Demonstration by immunofluorescence that the same cells from chick embryo aortas synthesize elastin and collagen types I and III

Cells were isolated from the aortas of 17-day old chick embryos and they were stained with fluorescent antibodies specific for Type I collagen, Type I procollagen, Type III collagen, elastin and prolyl hydroxylase. The results indicated that the same cells simultaneously synthesize Type I procollagen, Type III procollagen and elastin. The synthesis of procollagens, and the presence of prolyl hydroxylase, in the same cells which synthesize elastin may well explain why elastin contains hydroxy-proline.     

TGFbeta-induced fibrogenesis of the pancreas

The biological cause of fibrosis is the accumulation of excessive amounts of extracellular matrix (ECM) which leads to tissue dysfunction and organ failure. A strong correlation can be found between pancreatic diseases and fibrotic processes, in particular chronic pancreatitis and pancreatic cancer. There is growing evidence that pancreatic fibrosis represents a dysregulation of the normal repair processes after injury. This concept is based on the findings that fibrosis and tissue repair involve similar biological reactions regulated by the same group of molecules. The best characterized example for these regulatory molecules are the members of the transforming growth factor beta family (TGFβ). TGFβ1 represents the prototype of this family of highly similar growth factors, with the unique ability to stimulate the expression and deposition of extracellular matrix and to inhibit its degradation. Growth factor-induced fibrotic events are targeted by a myofibroblast-like cell called pancreatic stellate cell (PSC). These cells show enhanced expression of all-important ECM proteins after TGFβ stimulation including collagen, fibronectin and proteoglycans. At the same time TGFβ inhibits the degradation of ECM by blocking the secretion of proteases and stimulating the production of naturally occurring protease inhibitors.     

Serum markers for fibrosis and plasma transforming growth factor-β1 in patients with hepatocellular carcinoma in comparison with patients with liver cirrhosis

In order to elucidate collagen metabolism in hepatocellular carcinoma (HCC) tissue, we compared levels of different potential markers of collagen metabolism and plasma transforming growth factor-β₁ in patients with HCC and in patients with liver cirrhosis. Serum levels of prolyl hydroxylase and the tissue inhibitor of metalloproteinase-1 in patients with HCC were significantly higher than those in patients with liver cirrhosis and increased with the size of the HCC tumour, whereas the serum levels of procollagen type III propeptide and type IV collagen 7S domain were similar in the two groups. In HCC, the increased plasma transforming growth factor-β₁ levels were closely correlated with serum levels of prolyl hydroxylase and the tissue inhibitor of metalloproteinase-1. These findings suggest that, in HCC tissue, the intracellular biosynthesis of collagen is enhanced, whereas the secretion of procollagen is disturbed and the degradation of collagen is suppressed by the excess production of the tissue inhibitor of metalloproteinase-1. The results also suggest that plasma transforming growth factor-β₁ plays an important role in the altered metabolism of collagen in HCC.     

Collagen synthesis by cultured rat liver cells isolated from chronically alcohol-treated rats

Incorporation rates of 14C-proline into collagen hydroxyproline in cultured Ito cells and hepatocytes isolated from chronically alcohol-treated rats were studied in order to clarify the role of Ito cells in the development of alcoholic liver fibrosis pathogenesis. In the cultured Ito cells isolated from alcohol-treated rats, prolyl hydroxylase activity significantly increased. Total collagen synthesis tended to increase in the alcohol group, and the increase in intracellular intact collagen was statistically significant. More than half of the 14C-radioactivity in intact collagen in cultured Ito cells from control rats was found in collagen other than types I and III collagen (mainly type IV collagen). In Ito cells from alcohol-treated rats, synthesis of collagen other than type I and III significantly increased and type I collagen synthesis tended to be decreased. No significant difference was found in collagen synthesis between the cultured hepatocytes from alcoholic and control rats. These results suggest that chronic alcohol consumption stimulates collagen synthesis in Ito cells, especially type IV collagen. This stimulation of Ito cells may play a role in the development of alcoholic liver fibrosis.     

