Biosynthesis and degradation of collagen in X-irradiated mouse lung

Fibrosis, characterized by accumulation of collagen, is a delayed result of radiation injury in many tissues, including lung. To investigate its development, synthesis and degradation of collagen were measured in lungs of mice after X irradiation of the whole thorax. The ratio of type I (coarse fibered) to type III (meshwork) collagen was also determined. Synthesis of procollagen, measured as the activities of prolyl-4-hydroxylase and protein disulfide isomerase in lung tissue, was increased at 2 months after X-ray doses of 5, 7.5, and 9 Gy. Maximal increases were observed 6 to 7 months after doses of 9 Gy and persisted up to 15 months after exposure. Increases after 5 and 7.5 Gy were more gradual, but by 1 year after irradiation they had reached levels similar to those after 9 Gy. X irradiation had no effect on the degradation of collagen as assessed by collagenase activity in lung. The ratio of type I to type III collagen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of collagen-derived cyanogen bromide peptides, was the same in irradiated lungs as in age-matched controls. Therefore, increased synthesis of procollagen, rather than decreased degradation of collagen or changes in collagen type, is an important factor in the accumulation of collagen in irradiated lung.     

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Real time RT-RCR showed that both α1 and α2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-β1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-β receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of α2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.     

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.     

Heparan mimetic regulates collagen expression and TGF-beta1 distribution in gamma-irradiated human intestinal smooth muscle cells.

Radiation-induced intestinal fibrosis is characterized by collagen accumulation, a process in which TGF-beta1 plays a key role. We analyzed the effects of gamma radiation on collagen expression and TGF-beta1 distribution in human intestinal smooth muscle cells (HISM). We investigated the activity of a carboxymethylated and sulfated dextran (RG-1503), exhibiting antifibrotic properties and promoting in vivo intestinal tissue repair, on irradiated HISM. After (60)Co irradiation (10 Gy), HISM were labeled with [(3)H] proline (+/-RG-1503). Radiolabeled collagen I, III, and V were quantified by SDS-PAGE. TGF-beta1 was quantified by ELISA in culture medium, pericellular and intracellular compartments. Irradiation induced a specific 2.85-fold increase in collagen III production by HISM. Collagen V decreased by 80% 72 h after irradiation. Pericellular TGF-beta1 was increased (up to twofold) in irradiated HISM. RG-1503 added before or after irradiation reversed both mRNA and protein levels of collagen III and V to control values. RG-1503 decreased the amount of TGF-beta1 in the cell layer below the control values. Irradiation of HISM induced the development of a fibrotic phenotype in terms of collagen production and TGF-beta1 distribution. The antifibrotic RG-1503 restored HISM physiological characteristics and may represent a promising therapeutic approach for radiation-induced intestinal fibrosis.     

Vascular permeability and late radiation fibrosis in mouse lung

It has been suggested that fibrosis which develops after irradiation is caused by increases in vascular permeability. Plasma proteins leak into irradiated tissue where fibrinogen may be converted into fibrin which is gradually replaced by fibrous tissue. Vascular and fibrotic changes in mouse lung were investigated after X irradiation of the right hemithorax. Blood volume and accumulation of extravascular proteins were measured using indium (111In)-labeled red cells, iodinated (131I) albumin, and iodinated (125I) fibrinogen. Tracers were injected 1-47 weeks after irradiation and lungs were excised 24 or 96 hr later to determine radioactivity. The amount of collagen was estimated by measuring the hydroxyproline content. During the first few months after X rays, lung blood volume decreased to a plateau which depended on radiation dose (10-25 Gy). Small increases in extravascular albumin and fibrinogen occurred at 1-12 weeks after 10-25 Gy. Subsequently, protein returned to normal after 10 Gy, remained elevated after 15 Gy, and increased after 20 and 25 Gy. Hydroxyproline per gram of dry irradiated lung was increased at 18 weeks after 15-25 Gy. Subsequently it showed little change although both total hydroxyproline content and dry weight decreased after 20 and 25 Gy. Support for the hypothesis was that hydroxyproline per gram only increased after X-ray doses which caused marked extravasation of protein. There was no evidence, however, for deposition of 125I-fibrin or for a gradual increase in fibrosis corresponding to the prolonged excess of extravascular protein.     

