Differential expression of two tropoelastin genes in zebrafish.

Elastin is the extracellular matrix protein responsible for properties of extensibility and elastic recoil in large blood vessels, lung and skin of most vertebrates. Elastin is synthesized as a monomer, tropoelastin, but is rapidly transformed into its final polymeric form in the extracellular matrix. Until recently information on sequence and developmental expression of tropoelastins was limited to mammalian and avian species. We have recently identified and characterized two expressed tropoelastin genes in zebrafish. This was the first example of a species with multiple tropoelastin genes, raising the possibility of differential expression and function of these tropoelastins in elastic tissues of the zebrafish. Here we have investigated the temporal expression and tissue distribution of the two tropoelastin genes in developing and adult zebrafish. Expression was detected early in skeletal cartilage structures of the head, in the developing outflow tract of the heart, including the bulbus arteriosus and the ventral aorta, and in the wall of the swim bladder. While the temporal pattern of expression was similar for both genes, the upregulation of eln2 was much stronger than that of eln1. In general, both genes were expressed and their gene products deposited in most of the elastic tissues examined, with the notable exception of the bulbus arteriosus in which eln2 expression and its gene product was predominant. This finding may represent a sub-specialization of eln2 to provide the unique architecture of elastin and the specific mechanical properties required by this organ.

The endogenous vascular elastase that governs development and progression of monocrotaline-induced pulmonary hypertension in rats is a novel enzyme related to the serine proteinase adipsin.

We showed previously a cause and effect relationship between increased activity of an endogenous vascular elastase (EVE) and experimentally induced pulmonary hypertension in rats. We now report the isolation and characterization of EVE. Degenerate oligonucleotides synthesized to homologous sequences in serine elastases were used in a PCR with rat pulmonary artery (PA) cDNA. The PCR product hybridized to a 1.2-kb mRNA and the intensity of hybridization was threefold increased in RNA from rat hypertensive PA at a timepoint when EVE activity was increased. The PCR product was used to screen a cDNA library and sequences obtained encoded rat adipsin. We then used immunoaffinity to purify EVE. An antibody to the elastin-binding protein was used to remove this competitor of elastase from the PA extract and the elastolytic activity increased 100-fold. The enzyme was purified using an antibody that recognizes NH2-terminal sequences of serine proteinases and the eluate was further purified using an antibody raised against recombinant adipsin. A single band at 20 kD immunoreactive with the adipsin antibody was resolved as an active enzyme on an elastin substrate gel. Immunogold labeling with an antibody to an adipsin peptide sequence localized EVE to PA smooth muscle cells. This is the first isolation of EVE; it appears to be a novel enzyme related to the serine proteinase adipsin originally found in adipose tissue.

The extraction and partial characterization of proteins released by decalcification from calcified human aortic plaques.

Tissue from severely atherosclerotic human aortas was divided into heavily calcified (plaque) and less calcified (non-plaque) areas. After defatting and thorough extraction with neutral salt solutions, further proteinaceous material was released from plaque than non-plaque areas. Although organic phosphorous was associated with the EDTA-solubilized protein from both plaque and non-plaque areas, the phosphorous to protein ratio of extracts from plaque areas was 2 to 3 times that from non-plaque areas. Analysis of the major protein component of the extract of plaque areas indicated an acidic glyco-protein which appeared to be associated with an organic phosphorous moiety as well as cholesterol and cholesterol esters.

Self-aggregation characteristics of recombinantly expressed human elastin polypeptides.

Elastin is an extracellular matrix protein found in tissues requiring extensibility and elastic recoil. Monomeric elastin has the ability to aggregate into fibrillar structures in vitro, and has been suggested to participate in the organization of its own assembly into a polymeric matrix in vivo. Although hydrophobic sequences in elastin have been suggested to be involved in this process of self-organization, the contributions of specific hydrophobic and crosslinking domains to the propensity of elastin to self-assemble have received less attention. We have used a series of defined, recombinant human elastin polypeptides to investigate the factors contributing to elastin self-assembly. In general, coacervation temperature of these polypeptides, used as a measure of their propensity to self-assemble, was influenced both by salt concentration and polypeptide concentration. In addition, hydrophobic domains appeared to be essential for the ability of these polypeptides to self-assemble. However, neither overall molecular mass, number of hydrophobic domains nor general hydropathy of the polypeptides provided a complete explanation for differences in coacervation temperature, suggesting that the specific nature of the sequences of these hydrophobic domains are an important determinant of the ability of elastin polypeptides to self-assemble.

