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.     

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.     

Glucocorticoids coordinately regulate procollagens type I and type III synthesis

Antibodies to type I and type III procollagens were raised in rabbits and were made monospecific by chromatography on collagen and procollagen affinity columns. The antibodies were determined to be monospecific by the direct enzyme-linked immunosorbent assay and the enzyme-linked immunosorbent assay inhibition assay. Rats were treated with various doses of triamcinolone diacetate, pulse-labeled with radioactive proline for 20 min, and the procollagens were precipitated with procollagen antibodies. The degree of inhibition of procollagen type I and type III synthesis to corticosteroid treatment was the same. This coordinate effect of glucocorticoids on the synthesis of the two procollagens was reversible, dose-dependent, time-dependent, and observed in lung as well as in skin. These data indicate that glucocorticoids coordinately regulate the synthesis of type I and type III procollagen in skin and lung to the same extent.     

A semiquantitative probe for radiation-induced normal tissue damage at the molecular level

Sheep antibodies to bovine type I collagen were employed in the immunohistochemical detection of type I collagen in lung tissue sections of irradiated LAF1 mice. A video image digitizing system was developed to estimate collagen levels, by assigning a numerical value (0-63) to each of approximately 53,800 picture elements (pixels) in the microscope field, according to the collagen-dependent fluorescence intensity at each locus. For lungs harvested 52 weeks subsequent to graded doses of 60Co gamma radiation between 0 and 10 Gy, a dose-dependent increase in type I collagen was observed in the alveolar walls. A reproducible increase was evident for doses as low as 5 Gy: doses of 7 to 10 Gy elicited type I collagen levels significantly elevated with respect to those of age-matched controls. These results are consistent with a role for type I collagen in the development of radiation-induced pulmonary fibrosis. The assay system developed here will be used to explore the role of connective tissue macromolecules in the development of radiation pneumonitis and fibrosis.     

Bleomycin-induced synthesis of type I procollagen by human lung and skin fibroblasts in culture

Bleomycin is a chemotherapeutic agent sometimes associated with pulmonary fibrosis and skin lesions in patients undergoing treatment. We examined the mechanisms of increased collagen deposition on bleomycin-induced fibrosis by incubating human lung and skin fibroblast cultures with [14C]proline; the synthesis of [14C]hydroxyproline relative to DNA or cell protein was taken as an index of procollagen formation. Procollagen synthesis by lung cells in the presence of 0.1 and 1.0 microgram/ml bleomycin was significantly increased and similar results were obtained with skin fibroblasts. The relative synthesis of genetically distinct types of collagen was measured by isolating the newly synthesized type I and type III procollagens by DEAE-cellulose chromatography. The proportion of type III procollagen of total newly synthesized procollagen in control lung fibroblast cultures was 17.4 +/0 0.6% (mean +/- S.E.) while the corresponding value in cells incubated in 1 microgram/ml bleomycin was 12.5 +/- 0.6% (n = 6, P < 0.01). Similar results were obtained when the ratios of newly synthesized type I and type III collagens were estimated by interrupted polyacrylamide disc gel electrophoresis in sodium dodecyl sulfate after a limited proteolytic digestion with pepsin. The results indicate that the increased procollagen synthesis induced by bleomycin in fibroblast cultures is predominantly directed towards the synthesis of type I procollagen.     

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.     

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.     