Effect of ionizing radiation on the structure of the collagen fibers of human tendons

In studying the effect of ionizing radiation on the properties of human Achilles tendon collagen fibres, the following parameters were analyzed: hydrothermal contraction temperature, module of elasticity, the number of cross-links, free and bound water levels, acids-soluble fraction content, and ultrastructure. With radiation doses of 2-10 Gy no changes in the collagen status were noted. An increased (from 5 to 25 Gy) radiation dose caused changes in physicochemical properties which was indicative of the formation, in the connective tissue collagen, of radiation-induced intermolecular cross-links stabilizing the biopolymer structure.     

Extracellular matrix proteins: A positive feedback loop in lung fibrosis?

Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the development of bleomycin-induced lung fibrosis. We further report in vitro experiments clarifying both the effect of myofibroblast differentiation on this expression and the effect of extracellular elastin on myofibroblast differentiation.Lung fibrosis was induced in female C57Bl/6 mice by bleomycin instillation. Animals were sacrificed at zero to five weeks after fibrosis induction. Collagen synthesized during the week prior to sacrifice was labeled with deuterium. After sacrifice, lung tissue was collected for determination of new collagen formation, microarray analysis, and histology. Human lung fibroblasts were grown on tissue culture plastic or BioFlex culture plates coated with type I collagen or elastin, and stimulated to undergo myofibroblast differentiation by 0–10 ng/ml transforming growth factor (TGF)β₁. mRNA expression was analyzed by quantitative real-time PCR.New collagen formation during bleomycin-induced fibrosis was highly correlated to gene expression of elastin, type V collagen and tenascin C. At the protein level, elastin, type V collagen and tenascin C were highly expressed in fibrotic areas as seen in histological sections of the lung. Type V collagen and tenascin C were transiently increased. Human lung fibroblasts stimulated with TGFβ₁ strongly increased gene expression of elastin, type V collagen and tenascin C. The extracellular presence of elastin increased gene expression of the myofibroblastic markers α smooth muscle actin and type I collagen.The extracellular matrix composition changes dramatically during the development of lung fibrosis. The increased levels of elastin, type V collagen and tenascin C are probably the result of increased expression by fibroblastic cells; reversely, elastin influences myofibroblast differentiation. This suggests a reciprocal interaction between fibroblasts and the extracellular matrix composition that could enhance the development of lung fibrosis.     

MMP‐13 deletion decreases profibrogenic molecules and attenuates N‐nitrosodimethylamine‐induced liver injury and fibrosis in mice

Connective tissue growth factor (CTGF) is involved in inflammation, pathogenesis and progression of liver fibrosis. Matrix metalloproteinase‐13 (MMP‐13) cleaves CTGF and releases several fragments, which are more potent than the parent molecule to induce fibrosis. The current study was aimed to elucidate the significance of MMP‐13 and CTGF and their downstream effects in liver injury and fibrosis. Hepatic fibrosis was induced using intraperitoneal injections of N‐nitrosodimethylamine (NDMA) in doses of 10 μg/g body weight on three consecutive days of each week over a period of 4 weeks in both wild‐type (WT) and MMP‐13 knockout mice. Administration of NDMA resulted in marked elevation of AST, ALT, TGF‐β1 and hyaluronic acid in the serum and activation of stellate cells, massive necrosis, deposition of collagen fibres and increase in total collagen in the liver of WT mice with a significant decrease in MMP‐13 knockout mice. Protein and mRNA levels of CTGF, TGF‐β1, α‐SMA and type I collagen and the levels of MMP‐2, MMP‐9 and cleaved products of CTGF were markedly increased in NDMA‐treated WT mice compared to the MMP‐13 knockout mice. Blocking of MMP‐13 with CL‐82198 in hepatic stellate cell cultures resulted in marked decrease of the staining intensity of CTGF as well as protein levels of full‐length CTGF and its C‐terminal fragments and active TGF‐β1. The data demonstrate that MMP‐13 and CTGF play a crucial role in modulation of fibrogenic mediators and promote hepatic fibrogenesis. Furthermore, the study suggests that blocking of MMP‐13 and CTGF has potential therapeutic implications to arrest liver fibrosis.     

