Constituents Collagen Research Articles

Immunophenotyping of congenital myopathies: disorganization of sarcomeric, cytoskeletal and extracellular matrix proteins

We have studied the expression and distribution patterns of the intermediate filament proteins desmin and vimentin, the sarcomere components titin, nebulin and myosin, the basement membrane constituents collagen type IV and laminin, and the reticular layer component collagen type VI in skeletal muscle of patients with “classic” congenital myopathies (CM), using indirect immunofluorescence assays. In all biopsy specimens obtained from patients with central core disease (CCD), nemaline myopathy (NM), X-linked myotubular myopathy (XLMTM) and centronuclear myopathy (CNM), disease-specific desmin disturbances were observed. Vimentin was present in immature fibres in severe neonatal NM, and as sarcoplasmic aggregates in one case of CNM, while the amounts of vimentin and embryonic myosin, observed in XLMTM, decreased with age of the patients. Abnormal expression of myosin isoforms was found in several CM biopsies, although the organization of myosin and other sarcomere components was rarely disturbed. Basement membrane and reticular layer proteins were often prominently increased in severe cases of CM. We conclude that (i) desmin is a marker for individual types of CM and might be used for diagnostic purposes; (ii) the expression patterns of the differentiation markers desmin, vimentin and embryonic myosin in XLMTM, point either to a postnatal muscle fibre maturation or to a variable time-point of maturational arrest in individual patients; (iii) the correlation between the distribution patterns of extracellular matrix proteins and clinical presentation points to a role of these proteins in pathophysiology of CM.

Autogenous callo-osseous grafts for the repair of osteochondral defects

We describe a new method of biological repair of osteochondral defects. In rabbit knees an osteochondral defect was reconstructed with a callo-osseous graft made of a superficial sheet of medullary fracture callus attached to a base of cancellous bone. This was taken from the iliac bone of the same animal which had been osteotomised ten days earlier. The reparative tissues were evaluated for 24 weeks by quantitative histology, biochemical analysis of the uronic acid content, and immunohistochemical staining of collagen constituents. The callo-osseous graft provided significantly faster and better repair of the articular surface than an untreated defect or a callo-osseous graft in which the cells had been devitalised by irradiation before transplantation. Our findings indicate that the callo-osseous graft contributes to the repair process by providing both favourable extracellular matrices and pluripotential mesenchymal cells. Our study tested the hypothesis that early medullary callus generates hyaline cartilage instead of bone after transfer to an articular surface.

Targeting Platelets Containing Electro-encapsulated lloprost to Balloon Injured Aorta in Rats

Drugs can be electro-encapsulated within platelets and targeted to damaged blood vessels by exploiting the platelet's natural haemostatic properties to adhere to collagen and other vessel wall constituents revealed by injury. A rat aorta balloon angioplasty model has been used to study the effect on platelet deposition of giving iloprost loaded platelets i.v. during the balloon injury. After labelling the circulating platelets with 111-Indium before balloon injury, time course studies showed maximum platelet deposition on the injured aorta occurred at about 1 h post-injury and the deposition remained stable over the next 2-3 h. When iloprost-loaded platelets were given i.v. during injury and the circulating platelet pool labelled with 111-Indium 30 min later, platelet deposition, measured at 2 h postinjury, was substantially and significantly reduced compared with control platelet treatment. Some anti-proliferative effects of iloprost-loaded platelets given i.v. during injury have also been observed. Whereas the incorporation of [3H]-thymidine into aorta intima-media DNA at 3 days post injury was 62-fold higher in balloon injured rats than in control sham operated rats, thymidine incorporation into intima/media of rats which had received iloprost loaded platelets during injury was reduced as compared with rats subjected only to the injury procedure. The reduction was only of near significance, however, but at 14 days after injury the total DNA content of the aorta intima/media of rats given iloprost loaded platelets during injury was significantly reduced. Although iloprost loaded platelets can clearly inhibit excessive platelet deposition, other encapsulated agents may have greater anti-proliferative effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary excretion of pyridinolines crosslinks measured by immunoassay and HPLC techniques in normal subjects and in elderly patients with vitamin D deficiency

Hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are specific constituents of mature skeletal collagens excreted in urine. Their measurement represents a sensitive index of bone resorption. In this study, we have measured urinary excretion of pyridinolines crosslinks by immunoassay (ELISA) and HPLC methods in 80 patients with different bone resorption rates. We chose a sample of 44 healthy adults (30 men and 14 women) and a sample of 36 elderly patients (7 men and 29 women) presenting a secondary hyperparathyroidism due to a vitamin D deficiency. The correlation between HPLC ( x) and ELISA ( y) was judged satisfactory ( y=0.794 x+6.947, r=0.92). The sensitivity of pyridinolines estimation was 50 nmol/1 for immunoassay and 20 nmol/l for HPLC. The intra-assay and inter-assay coefficients of variation for the two analytical methods was <10%. The mean excretion of crosslinks (nmol/mmol of creatinine) measured by both methods in the sample of healthy adults was higher in women than in men. The amount of pyridinolines crosslinks excreted by elderly patients with vitamin D deficiency are three time higher than those of normal adults when measured by ELISA and HPLC methods. The distribution of different molecular forms of urinary pyridinoline crosslinks was investigated. Values of pyridinolines measured by HPLC in our samples of elderly patients have shown that free and peptide-bound pyridinolines with molecular weight (mol. wt.) smaller than 1000 Da represent approximately 80% of the total pyridinolines contained in urinary samples. A study on the evaluation of the antiserum used in the immunoassay for reacting with the different molecular forms isolated from urine showed a high affinity for free and peptide-bound pyridinolines with molecular weight smaller than 10000 Da and that do not react strongly with peptide-bound with molecular weight greater than 10000 Da. We conclude that, although this immunoassay does not measure total pyridinolines and does not distinguish between HP and LP, it seems convenient for diagnostic of metabolic bone diseases.

Thermal stability of cortical bone collagen in relation to age in normal individuals and in individuals with osteopetrosis

The thermal stability of cortical bone collagen was determined in iliac crest biopsies obtained from 41 normal individuals (21 women aged 20–96 years and 20 men aged 21–84 years) and 8 individuals with autosomal dominant osteopetrosis type I (4 women and 4 men aged 17–63 years). The cortical bone was decalcified and the bone matrix was cut into 80-μm-thick freeze sections parallel to the bone surface. Circular specimens punched out from the sections were used for determination of area shrinkage during gradual heating and shrinkage temperature, T s (representing the temperature for 50% of the area shrinkage). In normal men, T s was not found to decrease until the age of 60–65 years, but was markedly decreased in the elderly individuals. In normal women, T s varied considerably throughout the age range tested, without relationship to age. In contrast to agematched controls, T s decreased with age in men with osteopetrosis, whereas T s in affected women was neither related to age nor different from the highly variable values found in age-matched normal women. Previous findings in rats indicate that T s decreases with the chronological age of the bone collagen. The present results agree with these findings, which imply that a reduction in turnover rate of bone results in an increasing age and a reduced T s of the constituent collagen. Following this assumption, the turnover rate of bone seems to be more variable hi women than in men and reduced in osteopetrotic men. A T s gradient which was demonstrated through the cortical lamina in the osteopetrotic individuals may reflect the presence of older, unexchanged collagen, which is superimposed younger collagen. The present results demonstrate that the defective cortical bone resorption characteristic of osteopetrosis is accompanied by an accumulation with age of cortical bone collagen with reduced T s . The reduction in T s indicates that osteopetrosis implies an increasing proportion of abnormally aged cortical bone collagen.

Scattering of Light from Histologic Sections: A New Method for the Analysis of Connective Tissue

We have developed a light scattering technique that can be used to analyze the orientation and diameter of collagen fibers in histologic sections of connective tissue. Scattering patterns obtained by transmitting laser light through sections of tissue contain information both on the orientation, degree of alignment, and size of the constituent collagen fibers. Analysis of the azimuthal intensity distribution of scattered light yields numerical values of the degree of alignment by use of an orientation index, S, which is chosen to vary between 0 for randomly oriented fibers and 1 for a perfectly aligned arrangement. The average diameter of the collagen fibers is calculated from the scattering angle at which the intensity reaches its first minimum. These measurements are independent of the nature of histologic stain. The procedure is illustrated by measurements obtained with sections of the guinea pig dermis and of control scar. We conclude from our experiments that light scattering can complement the analysis of tissue architecture typically performed with the light microscope.