Characterization of proteins from the calcified matrix of atherosclerotic human aorta.

The nature of proteins occluded in the mineralized matrix of calcified areas of atherosclerotic human aortic tissue has been investigated. In spite of their reported presence in atherosclerotic lesions, neither plasma lipoproteins, immunoglobulin G or fibrinogen could be detected among the proteins specifically trapped in the mineralized matrix and released by decalcification. However, human serum albumin was present in the decalcifying extract in a persistent complex with a more acidic protein component. Also present in the decalcifying extract was an acidic protein component with a molecular weight between 6000 and 10000 which contained both serine phosphate and gamma-carboxyglutamic acid. This component closely resembled in amino acid composition the non-albumin moiety of the serum albumin complex, suggesting that this low molecular weight, gamma-carboxyglutamic acid-containing protein component was present in the calcified matrix both in the free form and in association with human serum albumin.

The effect of induced atherosclerosis on the synthesis of elastin in chick aortic tissue.

Atherosclerosis was induced in growing chickens by the administration of a diet containing elevated levels of cholesterol and vitamin D3. The effect of this diet on the accumulation of insoluble elastin and the synthesis of soluble and insoluble elastin in the thoracic aortas of these animals was measured. Although the diet resulted in significant increases in levels of cholesterol, 25-OH vitamin D3 and calcium in plasma, increased levels of cholesterol and calcium in aortic tissue, and histological evidence of aortic lipid deposition, there were no detectable differences between experimental and control animals in either the rate or the time course of accumulation of total insoluble elastin in the thoracic aorta, or in the rate and time course of synthesis of soluble and insoluble elastin. These data suggest that, at least in this model, any effect of atherosclerosis on aortic elastin production must be either small or so localized as to be not measurable by the methods used.

Identification and quantitation of alpha 2-HS-glycoprotein in the mineralized matrix of calcified plaques of atherosclerotic human aorta.

alpha 2-HS-glycoprotein (HSGP) is a minor constituent of plasma with negative acute-phase reactant properties. HSGP has been shown previously to accumulate in the mineralized matrix of bone and dentin to concentrations substantially higher than those present in plasma. In addition, HSGP has also been found in experimentally induced dermal calcifications in animals. Here we show that HSGP is occluded in the organic matrix of pathological calcifications of atherosclerotic human aortic tissue in a concentration approximately 7-fold greater than that found in plasma.

Type IV Ehlers-Danlos syndrome presenting as sudden infant death.

A previously healthy 5-month-old female infant presented with sudden death due to spontaneous subarachnoid hemorrhage associated with minor multifocal visceral hemorrhages. The clinical diagnosis had been sudden infant death syndrome. Although the family history was noncontributory and other features of type IV Ehlers-Danlos syndrome (EDS) were absent, the pattern of hemorrhage was consistent with this type of connective tissue disorder. The diagnosis was confirmed after postmortem analysis of skin and aorta showed less than 5% type III collagen (normal greater than 15%). Extensive literature review failed to find any other reported cases of sudden death in infancy due to intracranial hemorrhage in patients with previously unsuspected type IV EDS. The authors suggest that collagen analysis should be performed in cases of unexplained multifocal spontaneous hemorrhage in infancy so that this rare diagnosis will not be missed.

Durability of regenerated articular cartilage produced by free autogenous periosteal grafts in major full-thickness defects in joint surfaces under the influence of continuous passive motion. A follow-up report at one year.

