Establishment of a novel collagenase perfusion method to isolate rat pancreatic stellate cells and investigation of their gene expression of TGF-beta1, type I collagen,and CTGF in primary culture or freshly isolated cells

In studies of the pathogenesis of pancreatic fibrosis, pancreatic stellate cells (PSCs) have recently gained attention. In the present study, we established a new collagenase perfusion method through thoracic aorta cannulation to isolate PSCs, and we studied gene expression of TGF-beta1, type I collagen, and connective tissue growth factor using primary cultured PSCs. Our method facilitated PSC isolation, and by our new method, 4.3 +/- 1.2 x 10(6) PSCs were obtained from a rat. In comparing the expression of these genes with that of hepatic stellate cells (HSCs), we observed a similar pattern, although PSCs expressed type I collagen gene earlier than did HSCs. These results suggest that PSCs may play an important role in fibrosis of the pancreas, as HSCs do in liver fibrosis; in addition, PSCs may exist in a preactivated state or may be more easily activated than are HSCs. We also isolated the PSCs from a WBN/Kob rat, the spontaneous pancreatitis rat, and compared the gene expression with that from a normal rat.     

The Wistar Bonn Kobori rat, a unique animal model for autoimmune pancreatitis with extrapancreatic exocrinopathy

The male Wistar Bonn/Kobori (WBN/Kob) rat is known to be a unique animal model for chronic pancreatitis with widely distributed fibrosis and degeneration of parenchyma because of the infiltration of lymphocytes. In this report, we show that female (but not male) rats develop dacryoadenitis at 3 months of age, and that both male and female WBN/Kob rats develop sialoadenitis, thyroiditis, sclerotic cholangitis and tubulointerstitial nephritis over 18 months of age. The infiltration of CD8⁺ cells and the deposits of tissue-specific IgG2b were observed in the injured pancreas and lachrymal glands. Furthermore, the number of regulatory T cells (defined as CD4⁺ Forkhead box P3⁺ cells) decreased in the periphery of both male and female WBN/Kob rats, suggesting that the onset of these diseases is attributable, at least, to the failure in the maintenance of peripheral immune tolerance. These features show clearly that WBN/Kob rats are a useful animal model for autoimmune pancreatitis and Sjøgren-like syndrome or multi-focal fibrosclerosis in humans. We also show that these autoimmune diseases can be prevented by a newly devised strategy of bone marrow transplantation (BMT) in which bone marrow cells are injected directly into the bone marrow cavity: intrabone marrow–BMT.     

Fibrosis markers in heavy alcohol drinkers

Alcoholic intake has increased in society in recent years. gamma-GTP is used as a marker of liver damage by alcohol intake, but there is no reliable marker of pancreatic fibrosis. We used animal experiments and clinical data to identify a new reliable marker of early-stage pancreatic fibrosis. Pancreatic fibrosis is induced by intra-peritoneal injection of diethyldithiocarbamate. Pancreas tissue was extracted and measured. Human pure pancreatic juice was collected by endoscopic procedures. Prolyl hydroxylase in pancreas tissue is increased in the early stage of pancreatic fibrosis. Secretion of matrix metalloproteinase from pancreatic stellate cells is increased by diethyldithiocarbamate stimulation. Pancreatic stellate cells, prolyl hydroxylase and a tissue inhibitor of metalloproteinase in human pure pancreatic juice is increased in heavy alcohol drinkers and normalized in former alcohol drinkers. Active matrix metalloproteinase 2 is detected in pure pancreatic juice of chronic pancreatitis patients. Treatment with oral camostat increases pancreatic secretory trypsin inhibitor in chronic pancreatitis patients. Experimental and clinical data indicated that matrix metalloproteinase 2 and prolyl hydroxylase are candidates as markers of early-stage pancreatic fibrosis. Clinical data showed that tissue inhibitor of metalloproteinase and pancreatic secretory trypsin inhibitor in pure pancreatic juice had potential as markers of early-stage pancreatic fibrosis.     