Signal transduction in pancreatic stellate cells

Pancreatic fibrosis is a characteristic feature of chronic pancreatitis and of desmoplastic reaction associated with pancreatic cancer. For over a decade, there has been accumulating evidence that activated pancreatic stellate cells (PSCs) play a pivotal role in the development of pancreatic fibrosis in these pathological settings. In response to pancreatic injury or inflammation, quiescent PSCs undergo morphological and functional changes to become myofibroblast-like cells, which express α-smooth muscle actin (α-SMA). Activated PSCs actively proliferate, migrate, produce extracellular matrix (ECM) components, such as type I collagen, and express cytokines and chemokines. In addition, PSCs might play roles in local immune functions and angiogenesis in the pancreas. Following the initiation of activation, if the inflammation and injury are sustained or repeated, PSCs activation is perpetuated, leading to the development of pancreatic fibrosis. From this point of view, pancreatic fibrosis can be defined as pathological changes of ECM composition in the pancreas both in quantity and quality, resulting from perpetuated activation of PSCs. Because the activation and cell functions in PSCs are regulated by the dynamic but coordinated activation of intracellular signaling pathways, identification of signaling molecules that play a crucial role in PSCs activation is important for the development of anti-fibrosis therapy. Recent studies have identified key mediators of stimulatory and inhibitory signals. Signaling molecules, such as peroxisome proliferator-activated receptor-γ (PPAR-γ), Rho/Rho kinase, nuclear factor-κB (NF-κB), mitogen-activated protein (MAP) kinases, phosphatidylinositol 3 kinase (PI3K), Sma- and Mad-related proteins, and reactive oxygen species (ROS) might be candidates for the development of anti-fibrosis therapy targeting PSCs.     

Sequential connective matrix changes in experimental acute pancreatitis. An immunohistochemical and biochemical assessment in the rat

The effects of acute pancreatitis on the rat pancreatic connective tissue matrix were studied following intraductal pancreatic injection of trypsin solution and serial killing of the animals. Pancreatic tissue was examined using light microscopy, hydroxyproline measurement and indirect immunofluorescence, using antibodies against collagen types I, III, IV, procollagen III, fibronectin and laminin. Light microscopy revealed that acute pancreatitis was present for up to four days after injection and that perilobular and intralobular fibrosis appeared at four days and subsequently regressed. Immunofluorescence studies demonstrated an abnormal fibronectin deposit at one day in acute pancreatitis. At four days this deposit was co-located with fibrosis which was composed of collagen and procollagen type III. By eight days the immunofluorescence and light microscopic changes were minimal. Biochemical analysis confirmed a significant rise in hydroxyproline concentration at four days, which was maximal at eight days, subsequently decreasing. This peak at eight days probably reflects collagen breakdown products.     

Growth Arrest and Decrease of α-SMA and Type I Collagen Expression by Palmitic Acid in the Rat Hepatic Stellate Cell Line PAV-1

Liver fibrosis is characterized by an activation of hepatic stellate cells (HSC). During primary culture HSC evolve from a quiescent into an activated phenotype which is characterized by α-smooth muscle actin (α-SMA) up-regulation, increase in cell growth, and extracellular matrix secretion. HSC culture with trans-resveratrol can lead to deactivation of myofibroblast-like HSC. We used an HSC line, PAV-1, to check the role of retinol and palmitic acid in the deactivation process of HSC. Using mass and metabolic-based methods, Western blot and immunocytochemistry assays, we demonstrated that treatment with palmitic acid (75 μM) alone or in combination with retinol (2 μM) significantly decreased cell proliferation and α-SMA expression. We also established that the association of both compounds strongly decreased collagen type I expression. Our results suggest the potential use of palmitic acid alone or in combination with retinol to induce HSC deactivation.     