Type I collagen messenger RNA levels in experimental granulation tissue and silicosis in rats

A complementary DNA (cDNA) clone was constructed for chick pro alpha 2(I) collagen mRNA. This and previously constructed cDNA clones for chick and human pro alpha 1(I) collagen mRNAs were used to measure levels of type I procollagen messenger RNAs in two experimental models: viscose cellulose sponge-induced experimental granulation tissue and silica-induced experimental lung fibrosis in rats. Both Northern RNA blot and RNA dot hybridizations were used to quantitate procollagen mRNAs during formation of granulation tissue. The period of rapid collagen synthesis was characterized by high levels of procollagen mRNAs, which were reduced when collagen production returned to a low basal level. The rate of collagen synthesis and the levels of procollagen mRNAs during the period of rapid reduction in collagen production did not, however, parallel with each other. This suggests that translational control mechanisms are important during this time in preventing overproduction of collagen. In silicotic lungs, the early stages of fibroblast activation follow a similar path but appear faster. At a later stage, however, the RNA levels increase again and permit collagen synthesis to continue at a high rate, resulting in massive collagen accumulation.     

Temporal changes in renal endoglin and TGF-β1 expression following ureteral obstruction in rats

Chronic renal disease is characterized by the accumulation of extracellular matrix proteins in the kidney and a loss of renal function. Tubulointerstitial fibrosis has been reported to play an important role in the progression of chronic renal diseases. Transforming growth factor-beta1 (TGF-beta1) is a profibrotic cytokine playing a major contribution to fibrotic kidney disease. Endoglin is a membrane glycoprotein of the TGF-beta1 receptor system. The aim of this work was to determine the time-course expression of renal type I and IV collagens, endoglin and TGF-beta1 in a rat model of induced tubulointerstitial fibrosis at 1, 3, 10 and 17 days after unilateral ureteral obstruction (UUO). In 17 days-ligated (L)-renal samples, a marked interstitial fibrosis was detected by Masson's trichromic and Sirius red staining, accompanied by an increase in type I collagen expression as shown by immunohistochemical analysis. Northern blot studies revealed a progressive increase in collagen alpha2(I), TGF-beta1 and endoglin mRNA expression in L kidneys when compared with the corresponding non-ligated (NL) kidneys from the animals subjected to left UUO. Seventeen days after UUO, significant increases in collagen alpha2(I), collagen alpha1(IV), TGF-beta1 and endoglin mRNA levels were detected in L kidneys vs NL kidneys. Significantly higher levels of the protein endoglin were found in L kidneys than in NL kidneys 10 and 17 days following obstruction. A marked increase expression for endoglin and TGF-beta1 was localized in renal interstitium by immunohistochemical studies 17 days after obstruction. In conclusion, this work reports the upregulation of endoglin coincident to that of its ligand TGF-beta1 in the kidneys of rats with progressive tubulointerstitial fibrosis induced by UUO.     