Glomerular lesions in spontaneously occurring diabetic WBN/Kob rats

Glomerular lesions in WBN/Kob male rats with spontaneous diabetes were examined histopathologically. The glomerulopathy caused by diabetes was compared with lesions in chronic progressive nephropathy of non-diabetic SD and F344 male aged rats. Diffuse and global thickening of the mesangial area was observed only in WBN/Kob rats and showed a statistically significant correlation with serum glucose concentrations. Therefore, it suggested that the mesangial thickening was a result of hyperglycaemia. Fibrin cap-like lesions were seen in both WBN/Kob and non-diabetic SD or F344 male rats. The severity of these lesions was closely related to that of chronic progressive nephropathy and, therefore, the fibrin cap-like lesions were considered to be due to the chronic progressive nephropathy.     

Activation of Pancreatic Stellate Cells in Human and Experimental Pancreatic Fibrosis

The mechanisms of pancreatic fibrosis are poorly understood. In the liver, stellate cells play an important role in fibrogenesis. Similar cells have recently been isolated from the pancreas and are termed pancreatic stellate cells. The aim of this study was to determine whether pancreatic stellate cell activation occurs during experimental and human pancreatic fibrosis. Pancreatic fibrosis was induced in rats (n = 24) by infusion of trinitrobenzene sulfonic acid (TNBS) into the pancreatic duct. Surgical specimens were obtained from patients with chronic pancreatitis (n = 6). Pancreatic fibrosis was assessed using the Sirius Red stain and immunohistochemistry for collagen type I. Pancreatic stellate cell activation was assessed by staining for alpha-smooth muscle actin (alphaSMA), desmin, and platelet-derived growth factor receptor type beta (PDGFRbeta). The relationship of fibrosis to stellate cell activation was studied by staining of serial sections for alphaSMA, desmin, PDGFRbeta, and collagen, and by dual-staining for alphaSMA plus either Sirius Red or in situ hybridization for procollagen alpha(1) (I) mRNA. The cellular source of TGFbeta was examined by immunohistochemistry. The histological appearances in the TNBS model resembled those found in human chronic pancreatitis. Areas of pancreatic fibrosis stained positively for Sirius Red and collagen type I. Sirius Red staining was associated with alphaSMA-positive cells. alphaSMA staining colocalized with procollagen alpha(1) (I) mRNA expression. In the rat model, desmin staining was associated with PDGFRbeta in areas of fibrosis. TGFbeta was maximal in acinar cells adjacent to areas of fibrosis and spindle cells within fibrotic bands. Pancreatic stellate cell activation is associated with fibrosis in both human pancreas and in an animal model. These cells appear to play an important role in pancreatic fibrogenesis.     

Collagen stimulating factors in hepatic fibrogenesis.

Four factors which stimulate collagen synthesis and prolyl hydroxylase activity in cultures of human and mouse fibroblasts have been isolated by molecular sieve chromatography from animal and human fibrotic and cirrhotic livers. These factors do not stimulate protein or DNA synthesis or total DNA in these cultures. It has also been shown that these factors, designated collagen stimulating factors F1-F4, do not owe their activity to ascorbate or glutamine. Collagen stimulating factors are heat stable, and F1 and F2 have apparent molecular weights of about 4000 and 1000 respectively. Since these factors are not present in normal animal or human liver it is suggested that they may be responsible for increased collagen production in vivo in hepatic fibrosis and cirrhosis.     

Effects of chlorella phospholipid on the aortic collagen and elastin metabolism and on the serum lipid content in rats with experimental arteriosclerosis

Rats treated with 44,800 IU of vitamin D₂ for 4 consecutive days were fed an atherogenic diet in the presence or absence of 1% chlorella phospholipid. Control rats received a basal diet and were administered olive oil. After 2 months the animals were killed and aortic prolyl hydroxylase, lysyl oxidase activity, collagen, elastin, and serum lipid levels were determined. Aortic prolyl hydroxylase activity was significantly decreased in rats receiving the atherogenic diet in the absence of chlorella phospholipid. The aortic collagen and elastin content was lower in rats additionally treated with chlorella phospholipid. Aortic lysyl oxidase activity was significantly decreased in all rats receiving the atherogenic diet. The serum cholesterol level was significantly higher in rats on the atherogenic diet, especially the absence of chlorella phospholipid supplementation. The findings suggest that the administration of chlorella phospholipid may stimulate the degradation of collagen and elastin in the aorta of rats fed an atherogenic diet and that the serum cholesterol lowering effect of chlorella phospholipid is not ascribable to thyroid functions. Furthermore, the results suggest that the aortic degradation rate of elastin was reduced by the atherogenic treatment.     