An autogenous graft of tibial periosteum was sutured (with its cambium layer facing into the joint) to the base of a five by ten-millimeter full-thickness defect in the patellar groove of each of forty-five adolescent rabbits. The rabbits were randomly treated postoperatively by either four weeks of immobilization in a cast, intermittent active motion in a cage, or two weeks of continuous passive motion. One year postoperatively, the regenerated tissue from each rabbit was analyzed macroscopically, histologically, histochemically, and biochemically. Gross degenerative changes were seen in 57 per cent of the rabbits that had been immobilized in a cast, in 73 per cent of the rabbits that had been allowed intermittent active motion, and in 22 per cent of the rabbits that had been subjected to continuous passive motion (p less than 0.05). Out of a possible score of 7.0 points for the nature of the regenerated tissue, the scores for the three groups were: immobilization in a cast, 4.1 points; intermittent active motion, 4.0 points; and continuous passive motion, 5.9 points (p greater than 0.05). Out of a possible perfect combined score of 10.0 points for the structural characteristics of the regenerated tissue, the cast-immobilization group scored 3.8 points; the intermittent active-motion group, 2.5 points; and the continuous passive-motion group, 6.4 points (p less than 0.001). The total scores for freedom from cellular changes of degeneration, a perfect score being 5.0 points, were: immobilization in a cast, 2.4 points; intermittent active motion, 2.3 points; and continuous passive motion, 3.9 points (p less than 0.01). Degenerative changes in the adjacent cartilage, which were noted in 42 and 46 per cent of the knees in the immobilization and intermittent active-motion groups, respectively, were not found in the knees that had been subjected to continuous passive motion (p less than 0.05). The total indices, which were derived by combining the scores for all categories (maximum, 24.0 points), revealed that the index for the continuous passive-motion group was significantly better than the index for either of the other two groups: immobilization in a cast, 12.9 points; intermittent active motion, 11.2 points; and continuous passive motion, 19.2 points (p less than 0.0005).(ABSTRACT TRUNCATED AT 400 WORDS)

The chondrogenic potential of free autogenous periosteal grafts for biological resurfacing of major full-thickness defects in joint surfaces under the influence of continuous passive motion. An experimental investigation in the rabbit.

A rectangular graft of autogenous tibial periosteum was sutured (with its cambium layer facing into the joint) onto the base of a five by ten-millimeter full-thickness defect in the patellar groove of each of 143 adolescent and adult rabbits. The rabbits were managed postoperatively by either immobilization, intermittent active motion, continuous passive motion for two weeks, or continuous passive motion for four weeks. When the animals were killed four weeks postoperatively, the contour of the patellar groove had been restored in all of the rabbits in the group that had had four weeks of continuous passive motion, and the newly formed tissue in all of the defects in this group had the gross, histological, and histochemical appearance of smooth, intact hyaline articular cartilage. Histologically, the nature of the tissue that had formed, as well as its surface regularity, structural integrity, and bonding to the adjacent cartilage, were significantly better in the group that had had four weeks of continuous passive motion than in any of the other groups. The results were significantly worse when the orientation of the periosteal graft was reversed (that is, when it had been sutured into the defect with the cambium layer of the graft facing the subchondral bone rather than into the joint) or when no periosteal graft was used. Biochemical analyses revealed that, in the group that had had four weeks of continuous passive motion, the total hexosamine content, the levels of chondroitin sulphate and keratan sulphate, and the ratio of galactosamine to glucosamine were all comparable with the values for normal articular cartilage. In contrast, in the groups that were treated by immobilization, intermittent active motion, or two weeks of continuous passive motion, as well as in the adult rabbits, the content of the first three of these substances was significantly less than normal. In the groups that were treated by immobilization, intermittent active motion, or two weeks of continuous passive motion, 32 to 47 per cent of the total collagen was type II, while in the group that had had four weeks of continuous passive motion, 93 per cent of the total collagen was type II. These results demonstrate that, under the influence of continuous passive motion, free autogenous periosteal grafts can repair a large full-thickness defect in a joint surface by producing tissue that resembles articular cartilage grossly, histologically, and biochemically, and that contains predominantly type-II collagen.

Distinct non-collagen based cartilages comprising the endoskeleton of the Atlantic hagfish, Myxine glutinosa.

