Tensile forces attenuate estrogen-stimulated collagen synthesis in the ACL

The purpose of this study was to examine whether mechanical tensile forces affect estrogen regulation of collagen synthesis of anterior cruciate ligament fibroblasts at the mRNA level. Estrogen was studied at three physiologic levels, 10(-11), 10(-10), and 10(-9)M. The results revealed that estrogen alone stimulated Type I and III collagen synthesis at the mRNA level, and application of mechanical force decreased the expression of collagen Type I and III genes at all tested estrogen levels. These findings suggest that estrogen may directly regulate ligament structure and function by alteration of Type I and III collagen synthesis. This regulation is dependent on mechanical loading.     

Upregulation of HSP47 and collagen type III in the dermal fibrotic disease, keloid

Keloid is a dermal fibrotic disease characterized by excessive accumulation of mainly type I collagen in extracellular matrix of the dermis. We have studied the expression levels of collagen types I and III, and its molecular chaperone HSP47 in keloid lesions and surrounding unaffected skin using Northern and Western blotting and immunohistochemical analyses. Collagen types I and III mRNA levels were found to be upregulated 20-fold in keloid tissues, contradicting previous reports of nearly normal type III collagen levels in this disease. HSP47 expression in keloid lesions was also highly upregulated; eightfold at mRNA level and more than 16-fold at the protein level. Strong upregulation of these three proteins in keloid was confirmed by immunohistochemical staining. These results suggest that accumulation of both type I and type III collagen is important for the development of keloid lesions, and that HSP47 plays a role in the rapid and extensive synthesis of collagen in keloid tissues.     

Thermodynamic characteristics of collagen fibrils reconstructed in vitro at different temperatures and concentrations

The results of a calorimetric study of type I collagen fibrillogenesis were analyzed. The dependence of the half-width of the temperature transition of a collagen solution on the concentration and temperature of collagen formation was studied. It was demonstrated that, by varying temperature and collagen concentration, one can regulate the density of packing and dimensions of cooperative fibril blocks. At temperatures below the physiological level (25 degrees C and 30 degrees C), and a relatively low concentration of collagen (0.3 mg/ml), fibrils with the lowest density of packing are formed. The degree of order does not change as the collagen concentration increases twofold but grows as the concentration increases fourfold. It was shown that, at the physiological temperature (35 degrees C), fibrils with a dense packing of molecules are formed at all collagen concentrations studied. The value of fibril formation enthalpy is minimal at a temperature of 35 degrees C, pH 7.2, an ionic strength of 0.17 M and a concentration of 1.2 mg/ml. Based on the results obtained, a conclusion was made that the packing density of fibrils formed at physiological temperature does not depend on collagen concentration over the concentration range of 0.3 - 1.2 mg/ml.     

Rapid and reversible regulation of collagen XII expression by changes in tensile stress

We studied the expression of the fibril-associated collagen XII by fibroblasts cultured on attached (stretched) or floating (relaxed) collagen I gels. Accumulation of collagen XII in the medium as determined by semiquantitative immunoblotting was 8-16 times higher under stretched compared to relaxed conditions. Northern blot experiments showed that tensile stress controls collagen XII expression at the mRNA level. Tenascin-C mRNA levels were also influenced, whereas relative amounts of fibronectin and matrix metalloproteinase-2 mRNA were barely affected. The response to a change in tensile stress is rapid, since de novo biosynthesis of collagen XII was fully down-regulated 12 h after relaxation of a stretched culture. To demonstrate that the effect is also reversible, we mounted collagen gels with attached cells to movable polyethylene plugs. The cultures were relaxed or stretched at intervals of 24 and 48 h, and media samples were analyzed every 24 h. By ELISA, the amount of collagen XII secreted into the medium was found to increase or decrease in accordance with the tensile stress applied. This is evidence that the mechanical stimulus per se, rather than an indirect secondary effect, was responsible for the observed changes in collagen XII production.     

Selective inhibition of cysteine proteinases by Z-Phe-AlaCH2F suppresses digestion of collagen by fibroblasts and osteoclasts

Effects of the selective inhibitor of cathepsins B and L, Z-Phe-AlaCH2F were studied on the degradation of fibrillar collagen by fibroblasts and osteoclasts in cultured rabbit calvariae at the electron microscopic level. Periosteal fibroblasts from inhibitor-treated explants showed a dose-dependent increase of the volume fraction of vacuoles containing cross-banded collagen fibrils. This was a 7-fold increase over control fibroblasts and the ratio of intracellular and extracellular collagen increased from 2 to 43. The presence of collagen-containing vacuoles was also found in some osteoclasts from inhibitor-treated explants (1 microM or more). The inhibitor appeared to have cytotoxic effects at a concentration of 100 microM. It was concluded that this selective inhibitor exerts its effects intralysosomally in living cells, indicating possibilities for in vivo inhibition of protein degradation.     

