Collagen metabolism in mouse lung after X irradiation

Collagen and total protein synthesis rates have been determined in the lungs of CBA mice irradiated with single doses of X rays between 8 and 16 Gy. Mice were injected with [3H]proline accompanied by a large dose of unlabeled proline, and synthesis rates were measured at 2-month intervals from 8 to 31 weeks after irradiation. At 2 months after radiation treatment, collagen and total protein synthesis rates were significantly depressed but they had recovered by 4 months. By 6 months collagen synthesis rates had increased above control in a dose-dependent manner, so that in the 14-Gy dose group the fractional synthesis rate for collagen was 4.6 times higher than in control mice as measured by incorporation of [3H]proline. However, a significant net accumulation of collagen was seen only in the lungs of the highest dose group at 31 weeks, as indicated by total hydroxyproline measurements. There was a slight increase in the ratio of types I and III collagen. Late radiation damage in the CBA mouse lung is characterized by increased collagen metabolism, which may or may not lead to a net accumulation of collagen.     

Radiation-induced changes in collagen isotypes I, III, and IV in the lung of LAF1 mouse: effects of time, dose, and WR-2721

Alterations in the amount and distribution of pulmonary connective tissue are commonly observed subsequent to thoracic radiotherapy. The extent to which these changes are important in the expression of radiation damage and its repair remains unclear. We have quantitated changes in the parenchymal levels of collagen types I, III, and IV in the lungs of LAF1 mice at intervals to 1 year, following doses of 0-14 Gy, 300 kV X rays, or 0-18 Gy in the presence of the radioprotective compound, WR-2721. The method of quantitation, which involves video image analysis of fluorescent antibody stained, cryostat tissue sections, provides both quantitative and morphological information for the three collagen isotypes. Type I collagen peaked in tissue content at 15 and 30 weeks postirradiation (p.i.), with transient return to control values 20-25 weeks p.i. Type III collagen peaked at 15 and 25 weeks p.i. and declined in tissue content at 20 and 30 weeks. Type IV peaked 15-20 weeks following irradiation, returned to control levels at 25 weeks, and reached a plateau above control values after 30 weeks. Fluctuations in collagen levels in the parenchyma were dose dependent but were not simultaneous, indicating a radiation response characterized by alpha-chain-specific regulation of collagen biosynthesis and breakdown. In general, WR-2721, which enhanced postirradiation survival (DMF, 1.3), reduced the magnitude and altered the timing of collagen fluctuations; again, the effects were type specific. The results clearly demonstrate that the postirradiation response of the connective tissue is dose dependent, is specific to each macromolecule, and involves both deposition and removal of extracellular matrix. These processes are independently influenced by the presence during irradiation of WR-2721.     

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.     

The role of the tubulointerstitium in radiation-induced renal fibrosis

The functional and morphological response of the remaining hypertrophied kidney in unilaterally nephrectomized rats to single doses of 0-20 Gy X rays was investigated. Functional and histological end points were assessed serially 4-24 weeks postirradiation. Renal irradiation led to time- and dose-dependent reductions in renal function, seen in terms of a decreased glomerular filtration rate, increased blood urea nitrogen, and reduced hematocrit. These changes were accompanied by morphological changes in the glomerular, tubular and interstitial portions of the kidney. However, dose-dependent changes were observed only in terms of tubulointerstitial lesions. Significant increases in the degree of interstitial staining for collagen type III and fibronectin were observed 24 weeks postirradiation. These increases in extracellular matrix components were accompanied by a significant increase in interstitial alpha smooth muscle actin, suggesting activation of interstitial fibroblasts into myofibroblasts. There was no evidence of glomerular Tgfb after renal irradiation. A significant increase in tubular Tgfb staining was only seen 8 weeks postirradiation. In contrast, there was a shift of staining to the interstitium such that by 24 weeks postirradiation interstitial Tgfb staining was significantly greater than that seen in controls. These findings suggest that the tubule epithelial cell and the interstitial fibroblast are both active participants in the development and/or progression of radiation-induced renal fibrosis.     