Pancreatic fibrosis associated with age and ductal papillary hyperplasia

Little is known about the frequency, type and pathogenesis of fibrotic changes that may occur in the pancreas of persons without any clinically apparent or macroscopically visible pancreatic disease. We screened pancreas specimens for the presence and pattern of fibrosis, determined the relationship between fibrosis, age, and duct lesions, and studied the fibrogenic mechanisms. In 89 postmortem specimens from persons without any known pancreatic disease (age range 20–86 years), fibrosis was recorded and graded and the patients were divided into two age classes (younger or older than 60 years). In addition, we analyzed the association between ductal papillary hyperplasia [i.e., pancreatic intraepithelial neoplasia type 1B (PanIN-1B)] and fibrotic foci in the pancreatic tissue to determine the potential impact of obliterating duct lesions on pancreatic fibrosis. Finally, we studied the occurrence in the pancreas of myofibroblasts, identified on the basis of their α-SMA and desmin positivity, and determined their relationship to the fibrotic foci. Thirty-eight (44%) of 89 pancreata showed scattered foci of lobular fibrosis affecting peripheral lobuli. Fibrotic changes were significantly more common in individuals older than 60 years. Fibrotic foci were commonly associated (p<0.05) with ductal papillary hyperplasia in ducts draining fibrotic lobuli. Myofibroblasts were detected in the fibrotic foci. The “normal” pancreas develops a specific type of focally accentuated fibrosis that is highly age related. This patchy lobular fibrosis in the elderly (PLFE) was closely associated with PanIN-1B lesions in the ducts, suggesting that the narrowing of a duct due to papillary hyperplasia of the epithelium may hamper secretion and cause fibrosis of the drained lobule. The presence of myofibroblasts in association with the fibrotic foci indicates an ongoing fibrogenic process.     

Collagen production in fat-storing cells after carbon tetrachloride intoxication in the rat. Immunoelectron microscopic observation of type I, type III collagens, and prolyl hydroxylase

Monospecific antibodies directed against type I, type III collagens, and prolyl hydroxylase were used to clarify the process of liver fibrosis after CCl4 intoxication in rats by the direct immunoperoxidase method. In acute CCl4 intoxication, fat-storing cells (FSCs) were increased in number in the areas of necrosis around the central veins. These FSCs exhibited intense positive stainings for type I, type III collagens, and prolyl hydroxylase in well-developed rough endoplasmic reticula and Golgi apparatus. This was the direct evidence that the collagens formed after CCl4 intoxication are produced by FSCs. In chronic CCl4 intoxication, increased FSCs in and around the fibers also contained strong immunoreactive materials of both collagens and prolyl hydroxylase mainly in the rough endoplasmic reticula. These collagens were also present in the Golgi apparatus and vesicles close to the cytoplasmic membrane, demonstrating the exocytic process of collagen formation of FSCs. In contrast, faint immunoreactions of both collagens were found in the rough endoplasmic reticula and Golgi apparatus of hepatocytes during the process of fibrosis. These findings indicate that FSCs play an important role in fibrogenesis after acute and chronic CCl4 intoxication in the rat.     

Dynamics of the healing of skin wounds in the horse as compared with the rat.

The dynamics of skin wound healing were studied in three horses with either full thickness skin excision or with subcutaneously implanted polyvinyl alcohol sponges. Granulation tissue and reactive granuloma were harvested from three anatomical sites and were analyzed by morphological and biochemical methods at three time intervals. PVA sponges were also implanted in rats and studied by similar methods. No effect of wound or implant location on morphology or density of the sample tissue was found. In the horse, neutrophil infiltration was found in the tissue of wounds less than one day old. This tissue actively synthesized collagen and showed high activity of collagenase. The activity of prolyl hydroxylase (PH), however, was low. At later time sampling period (3, 7, 14 days), both granulation and granuloma tissue showed increasing PH activity, high activity of collagenase, and decreasing rate of collagen synthesis. Collagen content also increased with time. The reactive granuloma tissue found in rats showed less connective tissue reactivity than in the horse as seen by the dynamics of the morphological and biochemical changes of the tissue. We conclude that the healing in the horse is rather prompt and excessive and may tend toward abnormal repair reactions.     