Distribution of type III collagen in the pulp parenchyma of the human developing tooth

Summary The distribution of collagen type III throughout the pulp tissue from human developing tooth was studied using specific antibodies, immunofluorescence as well as immuno-peroxidase labelling for electron microscopy.Our results indicate that type III and type I collagen are present in the pulp. The staining intensity seems to correlate with the relatively high proportions of type III collagen biochemically found in pulp. In addition, type III collagen and reticulin fibres are similarly distributed, except that the Von Korff fibres were never detected with anti-type III collagen antibodies. Correspondingly, at the ultrastructural level, type III collagen appears as fine, branched filaments or electron dense material distributed throughout the tissue and particularly in close association with the plasma membrane of pulp fibroblasts. In contrast, type I collagen appears as typical coarse cross banded fibres.     

Stimulating effect of growth hormone on type IV collagen production by endothelial cells cultured in normal and high glucose

Collagen IV accumulation is characteristic of diabetic angiopathy. To test the possible contribution of GH, we studied its effects on collagen IV production by human umbilical vein endothelial cells at 5.5 and 16.7 mmol/l glucose. GH (100 ng/ml) markedly increased collagen IV level in the culture supernatant and in the insoluble extracellular matrix and cell fraction at both glucose concentrations. This stimulating effect of GH was additional to that of high glucose. It was more pronounced on collagen IV than on total protein synthesis. GH increased free latent gelatinase activity slightly at normal and markedly at high glucose. Using GF109203X, a PKC inhibitor, we observed that high glucose, but not GH, activated PKC. These two factors stimulating collagen IV production appear to work through different pathways, favoring an additivity of their effects. This supports the contribution of high plasma GH in diabetic vascular basement membrane thickening.     

Ultrastructural features of adult human tendon

Summary A variety of human tendons have been studied at the electron microscope level. The fibers of these tendons are composed of collagen fibrils that average 1,750 Å and 600 Å in diameter. A third population that measures 100 Å in diameter may represent immature collagen or filaments that are incorporated into tendon elastic fibers. The larger collagen fibrils vary in ratio with respect to one another, and are connected by interfibrillar bridges which in some cases appear to extend through the substance of the fibril. The collagen fibrils of the paratenon are less-well organized than those of the tendon proper and average 600 Å in diameter. Tendons that exhibit the property of lateral stretch (plantaris and palmaris) were compared at the ultrastructural level with tendons that do not have this property. No differences between the two tendon types could be determined in normal or spread preparations, indicating that the differences in physical characteristics are a result of fiber rather than fibril organization.Supported by Edward G. Schlieder Foundation GrantThe authors wish to thank Mrs. Janell Buck and Mrs. Eunice Schwartz for their excellent technical and secretarial assistance, and Mr. Garbis Kerimian for his excellent photographic work     

Distribution of type III collagen in the pulp parenchyma of the human developing tooth. Light and electron microscope immunotyping

The distribution of collagen type III throughout the pulp tissue from human developing tooth was studied using specific antibodies, immuno-fluorescence as well as immuno-peroxidase labelling for electron microscopy. Our results indicate that type III and type I collagen are present in the pulp. The staining intensity seems to correlate with the relatively high proportions of type III collagen biochemically found in pulp. In addition, type III collagen and reticulin fibres are similarly distributed, except that the Von Korff fibres were never detected with anti-type III collagen antibodies. Correspondingly, at the ultrastructural level, type III collagen appears as fine, branched filaments or electron dense material distributed throughout the tissue and particularly in close association with the plasma membrane of pulp fibroblasts. In contrast, type I collagen appears as typical coarse cross banded fibres.     

Biosynthesis and secretion of collagen in normal and SV-40 virus-transformed human embryonal fibroblasts

The biosynthesis and secretion of collagen proteins was studied in cultures of normal human embryo fibroblasts at different passages and growth stages as well as in cultures of human embryo fibroblasts transformed by oncogenic virus SV-40. It was found that normal fibroblasts maintain at a constant level the collagen synthesis throughout 20 passages, which is typical of proliferating and resting cells. Virus-transformed cells produce 3-4 times less collagen proteins on a per cell count. Normal and transformed fibroblasts do not differ in terms of total protein synthesis. Secretion of collagen and non-collagen proteins in transformed cell cultures appeared to be much lower than in normal cell cultures. Study of synthesized proteins by polyacrylamide gel electrophoresis showed that both types of cells secrete collagen proteins predominantly as polymers containing interchain S-S bonds of 3-helix molecules. Study of the protein-synthesizing activity of two polysomal fractions, i.e. membrane bound and free polysomes, isolated from the cells of both types in a cell-free system showed that membrane-bound polysomes from transformed fibroblasts synthesize collagen much less actively in comparison with normal cells. However, in transformed cells free polysomes, in contrast with normal cells, are active participants of a cell-free collagen protein synthesis.     