Altered expression of extracellular matrix molecules and their receptors in chronic pancreatitis and pancreatic adenocarcinoma in comparison with normal pancreas

Summary The aim of this study was to elucidate the expression and distribution patterns of both integrins and extracellular matrix (ECM) molecules in chronic pancreatitis (CP) and pancreatic adenocarcinoma (PC) compared with normal pancreas (NP). Expression of nine α-subunits (α2-α6, αv, αl, αm, and αx), four β-subunits (β1, β3-β5), and four ECM molecules (type IV collagen, laminin, fibronectin, and vitronectin) was investigated immunohistochemically. In CP, all integrins except αv showed nearly the same staining patterns compared with NP. Some acinar cells in CP expressed αv. Whereas α2, α3, and α6 expression was stronger and diffuse, no α5 expression was seen in PC. Basement membrane (BM) showed continuous staining in CP, whereas it showed discontinuous/absent staining in PC with antitype IV collagen, laminin, and vitronectin antibodies. Some carcinoma cells showed reverse correlation between α2, α3, and α6 expression and type IV collagen and laminin expression. Fibronectin showed diffuse stromal expression in CP and PC. Some acinar cells or duct cells in CP carcinoma cells in PC showed intracellular VN expression. These results suggest that these integrins and ECM molecules are involved in inflammatory and malignant processes in pancreas.     

Age-related atrial fibrosis

Many age-related diseases are associated with, and may be promoted by, cardiac fibrosis. Transforming growth factor (TGF)-β, hypoxia-induced factor (HIF), and the matrix metalloproteinase (MMP) system have been implicated in fibrogenesis. Thus, we investigated whether age is related to these systems and to atrial fibrosis. Right atrial appendages (RAA) obtained during heart surgery (n = 115) were grouped according to patients’ age (<50 years, 51–60 years, 61–70 years, or >70 years). Echocardiographic ejection fractions (EF) and fibrosis using Sirius-red-stained histological sections were determined. TGF-β was determined by quantitative RT-PCR and hypoxia-related factors [HIF1α, the vascular endothelial growth factor (VEGF)-receptor, CD34 (a surrogate marker for microvessel density), the factor inhibiting HIF (FIH), and prolyl hydroxylase 3 (PHD 3)] were detected by immunostaining. MMP-2 and -9 activity were determined zymographically, and mRNA levels of their common tissue inhibitor TIMP-1 were determined by RT-PCR. Younger patients (<50 years) had significantly less fibrosis (10.1% ± 4.4% vs 16.6% ± 8.3%) than older individuals (>70 years). While HIF1α, FIH, the VEGF-receptor, and CD34 were significantly elevated in the young, TGF-β and PHD3 were suppressed in these patients. MMP-2 and -9 activity was found to be higher while TIMP-1 levels were lower in older patients. Statistical analysis proved age to be the only factor influencing fibrogenesis. With increasing age, RAAs develop significantly more fibrosis. An increase of fibrotic and decrease of hypoxic signalling and microvessel density, coupled with differential expression of MMPs and TIMP-1 favouring fibrosis may have helped promote atrial fibrogenesis. No author has any conflicts of interest to disclose.     

Mechanisms of pancreatic fibrosis and applications to the treatment of chronic pancreatitis