甘草酸二铵对肝纤维化大鼠肝脏脂质过氧化与间质性胶原酶活性的影响

目的：探讨甘草酸二铵影响纤维化肝脏脂质过氧化与间质性胶原酶活性的抗肝纤维化作用机制。 方法： 以四氯化碳(CCl4)皮下注射与高脂肪低蛋白饮食复合因素诱导大鼠肝纤维化模型，而后予以甘草酸二铵口服治疗，正常大鼠与模型对照组给予等量生理盐水。HE染色与胶原染色观察大鼠肝组织炎性坏死与胶原沉积病理改变，按试剂盒方法检测血清肝功能（ALT、AST、Alb与总胆红素等）变化，检测肝组织主要过氧化损伤指标：超氧化物歧化酶（SOD）活性, 丙二醛（MDA）含量，谷胱甘肽（GSH）含量与谷胱甘肽过氧化物酶（GSH-Px）活性，水解法测定肝组织羟脯氨酸含量，酶底物反应法分析肝组织间质性胶原酶活性变化，RT-PCR法分析肝组织Ⅰ型前胶原基因表达。 结果： 与正常大鼠相比，模型大鼠肝脏有明显胶原沉积与肝纤维化，伴有不同程度的肝细胞炎性损伤坏死；甘草酸二铵药物组明显减轻模型大鼠肝组织损伤坏死与胶原沉积等病理变化。模型大鼠肝功能指标,包括血清总胆红素含量、ALT与AST活性、白蛋白含量均明显减少（P<0.01）。药物组大鼠血清总胆红素含量、AST与ALT活性显著低于模型组（P<0.05），而白蛋白含量高于模型组（P<0.05）。此外，药物组大鼠肝组织羟脯氨酸含量(151.3±37.3 μg/g)明显少于模型组(170.9±15.3 μg/g，P<0.05)，Ⅰ型前胶原基因表达弱于模型组（P<0.05）；肝组织MDA含量(1.96±0.23 μmol/g)低于模型组(2.44±0.32 μmol/L, P<0.05)，SOD水平(19.60±0.97 NU/g)高于模型组(20.60±0.33 NU/g，P<0.05)，GSH含量(47.0±9.1 g/g)与GSH-Px活性(53.1±4.1 U/g)高于模型组(41.2±3.5 g/g；46.7±6.1 U/g，P<0.05)；肝组织间质性胶原酶活性(43.89±7.74 U)高于模型组(32.01±2.75 U，P<0.05)。 结论： 甘草酸二铵有明显抗肝纤维化大鼠肝脏脂质过氧化损伤与提高肝组织间质性胶原酶活性的作用，该作用是药物抗肝纤维化的重要机制。     

Proteasome inhibition prevents development of experimental dermal fibrosis

Scleroderma is a chronic fibrotic disorder. Bortezomib, a proteasome inhibitor, is reported to attenuate experimentally induced renal and cardiac fibrosis. This study aimed to evaluate the preventive and therapeutic efficacies of bortezomib on a bleomycin (BLM)-induced scleroderma model. Dermal fibrosis was induced in Balb/c mice by subcutaneous BLM (100 μg/day) injections. Bortezomib (1.6 mg/kg twice/week) was applied intraperitoneally to BLM-injected mice during the first 3 weeks for preventive interventions and in the second 3 weeks for therapeutic interventions. IL-4 and TGF-β1 serum levels, dermal thicknesses, dermal inflammatory cell counts, and α-SMA-positive fibroblastic cell counts were determined, and type-I collagen, NF-κBp65, I-κBα, and JNK1 expressions were assessed. BLM applications increased serum IL-4 level, dermal inflammatory cell counts, α-SMA-positive cell counts, expression of type-I collagen, NF-κB, and JNK1, and dermal thickness in early stage of fibrosis, but serum IL-4 level and dermal inflammatory cell counts showed no increases in later stages. As a preventive intervention, bortezomib decreased dermal thickness, inflammatory cell infiltrations, fibroblastic activity, and expression of type-I collagen, NF-κB, and JNK1, but did not decrease fibroblastic activity and dermal thickness at later stages of fibrosis. Inflammatory status is prominent in the early stage of dermal fibrosis, but declines at later stages. In BLM-induced dermal fibrosis, bortezomib has a preventive anti-fibrotic and anti-inflammatory efficacy, but has no therapeutic anti-fibrotic efficacy in preexisting tissue fibrosis. These findings suggest that the effect of proteasome inhibition in early stages of dermal fibrosis may be related to its anti-inflammatory effects.     