Modulation of types I and III procollagen synthesis at various stages of arterial smooth muscle cell growth in vitro

Collagen synthesis was monitored in cultures of rabbit arterial smooth muscle cells (SMC). Both the rate of collagen synthesis per cell and collagen synthesis as a percent of total protein synthesis were measured at specific intervals from 1 to 14 days after inoculation of smooth muscle cells. The proportions of types I and III collagen present in the conditioned incubation medium and in the cell layer were also examined. After inoculation the cells displayed population expansion typical of SMC in which growth slowed but did not cease after the cells attained confluence. Collagen synthesis rates, expressed as [14C]hydroxyproline per cell, were eight-fold higher in preconfluent cells. In these cultures collagen accounted for more than 20% of the newly synthesized, 14C-labeled protein present as trichloroacetic acid (TCA)-insoluble material in 24 h culture media. In post-confluent cultures, this percentage was reduced to about 7% of the total protein synthesized. Synthesis rates of both collagen and non-collagen protein decreased with increasing time after inoculation. However, the rate of decline of collagen synthesis was three times greater than that seen for non-collagen protein. Early cultures synthesized relatively more type I than type III procollagen. The type I to type III ratio was highest at day 3 and declined after that time to day 14. While the synthesis of both types decreased with increasing age, type I declined at a greater rate resulting in a predominance of type III procollagen secretion by older cultures. We conclude that protein synthesis in general and collagen synthesis in particular are quantitatively and qualitatively dependent upon the growth stage of SMC in vitro.     

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.     

Developmental changes in collagen and elastin biosynthesis in the porcine aorta

Elastin and collagen are the principal scleroproteins of the aortic wall, and they largely determine its physical and mechanical properties. During perinatal development of the aorta, elastin and collagen accumulate rapidly, being present as inverse gradients by the time of birth. Elastin is most prevalent in the thoracic aorta, decreasing distally, while collagen shows the opposite trend. The present studies have determined the relative and absolute rates of collagen and elastin synthesis in the porcine aorta between 60 days of fetal development (mid-gestation) and 110 days after birth. Although there was measurable elastin synthesis in the upper thoracic aorta at the earliest time evaluated, there was a fourfold increase in relative elastin synthesis (from 4 to 16% of total protein synthesis) between 60 fetal days and birth. Elastin synthesis was maximal in successively distal segments between 1 and 3 weeks after birth. Relative collagen synthesis progressively increased in distal aortic regions between 90 fetal days and 60 days postpartum. Greater than twofold increases over thoracic levels were measured. Both elastin and collagen synthesis largely subsided by 110 days of development. When expressed as absolute rates of protein synthesis, these scleroproteins were maximally expressed in the first 3 postnatal weeks. Elastin mRNA levels were determined with a cloned sheep gene fragment by molecular hybridization. Gradients of elastin message were present at 60 fetal days and at 4 and 14 days after birth, elastin mRNA levels being maximal in the upper thoracic aorta at 14 days after birth. The differentiation of the aortic wall thus follows discrete patterns of phenotypic change which may be coupled to the rheologic stresses accompanying development of the circulatory system.     

Collagen metabolism in the regenerating forelimb of Notophthalmus viridescens: Synthesis, accumulation, and maturation

Collagen metabolism was studied in the regenerating forelimbs of adult newts (Notophthalmus viridescens) with respect to the pattern of accumulation relative to total protein accretion, maturation, and rate of biosynthesis. Measurements of collagen and noncollagen protein in regenerating limbs at various stages indicate that a preferential enrichment in collagen occurs at two periods correlating with (1) the onset of differentiation and chondrogenesis and (2) the initial period of elongation and outgrowth following morphogenesis. The maturation of collagen was determined by measuring the distribution of collagen into acetic acid soluble and insoluble forms. Soluble collagen increased to 30% during the differentiative period, remained at a high level during digit-formation, and decreased progressively following morphogenesis.Tracer studies were performed to determine whether the net accumulation of collagen resulted from a preferential increase in collagen biosynthesis. Separation of collagen and noncollagen proteins labeled in vivo with [³H]proline was performed enzymatically using purified clostridial collagenase. Rates of incorporation of proline into collagen relative to noncollagen proteins did not vary significantly during regeneration, although a threefold increase in incorporation rates into both species occurs at the onset of differentiation. Collagen synthesis constitutes 7–11% of the total protein synthesis in the regenerate. Estimates of variations in the absolute rates of protein synthesis, based on endogenous levels of proline, indicate that the highest rates of protein synthesis occur during morphogenesis. The relationship between protein content and relative rates of synthesis suggests that the net accumulation is governed by variations in rates of degradation. The relationship between collagen content and solubility also suggests that the rate of insolubilization plays a role in the net accumulation of collagen.     