Ionizing radiation induces early, sustained increases in collagen biosynthesis: a 48-week study in mouse skin and skin fibroblast cultures

Groups of 10 CF1 female mice, irradiated to the thorax with a dual-head 137Cs gamma-RAY source, received single doses of 0, 5, 10, 15, or 25 Gy. One to forty-eight weeks later collagen synthesis was measured in minced skin specimens incubated in medium containing [3H]proline and then assayed for radioactive hydroxyproline. A progressive, generally dose-dependent increase in collagen biosynthesis, up to 50% above control sites, was found 1, 4, and 12 weeks after radiation exposure. These changes showed further small fluctuations at 12-36 weeks, increasing again at the 48-week interval. At the same times throughout the study fibroblasts were cultured from skin explants. Following the second subculture, these cells were also incubated in medium containing [3H]proline, and collagen synthesis was again determined by [3H]hydroxyproline assay. At all radiation dose levels studied, collagen production increased threefold by 12 weeks postradiation and remained elevated for the 48-week duration of the study. In vitro radiation dose response differences were not observed.     

Increased procollagen mRNA levels in carbon tetrachloride-induced liver fibrosis in rats

Carbon tetrachloride-induced liver damage is a well-characterized experimental model for studying liver fibrosis. We used this model to examine alpha 1(I), alpha 1(III), and alpha 1(IV) procollagen mRNA levels during the development of liver fibrosis. Rats were given 0.5 ml of carbon tetrachloride/kg of body weight for 1-6 weeks. The liver tissue was assayed for collagen content by measuring total hydroxyproline content. Specific increases in procollagen mRNAs were assayed by slot blot hybridization. There was a significant increase in hydroxyproline content of liver tissue following 3 weeks of carbon tetrachloride treatment. The increase in tissue collagen content correlated with an increase in alpha 1(I) procollagen mRNA levels. At 5 and 6 weeks of treatment, there was an increase in alpha 1(III) procollagen mRNA levels. alpha 1(IV) procollagen levels increased slightly with five injections of carbon tetrachloride treatment. These results suggest that specific increases in procollagen mRNAs in liver fibrosis parallel, but do not precede, increases in tissue collagen content.     

ALK1 heterozygosity increases extracellular matrix protein expression,proliferation and migration in fibroblasts

Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-β1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-β1 with an important role in angiogenesis whose function in cellular biology and TGF-β signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1^+/+ and ALK1^+/− mouse embryonic fibroblasts (MEFs) after TGF-β1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-β/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.     

Mechanisms by which bradykinin promotes fibrosis in vascular smooth muscle cells: role of TGF-beta and MAPK

Accumulation of extracellular matrix (ECM) is a hallmark feature of vascular disease. We have previously shown that hyperglycemia induces the expression of B(2)-kinin receptors in vascular smooth muscle cells (VSMC) and that bradykinin (BK) and hyperglycemia synergize to stimulate ECM production. The present study examined the cellular mechanisms through which BK contributes to VSMC fibrosis. VSMC treated with BK (10(-8) M) for 24 h significantly increased alpha(2)(I) collagen mRNA levels. In addition, BK produced a two- to threefold increase in alpha(2)(I) collagen promoter activity in VSMC transfected with a plasmid containing the alpha(2)(I) collagen promoter. Furthermore, treatment of VSMC with BK for 24 h produced a two- to threefold increase in the secretion rate of tissue inhibitor of metalloproteinase 1 (TIMP-1). The increase in alpha(2)(I) collagen mRNA levels and alpha(2)(I) collagen promoter activity, as well as TIMP-1 secretion, in response to BK were blocked by anti-transforming growth factor-beta (anti-TGF-beta) neutralizing antibodies. BK (10(-8) M) increased the endogenous production of TGF-beta1 mRNA and protein levels. Inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD-98059 inhibited the increase of alpha(2)(I) collagen promoter activity, TIMP-1 production, and TGF-beta1 protein levels observed in response to BK. These findings provide the first evidence that BK induces collagen type I and TIMP-1 production via autocrine activation of TGF-beta1 and implicate MAPK pathway as a key player in VSMC fibrosis in response of BK.     