In vitro endothelialization of commercially available heart valve bioprostheses with cultured adult human cells

The possibility of improving the performance of heart valve bioprostheses by preendothelialization with autologous cells has been suggested. In this study we used cultured adult human vein endothelial cells to endothelialize cusps isolated from commercially available porcine valve bioprostheses. We also describe a method for primary endothelialization of the intact heart valve bioprosthesis. After detoxification of the glutaraldehyde, the cusps or valves were seeded at high density with cultured cells. To obtain an even distribution on the intact valve bioprosthesis, a device was designed which permits application of cells from different directions during rotation. Evaluation was performed by hematoxylin-eosin staining, scanning electron microscopy and immunohistochemistry of the von Willebrand factor and the human basement membrane constituent collagen IV. The endothelial cells were vital-stained during culture by the addition to the culture medium of carbocyanine dye. This made it possible to verify that the endothelium was derived from culture. A confluent lining of cultured endothelial cells in close proximity to a de novo formed basement membrane was observed on the isolated cusps 7 days after seeding. The intact heart valve bioprosthesis showed an even distribution of seeded cells with areas of cells spreading to confluency as evaluated after 24 h. However, 7 days after seeding only unspread and probably dead cells were observed.

Compositional analysis of the collagenous bone matrix. A study on adult normal and osteopenic bone tissue.

The collagenous constituents of mature bone of 30 individuals 22-93 years of age were studied by post-mortem morphological and biochemical analysis. Morphometric evaluation of the second lumbar vertebral body revealed striking interindividual differences in bone mass, mean trabecular density and mean trabecular thickness. Collagen extracted from vertebral trabecular bone by limited pepsin digestion consisted mainly of collagen I (92%) and collagen V (8%). Immunohistochemistry revealed a distinct distribution of these two collagen types within the bone matrix. The degree of lysyl hydroxylation of the alpha 2(I) collagen chain correlated inversely with the trabecular bone volume (TBV) and with the mean trabecular plate density. This correlation was statistically significant for the entire study group as well as for the female and male subgroups. Within the female subgroup, the lysyl hydroxylation/TBV ratio was higher in postmenopausal than in premenopausal women and was highest in women with established osteoporosis. No significant correlation was found between the level of lysyl hydroxylation and the age of the patients. The alpha 1(I) collagen chain showed a nearly constant degree of lysyl hydroxylation in all 30 samples. The results provide convincing evidence that morphometric changes associated with osteopenia in adult bone are accompanied by an altered level of lysyl hydroxylation of the alpha 2(I)-chain of collagen I. The biochemical alterations observed may be responsible for the deposition of a deficient bone matrix in osteopenic conditions.

Microscopical investigation of canine anterior cruciate ligament and patellar tendon: collagen fascicle morphology and architecture.

The collagen structure of the canine anterior cruciate ligament (ACL) and patellar tendon (PT) was examined by using light and scanning electron microscopy. The collagen waviness known as a crimping was found to occur in ACL and PT fascicles. This waviness, seen at the periphery of fascicles, is very smooth, and its amplitude seems to decrease from the periphery toward the fascicular center. It appears as a periodic collapse of the fascicle in two dimensions. Two models of the architectural patterns of the ACL and PT wavy fascicles are presented. The constituent collagen fibrils are either parallel or twisted relative to the fascicle axis, giving rise to planar and helical wave patterns, respectively. There is a distinct difference between the ACL and PT collagen structure. The helical wave pattern occurs in both PT and ACL while the planar waveform is found only in the centrally located ACL fascicles. In addition, there is less variability in fascicular size and density over the PT cross-section than in ACL.

Distribution of isoaccepting tRNAs and codons for proline and glycine in collagenous and noncollagenous chicken tissues

The relation between codon usage and tRNA content for proline and glycine, the major constituents of collagen, was studied in two tissues: the magnum of laying hen oviduct and the leg tendons of chick embryo where collagen is produced. Although the relative contents of tRNA GCC Gly and tRNA IGG Pro in tendons, as compared to magnum indicate a specialization of the tRNA population for collagen synthesis, the distribution of the preponderant codons in collagen mRNA is correlated but at a lesser extent to that of their cognate tRNAs.

Submucosal collagen changes in the normal colon and in diverticular disease.

Full thickness specimens of normal colon (n = 15), and colon from patients with diverticular disease (n = 5) were obtained at operation or autopsy. In the isolated submucosa the ultrastructure of the constituent collagen fibres was examined by transmission electron microscopy. Collagen fibrils in the left colon become smaller (p less than 0.001) and more tightly packed (p less than 0.001) than those in the right colon with increasing age. This difference is accentuated in diverticular disease (p less than 0.01). Factors which contribute to the development of colonic diverticulosis, such as raised intraluminal pressure, may be responsible for premature change in submucosal structure.