Ascorbate independent differentiation of human chondrocytes in vitro: simultaneous expression of types I and × collagen and matrix mineralization

In this study we describe the collagen pattern synthesized by differentiating fetal human chondrocytes in vitro and correlate type X collagen synthesis with an intracellular increase of calcium and with matrix calcification. We show that type II collagen producing fetal human epiphyseal chondrocytes differentiate in suspension culture over agarose into hypertrophic cells in the absence of ascorbate, in contrast to chicken chondrocytes which have been shown to require ascorbate for hypertrophic differentiation. Analysis of the collagen synthesis by metabolic labeling and immunoprecipitation as well as by immunofluorescence double staining with anti type I, II or X collagen antibodies revealed that type X collagen synthesis was initiated during the third week. After 4 weeks culture over agarose we identified cells staining for both type I and X collagen, indicating further differentiation of chondrocytes to a new type of 'post-hypertrophic' cell. This cell type, descending from a type X collagen producing chondrocyte, is different from the previously described 'dedifferentiated' or 'modulated' types I and III collagen producing cell derived from a type II collagen producing chondrocyte. The appearance of type I collagen synthesis in agarose cultures was confirmed by metabolic labeling and immunoprecipitation and challenges the current view that the chondrocyte phenotype is stable in suspension cultures. An increase in the intracellular calcium concentration from 100 to 250 nM was measured about one week after onset of type X collagen synthesis. First calcium deposits were detected by alizarine red S staining in type X collagen positive cell nodules after 4 weeks, again in the absence of ascorbate. From these observations we conclude a sequence of events ultimately leading to matrix calcification in chondrocyte nodules in vitro that begins with chondrocyte hypertrophy and the initiation of type X collagen synthesis, followed by the increase of intracellular calcium, the deposition of calcium mineral, and finally by the onset of type I collagen synthesis.     

Effect of cardiac lymph flow obstruction on cardiac collagen synthesis and interstitial fibrosis

The effect of chronic cardiac lymphatic obstruction on the myocardial synthesis of collagen type I and III was investigated in a rabbit model. In the lymphatic obstruction group (n=16), plasma C-terminal propeptide type I procollagen (PICP) and N-terminal propeptide type III procollagen (PIIINP) were elevated at 7, 14 and 30 days after the operation (p<0.05). The elevated PICP and PIIINP returned to the pre-operation values 60 days after the operation. The myocardial expression of collagen type I and III mRNA were also enhanced in the lymphatic flow obstruction group. Plasma PICP, PIIINP and myocardial collagen type I and III mRNA remained unchanged in the control group (n=16). We concluded that chronic obstruction of cardiac lymph flow leads to enhanced myocardial collagen synthesis in rabbits. The enhanced collagen synthesis starts within seven days after lymphatic obstruction and subsides after 60 days.     

液相色谱/质谱联用法分析不同年龄鼠皮肤中I型、III型胶原蛋白相对含量

哺乳动物皮肤真皮中胶原蛋白含量约为70%,主要为是I型、III型胶原蛋白,本实验利用稀酸溶解和酶法提取了大鼠皮肤中的总胶原蛋白,将胶原蛋白粗提品在60℃变性后用胰蛋白酶进行降解,液相色谱/质谱联用法分析了两种胶原蛋白的特征多肽,利用特征多肽比较了不同生长期大鼠皮肤中I型和III型胶原蛋白相对含量。结果表明,大鼠皮肤中的III型胶原蛋白的相对含量随生长期延长逐渐降低,而I型胶原蛋白的相对含量逐渐升高,8周后两种胶原蛋白的比例趋于稳定。本实验结果表明使用高效液相色谱/质谱联用法分析组织中的胶原蛋白类型及其动态变化具有可行性,为更好的临床应用提供了实验基础。     