Radiation injury in rat lung. IV. Modification by D-penicillamine

To determine whether D-penicillamine, known to reduce fibrosis in irradiated rat lung (W. F. Ward, A. Shih - Hoellwarth , and R. D. Tuttle , Radiology 146, 533-537, 1983), also ameliorates radiation injury in the pulmonary endothelium, we measured angiotensin-converting enzyme (ACE) activity, plasminogen activator (PLA) activity, and prostacyclin (PGI2) production in the lungs of penicillamine-treated (10 mg/day, po, continuous after irradiation) and untreated rats from 2 weeks to 6 months after a single dose of 25 Gy of 60Co gamma rays to the right hemithorax. Both ACE and PLA activity in the irradiated right lung of untreated rats decreased dramatically between the 1st and 2nd months after exposure, then reached a plateau through 6 months at approximately 25 and 50% of the normal level, respectively. For the first 2 months after irradiation, penicillamine-treated animals exhibited significantly (P less than 0.05) higher activities of both ACE and PLA than did untreated rats. From 3 to 6 months after irradiation, however, the only significant drug effect on these enzymes was a 25% increase in PLA activity at 6 months. PGI2 production by the irradiated lung of untreated rats increased continuously, and at 6 months was approximately 10 times higher than normal. Penicillamine significantly (P less than 0.05) reduced this hypersecretion, and at 6 months after irradiation, PGI2 production by the lungs of drug-treated rats was only half that of untreated animals. In contrast, the drug had no significant effect on enzyme activities in the lungs of sham-irradiated rats. Thus the antifibrotic agent D-penicillamine delays the onset of radiation-induced enzyme dysfunction in the pulmonary endothelium. In addition at 6 months after irradiation, the lungs of penicillamine-treated rats exhibit 25% more PLA activity and only half as severe a hypersecretion of PGI2 as do the lungs of untreated animals. The drug is most effective in ameliorating endothelial damage during the first 2 months after irradiation, preceding the development of interstitial fibrosis. However, the effect of this penicillamine regimen on pulmonary endothelial function is not as large as its effect on collagen accumulation in irradiated rat lung.     

Immunohistochemical quantitation of three collagen isotypes in perfused areas and nonperfused foci of the lungs of irradiated mice

Collagen isotypes I, III, and IV were quantitated by video image analysis of fluorescent-antibody-stained lung tissue sections from control and irradiated C57L/J and BALB/c mice. The perfusion status of lungs was determined by injecting colloidal carbon into the hepatic vein immediately prior to sacrificing the animals. Well-perfused parenchymal regions turned black, whereas nonperfused areas remained pale. Previous histological studies indicated substantial differences in the types of lesions found in the lungs of these two strains. C57L/J mice develop extensive and persistent contracted fibrosis. In lung sections of C57L/J mice examined 28 weeks after a dose of 11 Gy X rays, all three collagen isotypes were significantly elevated to levels 37-51% higher than age-matched control values in perfused regions of lung. In nonperfused areas, which had the histological appearance of contracted scar tissue, the three collagen isotype levels were further elevated to values 83-90% greater than controls. This finding suggests that in C57L/J mice, an elevation of each or all of the three collagen isotypes to levels approximately 45% greater than controls is consistent with continued pulmonary function during the intermediate phase of lung damage, whereas areas of parenchyma containing isotype levels in excess of 185% of control values coincide with functionally deficient regions. BALB/cCr//Alt. mice examined 28 weeks subsequent to 14.5 Gy X rays had a variety of visible lesion, most of which were nonperfused. In addition, one-quarter of nonperfused acini had no visible lesion. In perfused areas, the three isotypes were increased to 119-132% of control levels, with a further, significant (P less than 0.05) increase to 128-144% of control values in nonperfused parenchyma. Nonperfused areas were not characterized by contracted fibrosis; however, it would appear that the threshold level for collagen elevation associated with functional compromise during intermediate phase lies in the region of 130%. For BALB/c/J mice, 1 year after 9 Gy X rays, perfused areas of lung contained control levels of the three collagen isotypes, while nonperfused areas had isotype levels 119-131% of control values. Two of seven animals died at 41 weeks, but we were unable to ascertain collagen levels, since the lungs were not infused with colloidal carbon.     

Pro‐apoptotic Noxa is involved in ablative focal irradiation‐induced lung injury

Although lung injury including fibrosis is a well‐documented side effect of lung irradiation, the mechanisms underlying its pathology are poorly understood. X‐rays are known to cause apoptosis in the alveolar epithelial cells of irradiated lungs, which results in fibrosis due to the proliferation and differentiation of fibroblasts and the deposition of collagen. Apoptosis and BH3‐only pro‐apoptotic proteins have been implicated in the pathogenesis of pulmonary fibrosis. Recently, we have established a clinically analogous experimental model that reflects focal high‐dose irradiation of the ipsilateral lung. The goal of this study was to elucidate the mechanism underlying radiation‐induced lung injury based on this model. A radiation dose of 90 Gy was focally delivered to the left lung of C57BL/6 mice for 14 days. About 9 days after irradiation, the mice began to show increased levels of the pro‐apoptotic protein Noxa in the irradiated lung alongside increased apoptosis and fibrosis. Suppression of Noxa expression by small interfering RNA protected cells from radiation‐induced cell death and decreased expression of fibrogenic markers. Furthermore, we showed that reactive oxygen species participate in Noxa‐mediated, radiation‐induced cell death. Taken together, our results show that Noxa is involved in X‐ray‐induced lung injury.     