Distinct molecular interactions mediate neuronal process outgrowth on non-neuronal cell surfaces and extracellular matrices.

We have compared neurite outgrowth on extracellular matrix (ECM) constituents to outgrowth on glial and muscle cell surfaces. Embryonic chick ciliary ganglion (CG) neurons regenerate neurites rapidly on surfaces coated with laminin (LN), fibronectin (FN), conditioned media (CM) from several non-neuronal cell types that secrete LN, and on intact extracellular matrices. Neurite outgrowth on all of these substrates is blocked by two monoclonal antibodies, CSAT and JG22, that prevent the adhesion of many cells, including neurons, to the ECM constituents LN, FN, and collagen. Neurite outgrowth is inhibited even on mixed LN/poly-D-lysine substrates where neuronal attachment is independent of LN. Therefore, neuronal process outgrowth on extracellular matrices requires the function of neuronal cell surface molecules recognized by these antibodies. The surfaces of cultured astrocytes, Schwann cells, and skeletal myotubes also promote rapid process outgrowth from CG neurons. Neurite outgrowth on these surfaces, though, is not prevented by CSAT or JG22 antibodies. In addition, antibodies to a LN/proteoglycan complex that block neurite outgrowth on several LN-containing CM factors and on an ECM extract failed to inhibit cell surface-stimulated neurite outgrowth. After extraction with a nonionic detergent, Schwann cells and myotubes continue to support rapid neurite outgrowth. However, the activity associated with the detergent insoluble residue is blocked by CSAT and JG22 antibodies. Detergent extraction of astrocytes, in contrast, removes all neurite-promoting activity. These results provide evidence for at least two types of neuronal interactions with cells that promote neurite outgrowth. One involves adhesive proteins present in the ECM and ECM receptors on neurons. The second is mediated through detergent-extractable macromolecules present on non-neuronal cell surfaces and different, uncharacterized receptor(s) on neurons. Schwann cells and skeletal myotubes appear to promote neurite outgrowth by both mechanisms.

Mechanical properties of collagen from decalcified rat femur in relation to age and in vitro maturation.

Cortical bone collagen was obtained by decalcifying femoral bones from 2-, 5-, 15-, and 25-month-old male rats. Collagen specimens were cut longitudinal to the long axis of the femur and tested mechanically. The maximum load (ultimate strength) and maximum slope of the load-strain curve (maximum stiffness) were found to decrease with age. The age-related reduction in the mechanical parameters resulted from a change in the mechanical strength of the constituent collagen and a change in the morphology of the bone tissue. In vitro aging, produced by incubating for 0-5 months, did not change the mechanical strength of bone collagen specimens obtained from 2-month-old rats. The changes in the mechanical characteristics of bone collagen, accompanying in vivo and in vitro aging are the opposite of those observed in soft tissue collagen. In the latter there is an increase in the mechanical strength during in vivo and in vitro aging.

High-Altitude Pulmonary Edema

To evaluate the cellular and biochemical composition of bronchoalveolar fluid in high-altitude pulmonary edema (HAPE), we performed bronchoalveolar lavage in three climbers with HAPE in a research facility at 4400 m on Mount McKinley. Three healthy climbers were used as controls. The HAPE fluids contained marked increases in high-molecular-weight proteins, erythrocytes, and leukocytes, most of which were alveolar macrophages. The HAPE fluids also contained detectable amounts of leukotriene B4 and other lipoxygenase products of arachidonic acid metabolism, complement fragments (C5a), inhibitors of neutrophil chemotaxis, and acid proteases but not hydroxyproline, a constituent of collagen. The data from this study indicate that HAPE involves a transient "large pore" leak in the pulmonary circulation. Despite the presence of two potent mediators of inflammation, leukotriene B4 and C5a, HAPE is not characterized by the intense neutrophil accumulation that is typical of other forms of acute lung injury.

The structure of rat tail tendon fascicles.

Evidence is presented to support the view that the surface of a fascicle reflects the wave pattern of its constituent collagen fibrils. The wave pattern shows considerable variation within and between fascicles. Smooth continuous phase shifts in crimp angles and crimp lengths across the width and along the length of a fascicle are seen as explaining many of the varied crimp patterns seen. It is proposed that collagen fibril packing is a simple side-by-side aggregation with a degree of freedom in any direction in the register of the wave patterns.