Previous evidence from our laboratories showed that collagen is not the major matrix protein of the cartilaginous endoskeleton of the lamprey (Petromyzon marinus). Here we have characterized the cartilage matrix proteins of the only other extant agnathan, the hagfish (Myxine glutinosa). Using morphological, immunochemical and biochemical methods, we show that the structural proteins of the cartilaginous endoskeleton of the hagfish are also non-collagenous in nature. Although these hagfish cartilage proteins share properties both with each other and with lamprey cartilage proteins, including resistance to solubilization with cyanogen bromide and an usual amino acid composition rich in glycine and non-polar amino acids, it is clear that at least two and probably more hagfish cartilage proteins can be distinguished, with distinct distributions in different cartilage structures. Furthermore, in spite of their similarities, matrix proteins from hagfish cartilage are not identical to the proteins we have previously characterized in lamprey cartilage. These results suggest the existence of a larger family of similar but not identical proteins that form the major structural elements of cartilage tissues of agnathans. These data also support our previous conclusion that type II collagen became the predominant structural protein of cartilage only after the divergence of the agnathans from the ancestral line of the vertebrates.

Spatial and temporal distribution of lamprin mRNA during chondrogenesis of trabecular cartilage in the sea lamprey.

The temporal and spatial expression patterns of lamprin, the principle structural protein in lamprey cartilages, were examined by in situ hybridization during chondrogenesis of trabecular cartilage in day 17-33 post-fertilization prolarval lampreys. Lamprin mRNA transcripts were first detected during day 19, concomitant with the end of the condensation phase of chondrogenesis and the initiation of matrix synthesis as indicated by light microscopic examination. In the stages which followed, the hybridization signal increased with progressive intensity, paralleling matrix synthesis, suggesting transcriptional control of lamprin gene expression. Spatially, lamprin expression patterns mirrored the rostrocaudal development of the trabecular cartilage rudiment. No signal was detected over adjacent tissues or control sections. Some similarities exist between the temporal patterns of lamprin expression and the expression of matrix proteins such as elastin and collagen of higher vertebrates. It is concluded that certain aspects of chondrogenesis are critical to the normal development of a functional cartilaginous matrix and are conserved throughout the vertebrate taxa.

Identification and characterization of a serpin with differential expression during the life cycle of the sea lamprey.

We have cloned a member of the serine proteinase inhibitor gene superfamily from the sea lamprey, Petromyzon marinus. The predicted translation product contains a putative signal peptide and mRNA expression is localized mainly to the liver. Northern blot analysis indicates that the mRNA increases in the larvae and peaks in late larval life. At the onset of metamorphosis there is a approximately 10-fold drop after which it remains low. These changes correspond with levels of circulating thyroid hormone suggesting that this serpin is involved in or regulated by molecular signals that induce metamorphosis in the lamprey. Use of alignments and structural information from other serpins indicates that the lamprey serpin has the potential to be inhibitory. In addition the lamprey serpin contains methionine and serine at the P1 and P1' positions, respectively. Appropriate residues at positions important in allowing the insertion of strand s4A into beta-sheet A that occurs upon cleavage in inhibitory serpins are also found in the lamprey serpin.

Fetal tibial bone healing in utero: the effects of miniplate fixation.

Although clinical and experimental findings have demonstrated that fetal soft-tissue wounds heal without scarring, very little is known about the process of fetal bone healing. This study examined fetal long bone healing in utero, both histologically and biochemically, with and without fracture fixation in a fetal sheep model. Our study group consisted of 25 live fetuses (from 16 ewes). There were 50 fetal tibias in this group; 12 were control, 17 were fixed (miniplate fixation), and 21 were nonfixed. A midshaft osteotomy of the tibia, either fixed or non-fixed, was performed on fetal sheep at 95 days' gestation (term = 145 days) in utero. The sheep were then killed at one of five postoperative time intervals (weeks 1, 2, 3, 4, and 7), and fetal bone healing was examined. The variables reviewed included gross morphology, histology, radiology, and collagen analysis (proportions of types II to I and III to I collagen). Fetal bone healing without fixation was accompanied by a large callus with rapid and abundant cartilage and collagen deposition. Bone healing was characterized by malunion or nonunion at 7 weeks. However, with miniplate and screw fixation, callus formation was minimal; primary bone healing occurred by 3 weeks and did not adversely affect long bone growth. Analysis of callus samples revealed a minimal amount of type III collagen, whereas the proportion of type II collagen was variable and proportional to the content of callus cartilage.

Detection of lamprin mRNA in the anadromous sea lamprey using in situ hybridization.