Characterization of type I,III and V collagens in high-density cultured tenocytes by triple-immunofluorescence technique

The purpose of this study is to examine the intracellular distribution of collagen types I, III and V in tenocytes using triple-label immunofluorescence staining technique in high-density tenocyte culture on Filter Well Inserts (FWI). The tenocytes were incubated for 4 weeks under monolayer conditions and for 3 weeks on FWI. At the end of the third week of high-density culture, we observed tenocyte aggregation followed by macromass cluster formation. Immunofluorescence labeling with anti-collagen type I antibody revealed that the presence of collagen type I was mostly around the nucleus. Type III collagen was more diffused in the cytoplasm. Type V collagen was detected in fibrillar and vesicular forms in the cytoplasm. We conclude that, the high-density culture on FWI is an appropriate method for the production of tenocytes without loosing specialized processes such as the synthesis of different collagen molecules. We consider that the high-density culture system is suitable for in vitro applications which affect tendon biology and will improve our understanding of the biological behavior of tenocytes in view of adequate matrix structure synthesis. Such high-density cultures may serve as a model system to provide sufficient quantities of tenocytes to prepare tenocyte-polymer constructs for tissue engineering applications in tendon repair.     

Specific immunohistochemical localization of Type I collagen in porcine periodontal tissues using the peroxidase-labelled antibody technique

Synopsis Antibody against Type I collagen was raised in rabbits and purified by immunoadsorption on Sepharose-conjugated Types I and III collagen. The cross-reactivity of purified antibody to Type III collagen was found to be less than 0.5% by passive haemagglutination and less than 1.5% by radioimmunoassay. When paraffin sections of fixed and decalcified pig molars were incubated with purified antibody to Type I collagen, varying degrees of staining were observed in the ligament, gingiva, bone and cementum. The periodontal ligament adjacent to bone was more widely stained than that adjacent to cementum in some regions, whereas in others, no difference in staining could be discerned between the two halves of the ligament. The lamina propria of gingiva was stained, and this appeared to be most intense in the vicinity of the overlying epithelium. The fibrous component in the endosteal spaces, the dentine and the extracellular coronal elements in the pulp were generally stained. The impression obtained from the staining pattern is that Type I collagen is not restricted to particular regions of the periodontal ligament or the lamina propria of the gingiva.     

The stability of the collagen phenotype during stimulated collagen,glycosaminoglycan, and DNA synthesis by articular cartilage organ cultures

Rabbit articular cartilage slices were grown in organ culture for 9 weeks. Eightfold increases in the synthesis of both glycosaminoglycan and collagen were observed at 1 and 3 weeks, respectively. These levels of synthesis gradually declined in parallel to fourfold at 9 weeks. DNA synthesis was stimulated more than 30-fold at 3 weeks and then declined to sevenfold at 9 weeks. In contrast, the content of glycosaminoglycans and collagen per milligram of original wet slices did not vary significantly, while the number of cells increased 1.7-fold by the end of the study. The collagen phenotype of these cultures was determined by sodium dodecyl sulfate electrophoresis of recently synthesized, [³H]proline-labeled intact collagen chains and CNBr peptides. Throughout the study the major collagen synthesized was type II, ranging from 95 to 68% of the collagen synthesized at 0 and 5 weeks, respectively. Increases in the proportions of X₂Y and type III collagen were first observed at 3 weeks in culture. The synthesis of type I collagen was detected only after 5 weeks in culture and never represented more than 11% of the total collagen synthesized. The synthesis of type I trimer could not be verified at any time. This study demonstrates that in vitro organ culture of articular cartilage slices allows chondrocytes to maintain the normal chondrocyte collagen phenotype of predominantly type II synthesis while stimulating their proliferation and matrix synthesis.     