Measurements of enzymes of collagen synthesis in rats with experimental silicosis

The levels of two enzymes of collagen biosynthesis namely lysyl oxidase and prolyl hydroxylase were measured in the lungs of rats 3 and 6 weeks after receiving a single intratracheal instillation of silica or sterile saline. Circulating levels of lysyl oxidase were also estimated. Significant increased lung-enzyme activities were observed in the silica-exposed rats at both time intervals. Plasma levels of lysyl oxidase were also found to be raised in the silica-exposed rats. These changes were not, however, accompanied by profound pathological alterations. These results demonstrate that after exposure to silica histologically detectable fibrosis is preceded by significant changes in the activities of enzymes associated with collagen synthesis.     

Measurements of enzymes of collagen synthesis in rats with experimental silicosis.

The levels of two enzymes of collagen biosynthesis namely lysyl oxidase and prolyl hydroxylase were measured in the lungs of rats 3 and 6 weeks after receiving a single intratracheal instillation of silica or sterile saline. Circulating levels of lysyl oxidase were also estimated. Significant increased lung-enzyme activities were observed in the silica-exposed rats at both time intervals. Plasma levels of lysyl oxidase were also found to be raised in the silica-exposed rats. These changes were not, however, accompanied by profound pathological alterations. These results demonstrate that after exposure to silica histologically detectable fibrosis is preceded by significant changes in the activities of enzymes associated with collagen synthesis.     

Effects of sensory denervation by neonatal capsaicin administration on experimental pancreatitis induced by dibutyltin dichloride

Increase in the number of intrapancreatic sensory nerve fibers has been implicated in the generation of pain in chronic pancreatitis. Because some sensory neurotransmitters (e.g., substance P) are known to have proinflammatory effects, we hypothesized that denervation of intrapancreatic nerves might influence not only pain generation but also inflammation. Neonatal Lewis rats were injected with capsaicin (50 mg/kg or 0 mg/kg), a neurotoxin, to induce denervation of primary sensory neurons. When rats reached 170–190 g body weight, experimental pancreatitis was induced by a single administration of dibutyltin dichloride (7 mg/mg). The severity of pancreatitis was evaluated in both groups in the acute phase (at 3 and 7 days) and chronic phase (at 28 days). At day 7, the sensory denervation induced by neonatal capsaicin administration inhibited pancreatic inflammation on both histological (determination of interstitial edema, expansion of interlobular septa and intercellular spaces, and inflammatory cell infiltration) and biochemical (intrapancreatic myeloperoxidase activity) evaluation. Furthermore, at day 28, glandular atrophy, pseudotubular complexes, and rate of fibrosis were each significantly lower in the capsaicin-pretreated group than in the vehicle-pretreated group. Our findings provide in vivo evidence that primary sensory neurons play important roles in both acute pancreatitis and chronic pancreatic inflammation with fibrosis.     

Effect of long-term sympathectomy on the arterial wall in rabbits and rats

The effect of chemical sympathectomy with 6-hydroxydopamine (6-OH-DA) on collagen formation in the aortic wall was investigated in rabbits and rats. Eight weeks after 6-OH-DA treatment of rabbits, there was a significant increase an collagen content in aortas and histologic changes in the elastic elements within the media. The possibility of a direct effect of 6-OH-DA on connective tissue formation was investigated in a subsequent experiment in rats. The rates of collagen synthesis and prolyl hydroxylase activity (PHA) were determined in aortas and in the fibrotic granuloma around subcutaneously implanted polyvinylalcohol sponges. Rates of collagen synthesis and PHA were significantly increased in the aortas of 6-OH-DA treated rats, but not in fibrotic granuloma, confirming the changes seen in the aorta of rabbits and suggesting that 6-OH-DA does not directly affect collagen synthesis. We conclude that the sympathetic nervous system influences the metabolic activity of the aorta. Our data indicate that when the aortic wall is deprived of adrenergic nervous stimulation, changes occur which resemble those seen in natural aging of the aorta. It is plausible to assume that such a metabolic derangement in the vessel wall will make these vessels more vulnerable to additional stresses.     