Subpopulations of rat lung fibroblasts with different amounts of type I and type III collagen mRNAs

A fluorescence-based method using the cell sorter has been devised to separate rat lung fibroblasts into subpopulations. Type I or type III collagen antiserum was used as the primary antibody to react with parent rat lung fibroblasts. This was followed by a fluorescein-conjugated secondary antibody. Specificity of the primary collagen antibody was determined using a monoclonal beta-actin antibody and purified IgG as the primary antibodies. The fluorescent shift of parent rat lung fibroblasts was optimized for the amount of primary collagen antibody and secondary fluorescein-conjugated antibody. An increase in slot blot intensity was observed for pro-alpha 1(I), pro-alpha 2(I), and pro-alpha 1(III) mRNAs with increasing amounts of cellular RNA. When precipitating with type I collagen antibodies, the total cellular steady-state levels of type I procollagen mRNAs were increased in the high intensity cells as compared with the low intensity cells. Alternately, when the type III collagen antibodies were used to precipitate the rat lung fibroblasts, the low intensity cells had increased type I procollagen mRNAs while the high intensity cells had increased type III procollagen mRNA. The subpopulations of rat lung fibroblasts after isolation using the fluorescent cell sorter were readily propagated for at least four passages.     

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.     

Protein synthesis in rat lung. Measurements in vivo based on leucyl-tRNA and rapidly turning-over procollagen I.

The relationships of the specific radioactivities of leucine in serum, leucine acylated to tRNA and leucine in procollagen I, procollagen III and total protein in lungs of unanaesthetized young male rats in vivo were assessed as a function of time during constant intravenous infusion of radiolabelled leucine. The specific radioactivity of free leucine in plasma reached a steady-state plateau value within 30 min of initiation of [3H]leucine infusion. Leucine acylated to tRNA isolated from lungs had the same specific radioactivity as free serum leucine. Leucine in procollagen I rapidly achieved a specific radioactivity equal to that of serum leucine and leucyl-tRNA, indicating that serum leucine and leucyl-tRNA isolated from total lung were in rapid equilibrium with the precursor leucine pool for procollagen I synthesis. On the basis of leucyl-tRNA or free serum leucine as the precursor, half-times of fractional conversion of procollagen I and III were calculated as 9 and 38 min respectively. The incorporation of leucine into mixed lung proteins calculated from the tracer studies was 6.8 mumol/day for the first 30 min of the infusion, after which the calculated rate increased to 15.0 mumol/day. This apparent increase correlated with the appearance of rapidly labelled plasma proteins trapped in the lungs. On the basis of short infusions lasting 30 min or less, followed by vascular perfusion of the lung, the average fractional synthesis rate of mixed pulmonary proteins in young male rats was 20%/day.     

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.     

Processing of procollagen types III and I in cultured bovine smooth muscle cells

The processing of type III and type I procollagen molecules in cultured bovine aortic smooth muscle cells was investigated. The molecular identities of the processing intermediates of type III and type I procollagen were characterized by analysis of the radioactive collagenous components using mammalian collagenase and pepsin digestions and cyanogen bromide peptide mapping. The results indicate that the processed intermediates for procollagen type III and type I are their respective pC components. Although the processing pathways for both collagen types are the same, data from pulse-chase experiments suggest that the rates at which the processing occurs are different. Type I procollagen is processed more rapidly to its intermediate than is type III procollagen. The type I pC intermediate is almost completely processed to alpha-chains and a significant portion of these fully processed molecules remains in a soluble form even after 11 h. In the same time period, the type III pC intermediate is slowly converted to alpha-chains. Since beta-aminopropionitrile was not employed in these studies, significant accumulation of collagen chains into the insoluble extracellular matrix was observed during the chase period.