type of voltage-sensitive K+ channel in guinea-pig hippocampus. A biologically active preparation of 125I-labelled dendrotoxin has been cross-linked to its high-affinity protein acceptor in synaptic plasma membranes from rat cerebral cortex. On SDS gel electrophoresis, a complex with a Mr of 72,000 was observed which, assuming one toxin molecule is attached, yields an apparent size of 65,000 for this subunit of the acceptor. Unlike dendrotoxin, low concentrations of beta-bungarotoxin, another pre-synaptically active toxin, do not inhibit its labelling.

Properties of monoclonal antibodies to nicotinic acetylcholine receptor from chick muscle.

Four stable, hybrid-cell lines secreting monoclonal antibodies to distinct determinants on the nicotinic acetylcholine receptor from chick muscle have been established. These were characterised by the following criteria: immunoglobulin isotype, ability to produce experimental autoimmune myasthenia gravis in mice and reactivity towards homologous and heterologous acetylcholine receptor proteins. Two monoclonal antibodies were found to inhibit the reaction of alpha-bungarotoxin with homologous acetylcholine receptor; in addition one of these, on binding to receptor-toxin, induced a rapid dissociation of the complex (t1/2 = 0.5 h at 23 degrees C). Three of the antibody preparations recognised epitopes on this receptor from muscle of other species and two of these caused experimental autoimmune myasthenia gravis in BALB/c mice following passive transfer. The latter two recognised to significant extents the alpha-bungarotoxin binding component purified from chick optic lobe and brain cortex. Sedimentation analysis demonstrated that two of the monoclonal antibodies form a distinct size (s20, w = 12S) of complex with the receptor of chick muscle which most probably corresponds to a 1:1 attachment of antibody and receptor; this may involve cross-linking of two determinants within the same oligomer. A similar observation was made with the alpha-bungarotoxin binding component from optic lobe using one of the cross-reacting antibodies. Another monoclonal antibody was found to be capable of forming much heavier complexes with the receptor from chick muscle, these are thought to involve inter-molecular cross-linking of oligomers. The observed properties of these antibodies are discussed in relation to their myasthenogenicity and with reference to the extent of structural similarities between the peripheral nicotinic acetylcholine receptor and the alpha-bungarotoxin binding protein from brain.

Botulinum neurotoxin and dendrotoxin as probes for studies on transmitter release.

Acceptors for BoNT have been detected autoradiographically on the terminal membrane of motor nerves at a density of approximately 150/micron2 and shown to mediate toxin internalization, a process deemed essential for its inhibition of transmitter release. DTX, a protein with pronounced central neurotoxicity, was shown to induce convulsive states in hippocampal slices from guinea-pig. Synaptic transmission was facilitated and spontaneous epileptiform activity produced in intact cell populations. Voltage clamp analysis of hippocampal neurones revealed that DTX specifically attenuated a transient voltage-dependent K+ conductance (A-current) and this could account for the excitatory effects observed. Proteinaceous acceptors with high affinity for DTX were identified on brain synaptosomal membranes and found to contain a 65 000 Mr polypeptide. Their location in rat brain regions was established and contrasted with that of the binding sites for beta-bungarotoxin. These findings indicate the usefulness of DTX as a probe for a protein associated with one variety of K+ channel while the larger subunit of BoNT was found to interact with a membraneous component that resides at cholinergic nerve terminals and, hence, is likely to have a unique role.

Biochemical and electrophysiological properties of antibodies against pure acetylcholine receptor from vertebrate muscles and its subunits from Torpedo in relation to experimental myasthenia.

Immunization of rabbits with homogeneous preparations of acetylcholine receptor from denervated muscle of cat and chicken, which contained single or multiple sizes of polypeptides respectively, induced myasthenic-like symptoms. One of the resultant antisera, and the IgG fraction thereof, reduced significantly and irreversibly the amplitude of miniature endplate potentials in murine muscle; the effect was not abolished by heat inactivation of complement. This antiserum also retarded the binding of ?-bungarotoxin to a solubilised extract of denervated muscle containing homologous receptor. The other five antibody preparations were unable to affect these miniature potentials but many of them did reduce the binding of ?-bungarotoxin to denervated muscle receptor in solution and, in some cases, decreased the effectiveness of the latter in blocking neuromuscular transmission. Although inoculation with each of the four individual subunits from the receptor of Torpedo marmorata electroplax did not produce muscle weakness in rabbits, antibodies to ?- or ?-polypeptides lowered, to a significant extent, the amplitude of spontaneous synaptic potentials in mouse diaphragm muscle. It is concluded that antibodies with direct blocking actions on the receptor-ion channel complex are not common in such immunized animals and their presence cannot be correlated readily with the induction of physical disability. The majority of the antibody species bind to loci distant from the acetylcholine recognition site. Antiserum from one of the immunized rabbits reacted preferentially with receptor from denervated rather than innervated cat and rat muscle, indicating some dissimilarity.

Similarity of acetylcholine receptors of denervated, innervated and embryonic chicken muscles. 1. Molecular species and their purification.

Acetylcholine receptors were purified to homogeneity from chicken embryonic, adult innervated and denervated muscles, by bio-specific chromatographies using immobilised alpha-neurotoxin and lentil lectin. A minimum specific activity for the pure receptor was estimated to be 6000 nmol alpha-toxin binding sites/g protein. For analysis, the receptors were radio-iodinated or tritiated to high specific radioactivity with succinimidyl-[2,3-3H]propionate. All of the iodinated protein present in the purified receptor preparation reacted with antibody against the pure acetylcholine receptor from Torpedo marmorata electric organ. In the case of all three muscle types used the same oligomeric forms were obtained. The principal form has a sedimentation coefficient of about 9 S, while a minor species (approximately 5S) was also appreciable in crude preparations of embryonic and denervated muscles. Immunization of rabbits with the homogenous receptor from chicken denervated muscle produced muscle weakness characteristic of experimental autoimmune myasthenia gravis. These antisera were equally reactive towards the receptor --125I-alpha-bungarotoxin complexes from chick innervated and denervated muscles. Likewise, the electrophoretic mobilities of the receptors (9-S form) from all three muscle types were identical, as were the isoelectric points of their complexes with 125I-alpha-bungarotoxin. Collectively, these findings and associated ones on subunit structure denote that the 9-S receptor molecules from junctional and extra-junctional area and embryonic stage of chicken muscle are indistinguishable by all criteria yet applied to them.

Nicotinic acetylcholine receptor from chick optic lobe.

An alpha-bungarotoxin-sensitive nicotinic cholinergic receptor from chick optic lobe has been completely purified. Its standard sedimentation coefficient is 9.1 S. The value near 12 S reported for the related component from other brain regions can be reproduced when the initial extraction is by Triton X-100 (rather than Lubrol PX), but other protein is then complexed with it. A single subunit of apparent molecular weight 54,000 is detected, and this subunit is specifically labeled by bromo-[3H]acetylcholine, but only after disulfide reduction. The same size subunit likewise is labeled in the protein (purified similarly) from the rest of the chick brain which can also bind alpha-bungarotoxin and nicotinic ligands. Immunological crossreactivity is demonstrated between both of these proteins with an antiserum to pure acetylcholine receptor from skeletal muscle. The acetylcholine receptor from chick optic lobe and the alpha-bungarotoxin-binding protein from the rest of the brain appear similar or identical by a series of criteria and are related to (but with differences from) peripheral acetylcholine receptors.