Step-by-step analysis of adhesion of human platelets to a collagen-coated surface defect in initial attachment and spreading of platelets in Von Willebrand's disease

Adhesion of platelets from the platelet-rich plasma (PRP) of patients with von Willebrand's disease (vWD) and healthy donors has been studied in a simple model system - wells of multiwell tissue culture plates coated with fibrillar calf skin collagen (CSC). This model is characterized by: (i) the presence of only one constituent of the vessel wall connective tissue matrix (collagen), (ii) the absence of surface-bound aggregates and thrombi, (iii) absence of overlapping of neighbouring spread platelets. A morphometric quantitation of adhesion by scanning electron microscopy (SEM) has been carried out. It allows to subdivide this process into three stages: 1) initial attachment of unspread platelets to the substrate, 2) platelet spreading on the substrate, and 3) attachment of unspread platelets to the upper surface of spread platelets. It was established that the PRP of vWD patients, compared to that of healthy donors, is characterized by a decreased total adhesion of platelets to a CSC-coated surface, which is manifested in the impairement of both the initial attachment and subsequent spreading of platelets. Addition of platelet-free plasma from healthy donors to the vWD PRP completely restores platelet spreading on collagen but little affects the initial attachment. These experiments performed on isolated collagen preparations provide further evidence for the initial attachment and spreading of platelets on collagenous constituents of the subendothelium being factor VIII/von Willebrand factor (FVIII/vWF)-dependent. In contrast to the adhesion on the collagen substrate, the adhesion of platelets from vWD PRP to a foreign surface, polystyrene plastic of uncoated wells, is the same as that of the normal PRP and, thus, FVIII/vWF-independent.

COLLAGEN POLYMORPHISM: TYPE V COLLAGEN IN NEUROFIBROMA

Three types of collagen were found in neurofibroma tumor tissue from a patient with neurofibromatosis (Von Recklinghausen's disease). Two of them were determined to be type I and type III collagen by CM-cellulose chromatography and SDS-polyacrylamide gel electrophoresis. The third matched a membrane type collagen from amnionic and chorionic membrane of human placenta in its elution profile during CM-cellulose chromatography and its electrophoretic patterns on SDS-polyacrylamide gel. This collagen was precipitated with 3.6-4.0 M NaCl at neutral pH after limited pepsin digestion. It was composed of two components with molecular weights of approximately 110K and 125K daltons. These collagenous constituents had a high content of hydroxylysine linked disaccharide units (glucosylgalactose) and were resistant to reduction. They were digested by bacterial collagenase (EC 3.4.24.3) but resisted tadpole collagenase (EC 3.4.24.7).

Ultrastructural aspects after cryoultramicrotomy of bone tissue and sutural cartilage in neonatal mice calvaria.

Using transmission electron microscopy after cryoultramicrotomy, mineralized as well as unmineralized bone tissues and sutural cartilage were observed in neonatal mice calvaria. A good definition of osteoblasts (nucleus, membranes, organelles) and extracellular constituents (collagen fibrils, matrix vesicles, mineral substance) was obtained. The sutural zone was composed of an unmineralized cartilaginous tissue with more or less hypertrophic cells surrounded by a finely fibrillar matrix.

Collagen of fibrocartilage: a distinctive molecular phenotype in bovine meniscus

Fibrocartilage of bovine knee meniscus was analyzed for major and minor collagen constituents. The main fraction (∼98%) of pepsin-solubilized collagen consisted of type I with a small proportion (< 10%) of type III molecules. The minor fraction (1–2%) isolated by salt precipitation could be further resolved into type V collagen that consisted of α1(V) and α2(V) chains and a type II-like molecule with chains that had all the characteristics of the 3α variant of α1(II) found in hyaline cartilage. The articular surface zone of the meniscus appeared 2–3-fold enriched in these minor collagens compared with deeper tissue, though qualitatively the same distinctive collagen phenotype was evident throughout.

An ultrastructural study of glycosaminoglycans associated with collagen and other constituents of the cat anulus fibrosus.

It has been confirmed that glycosaminoglycan is associated with collagen and other constituents of the anulus fibrosus. In cells, it appears to be present on the rough endoplasmic reticulum and is removed by hyaluronidase digestion and thus may consist of chondroitin sulphate. In pericellular material it appears as fibrils and granules, is unaffected by hyaluronidase and may consist of keratan sulphate or dermatan sulphate. Collagen fibrils contain hyaluronidase-digestable glycosaminoglycan (such as chondroitin sulphate) in the form of peripheral rings that appear as bands in longitudinal sections of the fibrils.