Collagen denaturation in spread monolayers at the air-water interface: Experiments and a possible model of the process

Spread monolayers of the fibril protein collagen were studied at the air-water interface in the presence of denaturants, urea and thiourea. The most prominent feature of spread collagen monolayers at the air-water interface is the ability to form supramolecular structures (fibrils), which themselves can form monolayers with collapse points of their own. The surface pressure isotherms of collagen monolayers have two “quasi-linear” centers, which are separated by a plateau and correspond to liquid-expanded and liquid-condensed states; this unique capability makes collagen different from other proteins. When in monolayer, collagen acquires the same level of structural organization as in the bulk. In the presence of denaturants, subphase characteristics of collagen monolayers change rapidly and irreversibly. Thiourea exerts more pronounced denaturing action on collagen monolayers than urea; this effect increases with exposure time and denaturant concentration. A hypothetical mechanism of thiourea-induced denaturation of fibril proteins is proposed according to which interactions between hydrophobic C=S groups of thiourea and nonpolar surface groups of the protein lead to reorientation of carbonyl groups to formation of intrinsic hydrogen bonds with NH₂-groups of thiourea eventually resulting in the rupture of intrinsic hydrogen bonds and denaturation of the protein.     

Discoidin Domain Receptor 1 Protein Is a Novel Modulator of Megakaryocyte-Collagen Interactions

Growing evidence demonstrates that extracellular matrices regulate many aspects of megakaryocyte (MK) development; however, among the different extracellular matrix receptors, integrin α2β1 and glycoprotein VI are the only collagen receptors studied in platelets and MKs. In this study, we demonstrate the expression of the novel collagen receptor discoidin domain receptor 1 (DDR1) by human MKs at both mRNA and protein levels and provide evidence of DDR1 involvement in the regulation of MK motility on type I collagen through a mechanism based on the activity of SHP1 phosphatase and spleen tyrosine kinase (Syk). Specifically, we demonstrated that inhibition of DDR1 binding to type I collagen, preserving the engagement of the other collagen receptors, glycoprotein VI, α2β1, and LAIR-1, determines a decrease in MK migration due to the reduction in SHP1 phosphatase activity and consequent increase in the phosphorylation level of its main substrate Syk. Consistently, inhibition of Syk activity restored MK migration on type I collagen. In conclusion, we report the expression and function of a novel collagen receptor on human MKs, and we point out that an increasing level of complexity is necessary to better understand MK-collagen interactions in the bone marrow environment.     

Collagen types I, III, and V in human embryonic and fetal skin

The dermis of human skin develops embryonically from lateral plate mesoderm and is established in an adult-like pattern by the end of the first trimester of gestation. In this study the structure, biochemistry, and immunocytochemistry of collagenous matrix in embryonic and fetal dermis during the period of 5 to 26 weeks of gestation was investigated. The dermis at five weeks contains fine, individual collagen fibrils draped over the surfaces of mesenchymal cells. With increasing age, collagen matrix increases in abundance in the extracellular space. The size of fibril diameters increases, and greater numbers of fibrils associate into fiber bundles. By 15 weeks, papillary and reticular regions are recognized. Larger-diameter fibrils, larger fibers, denser accumulations of collagen, and fewer cells distinguish the deeper reticular region from the finer, more cellular papillary region located beneath the epidermis. The distribution of collagen types I, III, and V were studied at the light microscope level by immunoperoxidase staining and at the ultrastructural level by transmission (TEM) and scanning electron microscopy (SEM) with immunogold labeling. By immunoperoxidase, types I and III were found to be evenly distributed, regardless of fetal age, throughout the dermal and subdermal connective tissue with an intensification of staining at the dermal-epidermal junction (DEJ). Staining for types III and V collagen was concentrated around blood vessels. Type V collagen was also localized in basal and periderm cells of the epidermis. By immuno-SEM, types I and III were found associated with collagen fibrils, and type V was localized to dermal cell surfaces and to a more limited extent with fibrils. The results of biochemical analyses for relative amounts of types I, III, and V collagen in fetal skin extracts were consistent with immunoperoxidase data. Type I collagen was 70-75%, type III collagen was 18-21%, and type V was 6-8% of the total of these collagens at all gestational ages tested, compared to 85-90% type I, 8-11% type III, and 2-4% type V in adult skin. The enrichment of both types III and V collagen in fetal skin may reflect in part the proportion of vessel- and nerve-associated collagen versus dermal fibrillar collagen. The accumulation of dermal fibrillar collagen with increasing age would enhance the estimated proportion of type I collagen, even though the ratios of type III to I in dermal collagen fibrils may be similar at all ages.     

Changes in collagen metabolism during the chronic electrical stimulation of the midbrain reticular formation

The content of 11-hydroxycorticosteroids, free and bound hydroxyproline in the peripheral blood, as well as the level of free and net hydroxyproline in the aortic wall and myocardium of rabbits were studied in chronic prolonged electrical stimulation of the mesencephalon reticular system. The experiments have shown that electrical stimulation leads to the activation of adrenal cortex function and is accompanied by alterations in collagen metabolism, accumulation of collagen in the aorta and decreased collagen level in the myocardium.