Increased serum type IV collagen peptide in carbon tetrachloride-treated rats

We developed a competitive enzyme-immunoassay for serum type IV collagen peptide as a marker of fibrogenesis, and examined the relationship between serum type IV collagen peptide and hepatic disorder in CCl4-treated rats. The rats were treated for 8 weeks and signs of liver damage began to appear from about week 2. With the progression of these signs to liver fibrosis, type IV collagen increased in the fibrous septa and especially in the perisinusoidal walls, where the increase was manifested as development of a real basement membrane beneath the sinusoidal endothelial cells. In CCl4-treated rats, serum type IV collagen peptide significantly increased with the progression of liver fibrosis. When CCl4 administration was stopped, the collagen peptide rapidly decreased without any rebound rise. An intimate relationship was found between the production of serum type IV collagen peptide and liver prolyl hydroxylase activity and the amount of collagen deposited in the liver. These results suggest that serum type IV collagen peptide will be a useful biochemical marker for the early detection of fibrogenesis in the liver.     

Hepatic collagen synthesis in a rat model of cirrhosis, and its modification by colchicine

Carbon tetrachloride inhalation in phenobarbitone treated rats caused a rapid rise in hepatic prolyl hydroxylase activity which was followed by an increase in hepatic collagen and free proline concentrations. Colchicine in a dose of 5 micrograms/day largely prevented the increase in hepatic collagen. This effect was not mediated by impairment of prolyl hydroxylase activity. Colchicine is of potential therapeutic value in preventing the progression of chronic liver disease to cirrhosis.     

Increased mRNAs for procollagens and key regulating enzymes in rat skeletal muscle following downhill running

 The purpose of the study was to investigate pre-translational regulation of collagen expression after a single bout of exercise. We analysed steady-state messenger ribonucleic acid (mRNA) levels for collagen types I, III and IV, α- and β-subunits of prolyl 4-hydroxylase and lysyl oxidase (enzymes modifying procollagen chains), and enzyme activity of prolyl 4-hydroxylase from rat soleus muscle (MS) and the red parts of quadriceps femoris muscle (MQF) after 12 h and after 1, 2, 4, 7 and 14 days of downhill (–13.5°) treadmill running at a speed of 17 m·min^–1 for 130 min. Histological and biochemical assays revealed exercise-induced muscle damage in MQF but not MS. Steady-state mRNA levels for the α- and β-subunits of prolyl 4-hydroxylase in MQF, lysyl oxidase in MS and MQF were increased 12 h after running, whereas prolyl 4-hydroxylase activity did not increase until 2 days after exercise. The mRNA levels for the fibrillar collagens (I and III) and basement membrane type IV collagen significantly increased 1 day and 12 h after exertion, respectively. Peak mRNA levels were observed 2–4 days after running, the increases being more pronounced in MQF than in MS. No significant changes were observed in types I or III collagen at the protein level. Strenuous downhill running thus causes an increase in gene expression for collagen types I and III and their post-translational modifying enzymes in skeletal muscle in a co-ordinated manner. These changes, together with the increased gene expression of type IV collagen, may represent the regenerative response of muscle extracellular matrix to exercise-induced injury and an adaptive response to running exertion. Received: 20 July 1998 / Received after revision: 30 November 1998 / Accepted: 26 January 1999     

Collagenase and diffuse interstitial pneumopathy

Chronic elastolytic activity in the lung is currently believed to be a major factor in pathogenesis of emphysema. Collagenase may have similar role in disorganizing lung collagen network, leading to fibrotic lung diseases (FLD). The possible involvement of collagenase in FLD is suggested by: 1) an increase collagenolitic activity in bronchoalveolar lavage fluid from patients with idiopathic pulmonary fibrosis or adult respiratory distress syndrome; 2) the accumulation and the activation of cells able to produce collagenases in FLD: fibroblasts, macrophages, neutrophils and eosinophils. However the exact role of collagenase in FLD is still unknown: it could inhibit neocollagen deposition, limiting fibrotic process or lead to further destruction of collagen network. Recent data suggest that genomic macrophage activation (such the proto oncogene c-SIS) may lead to several cellular events: 1) increase number and activation of fibroblasts with collagen synthesis; 2) increase collagenase production resulting of accumulation and activation of fibroblasts, macrophages, neutrophils. So we conclude that such a genomic macrophage activation may be the major factor contributing to the collagen network damage leading to lung tissue fibrosis.