An optimal in situ hybridization protocol is described for the detection of gene expression of a structural protein unique to lampreys, lamprin, in the cartilages of prolarval, metamorphic and adult sea lamprey, Petromyzon marinus. A 156 bp antisense RNA probe labeled with (35)S-UTP was transcribed in vitro from a recombinant plasmid containing a cDNA insert homologous to the largest (1.8 kb) of three known mRNAs for the lamprin gene and hybridized to 6 mu m paraffin sections. Optimal signal to noise ratio was achieved by fixing tissues 30 min in 4% paraformaldehyde and prehybridizing with a probe incorporating a nonradioactive S-UTP. Strong signals were visualized in all cartilaginous elements of the lamprey neurocranium; however, lamprin mRNA transcripts were not detected in branchial and pericardial cartilages suggesting differential expression of the lamprin gene. No signals were observed in tissue sections that had been treated with RNase A prior to hybridization or in sections hybridized with sense RNA probes. This technique has great potential for use in studies of the spatial and temporal distribution of cartilaginous components during developmental stages of lampreys.

Biochemical analysis of normal articular cartilage in horses.

Articular cartilage specimens from the distal articular surface of 32 radiocarpal bones from 24 2- to 5-year-old horses were analyzed. The total collagen content was determined on the basis of the 4-hydroxyproline content, using a colorimetric method. A method for estimating the proportions of types-I and -II collagen by measuring spectrophotometric densities of specific cyanogen bromide peptide bands from mixtures of types-I and -II collagen on sodium dodecyl sulfate-polyacrylamide gels was used. The cyanogen bromide peptides representative of each collagen types-I and -II were identified. The peptide ratios were then computed for each of several standards of type-I and -II mixtures. A standard curve was derived from the correlation between these ratios and the corresponding proportions of type-II collagen in standard mixtures. Galactosamine and glucosamine content (hexosamines) were measured by ion chromatography. The galactosamine-to-glucosamine ratio, chondroitin sulfate and keratan sulfate values, and total glycosaminoglycan content were derived from the measured hexosamine content. The total collagen content averaged 556 mg/g (55.6 mg/100 mg) of tissue (dry weight, [dw]). Type-II collagen was the major collagen type in normal articular cartilage specimens. The ratio of the area under the alpha 1 (II)CB10 peak to the area under the alpha 1 (I)CB 7,8 + alpha 1 (II)CB11 peak was a second-order polynomial function of the proportion of type-II collagen in the specimens. The mean galactosamine and glucosamine content were 20.6 mg/g and 7.9 mg/g (dw), respectively. The mean galactosamine-to-glucosamine ratio was 3.74 +/- 0.62.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical study of repair of induced osteochondral defects of the distal portion of the radial carpal bone in horses by use of periosteal autografts.

Periosteal autografts were used for repair of large osteochondral defects in 10 horses aged 2 to 3 years old. In each horse, osteochondral defects measuring 1.0 x 1.0 cm2 were induced bilaterally on the distal articular surface of each radial carpal bone. Control and experimental defects were drilled. Periosteum was harvested from the proximal portion of the tibia and was glued into the principal defects, using a fibrin adhesive. Control defects were glued, but were not grafted. Sixteen weeks after the grafting procedure, the quality of the repair tissue of control and grafted defects was assessed biochemically. Total collagen content and the proportion of type-II collagen were determined. Galactosamine and glucosamine contents also were determined. From these measurements, contents of chondroitin and keratan sulfate and total glycosaminoglycan, and galactosamine-to-glucosamine ratio were calculated. All biochemical variables were compared with those of normal equine articular cartilage taken from the same site in another group of clinically normal horses. Total collagen content was determined on the basis of 4-hydroxyproline content, using a colorimetric method. The proportions of collagen types I and II in the repair tissue were assessed by electrophoresis of their cyanogen bromide-cleaved peptides on sodium dodecyl sulfate slab gels. Peptide ratios were computed and compared with those of standard mixtures of type-I and type-II collagens. Galactosamine and glucosamine contents were determined by use of ion chromatography. In general, the biochemical composition of repair tissue of grafted and nongrafted defects was similar, but clearly differed from that of normal articular cartilage.(ABSTRACT TRUNCATED AT 250 WORDS)