Collagen production by rat liver fat-storing cells in primary culture

Morphological changes, proliferation and collagen synthesis of fat-storing cells (FSC) in primary culture were examined. FSC, isolated from rats treated with vitamin A, showed numerous large lipid droplets in the cytoplasm and positive desmin staining. After 4-7 days culture, these cells were transformed into fibroblast-like cells with a gradual depletion of lipid droplets and with abundant well-developed rough endoplasmic reticulum. The proliferation analysis revealed that DNA synthesis preceded the increase of cell number. Enhancement of the collagen synthesis by FSC were associated with the morphological change of the cells. Quantitative analysis revealed that these cells produced mainly type I collagen (84%) and a small amount of type III collagen (16%).     

Connective tissue elements in rat bone marrow: immunofluorescent visualization of the hematopoietic microenvironment

Immunofluorescent staining of frozen sections of rat bone marrow for collagen types I and III revealed the presence of a distinctive, collagen-producing cell type. Morphologically, these cells closely resembled reticular cells. They were large, with branching cytoplasm and were closely related to an extensive intercellular matrix of collagenous material that surrounded the hematopoietic cells of the marrow. Biochemical studies demonstrated synthesis of collagen types I and III, in a ratio of 4:1, by fresh rat bone marrow cells.     

Hindlimb unloading induces a collagen isoform shift in the soleus muscle of the rat

To determine whether hindlimb unloading (HU) alters the extracellular matrix of skeletal muscle, male Sprague-Dawley rats were subjected to 0 (n = 11), 1 (n = 11), 14 (n = 13), or 28 (n = 11) days of unloading. Remodeling of the soleus and plantaris muscles was examined biochemically for collagen abundance via measurement of hydroxyproline, and the percentage of cross-sectional area of collagen was determined histologically with picrosirius red staining. Total hydroxyproline content in the soleus and plantaris muscles was unaltered by HU at any time point. However, the relative proportions of type I collagen in the soleus muscle decreased relative to control (Con) with 14 and 28 days HU (Con 68 +/- 5%; 14 days HU 53 +/- 4%; 28 days HU 53 +/- 7%). Correspondingly, type III collagen increased in soleus muscle with 14 and 28 days HU (Con 32 +/- 5%; 14 days HU 47 +/- 4%; 28 days HU 48 +/- 7%). The proportion of type I muscle fibers in soleus muscle was diminished with HU (Con 96 +/- 2%; 14 days HU 86 +/- 1%; 28 days HU 83 +/- 1%), and the proportion of hybrid type I/IIB fibers increased (Con 0%; 14 days HU 8 +/- 2%; 28 days HU 14 +/- 2%). HU had no effect on the proportion of type I and III collagen or muscle fiber composition in plantaris muscle. The data demonstrate that HU induces a shift in the relative proportion of collagen isoform (type I to III) in the antigravity soleus muscle, which occurs concomitantly with a slow-to-fast myofiber transformation.     

Changes in the content of the fibrillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing

Summary The menisci are first seen as triangular aggregations of cells in the 20-day rabbit fetus. At 25-days, a matrix that contains types I, III and V collagens has formed. These collagens are also found in the 1-week neonatal meniscus, but by 3 weeks, type II collagen is present in some regions. By 12 to 14 weeks, typically cartilaginous areas with large cells in lacunae are found and by 2 years, these occupy the central regions of the inner two-thirds of the meniscus. The surface layers of the meniscus contain predominantly type I collagen. From 12 to 14 weeks onwards, there is little overlap between the regions with types I or II collagens, that is, these are discrete regions of type I-containing fibrocartilage and type II-containing cartilage. Types III and V collagens are found throughout the menisci, particularly in the pericellular regions. All the cells in the fetal and early neonatal menisci express the mRNA for type I collagen. At 3 weeks postnatal, cells that express type I collagen mRNA are found throughout the meniscus, but type II collagen mRNA is expressed only in the regions of developing cartilage. At 12- to 14-weeks, only type II collagen mRNA is expressed, except at the periphery next to the ligament where a few cells still express type I collagen mRNA. Rabbit menisci, therefore, undergo profound changes in their content and arrangement of collagens during postnatal development.