Effect of fibroblast implants on wound healing of irradiated skin: assay of wound strength and quantitative immunohistology of collagen

The role of dermal fibroblasts in the expression of radiation-induced damage to the skin was studied. Fibroblasts from neonatal mice were cultured, harvested, and injected into full-depth surgical incisions in the dorsal area of mouse skin, which had been previously locally irradiated by 18 Gy X rays. As a control, cells irradiated with a dose of 20 Gy were also injected. The effect of radiation and fibroblast implants on the gain of skin wound strength was assayed. In an additional experiment freshly isolated cells were implanted. Two weeks following wounding the irradiated skin had reached only about a third of the strength of unirradiated skin. A significant increase of wound strength in irradiated skin was observed when 1.5-2 x 10(6) cultured fibroblasts or freshly isolated fibroblasts were injected into the 20-mm-long wound bed. Irradiated cells had significantly less effect. This suggests that implanting isolated syngeneic cells may "rescue" wounds from the effect of prior irradiation. Semiquantitative immunohistology of types I and III collagen was performed in parallel using a video image digitizing system. Levels of both types I and III collagen were altered in the dermis and the wound tissues in irradiated skin, but the implant of cultured fibroblasts did not affect notably the total levels and the disposition of the two collagen isotypes.     

Dynamics of structural parameters of mast cells and accumulation of collagen fibers during radiation-induced pneumosclerosis in rats (a quantitative study)

The methods of stereometry were used to study dynamics of accumulation of collagen fibres and alteration of the number, sizes and state of mast cells of rat lung during the period of 12 months after single local X-irradiation with doses of 10, 14.3 and 20 Gy. A statistically significant correlation between the number of mast cells and spatial density of collagen fibres in the exposed pulmonary tissue was shown. Severity of changes in the structural characteristics of mast cells and the degree of collagen fibres accumulation were a function of radiation dose.     

Collagen metabolism in the murine colon following X irradiation.

Female CBA mice, aged 16 weeks, were irradiated to the total pelvic region with either single doses (5-20 Gy) or two equal fractions (10- to 30-Gy total dose, 24-h interval) of 240 kV X rays. Total protein and collagen synthesis rates, collagen breakdown, and net collagen content of the colon were measured at various times postirradiation using a radioisotope incorporation method and HPLC analysis. Immunohistochemical staining and computerized image analysis were used to assess the relative amounts of collagen types I and III at various times postirradiation, in various regions of the colon. Total protein and collagen synthesis rates were elevated above control levels at 4 and 8 weeks postirradiation, as was collagen degradation. Values had returned to control levels by 16 weeks postirradiation, and there were no further changes up to 71 weeks postirradiation. The net amount of collagen in the colon did not change relative to controls at any time during the investigation. There was, however, increased immunohistochemical staining for collagen type I 52 weeks postirradiation in all regions of the colon and decreased staining of type III in the circular muscle layer and villi. Altered ratios of these two collagen isotypes are consistent with changes in mechanical properties of the tissue.     

An animal model of pulmonary radiation fibrosis with biochemical, physiologic, immunologic, and morphologic observations

An animal model of pulmonary radiation fibrosis was established, using male CBA/j mice. Both lungs of each mouse in one group (DL) were irradiated with two doses of 8.5 Gy each, separated by 30 days. A control group (CG) was sham-irradiated. There was a small but significant difference (P less than 0.03) in average breathing rate between DL and CG 27 weeks after the second irradiation which increased until the 34th week followed by a plateau. The accumulated hydroxyproline content of the irradiated mouse lung was 40% greater (P less than 0.02) than that of the sham-irradiated lung at 42 weeks and thereafter. Anticollagen antibodies assayed 52 weeks after irradiation by enzyme-linked immunosorbent assay were elevated by 49% in sera from the irradiated mice compared to sera from sham-irradiated mice. Mortality during the 52-week period following the second irradiation was low (13%) for both groups. Histological comparison of irradiated and control mouse lungs fixed under uniform inflation pressure indicated no significant differences. The model has unique features including an increase in collagen deposition, no acute changes attributable to radiation, a small but statistically significant abnormality in pulmonary function, an immunologic response to collagen, and low mortality.