Pancreatic stellate cells (PSCs) play a crucial role in pancreatic fibrogenesis in chronic pancreatitis and in the desmoplastic reaction of pancreatic cancer. When PSCs are stimulated by oxidative stress, ethanol and its metabolite acetaldehyde, and cytokines, the phenotype of quiescent fat-storing cells converts to myofibroblastlike activated PSCs, which then produce extracellular matrix, adhesion molecules, and various chemokines in response to cytokines and growth factors. Recent data suggest that PSCs have a phagocytic function. Plateletderived growth factor is a potent stimulator of PSC proliferation. Transforming growth factor β, activin A, and connective tissue growth factor also play a role in PSC-mediated pancreatic fibrogenesis through autocrine and paracrine loops. Following pancreatic damage, pathophysiological processes that occur in the pancreas, including pancreas tissue pressure, hyperglycemia, intracellular reactive oxygen species production, activation of protease-activated receptor 2, induction of cyclooxygenase 2, and bacterial infection play a role in sustaining pancreatic fibrosis through increased PSC proliferation and collagen production by PSCs. Targeting PSCs might be an effective therapeutic approach in chronic pancreatitis. Various substances including vitamin A, vitamin E, polyphenols, peroxisome proliferator-activated receptor γ ligands, and inhibitors of the renin-angiotensin system show great promise of being useful in the treatment of chronic pancreatitis.     

Increased prolyl hydroxylase activity and collagen synthesis in hepatocyte cultures exposed to superoxide

Primary monolayer cultures of rat hepatocytes at confluence were exposed to an exogenously added source of superoxide, and its influence on collagen synthesis was examined. Superoxide was generated by the addition of dihydroxyfumarate to the culture medium. Exposure of hepatocytes to dihydroxyfumarate greatly stimulated the activity of prolyl hydroxylase and the synthesis of collagen. A significant increase in prolyl hydroxylase activity was observed with 5 micrograms per ml dihydroxyfumarate in 24 hr relative to that in the untreated cultures. Maximum stimulation of greater than 3-fold compared to the control value was elicited by 25 micrograms per ml dihydroxyfumarate. When scavengers of superoxide such as superoxide dismutase and Cu(Lys)2 were added in the medium, the increase in prolyl hydroxylase activity induced by dihydroxyfumarate was nearly abolished. Experiments with actinomycin D indicated that synthesis of new RNA was involved in the stimulation of prolyl hydroxylase activity. Analysis of collagen synthesis in cultures exposed to dihydroxyfumarate also showed a marked increase compared to that of the untreated cultures. The presence of superoxide dismutase in the medium significantly reduced the increase in collagen synthesis. Our results indicate that superoxide mediates the stimulation of collagen synthesis in hepatocytes. These findings may provide a possible explanation for excess collagen formation during induced liver fibrosis.     

Alteration of the langerhans islets in pancreatic cancer patients

Summary An abnormal glucose metabolism occurs in up to 80% of pancreatic cancer patients shortly or a few months before the first clinical admission. Reasons for this abnormality are obscure. We investigated immunohistochemically the pattern of islets in 14 pancreatic cancer specimens and used 14 chronic pancreatitis samples and 10 normal pancreata as controls. To study the topographical relationship of these islets to the cancer, islets in four different arbitrary zones within and around the cancer were evaluated. Ten out of 14 cancer specimens showed a significant loss of β cells (p<0.005) and eight of them also showed a significant increase of α cells (p<0.005), all of them from hyperglycemic patients. Most affected islets were found within zone 1 (intratumoral) and zone 2 (peritumoral), to a lesser extent in zone 3 (acini close to tumor) and none in zone 4 (acini remote from tumor). No comparable changes were found in chronic pancreatitis patients. The incidence of 72% with alteration of islets in our material correlates with the frequency of abnormal glucose levels in human pancreatic cancer patients. Our findings support the notion that islet cell abnormalities is likely caused by substances released from cancer cells.     

Ellagic Acid Inhibits Pancreatic Fibrosis in Male Wistar Bonn/Kobori Rats

The key pathological features of chronic pancreatitis are chronic inflammation, acinar atrophy, and pancreatic fibrosis. We have previously shown that ellagic acid, a plant-derived polyphenol found in fruits and nuts, inhibited activation of pancreatic stellate cells, a major profibrogenic cell type in the pancreas, in vitro. Here we examined whether ellagic acid inhibited the development of pancreatic fibrosis in vivo. Ellagic acid was administered orally in the diet to ten-week-old male Wistar Bonn/Kobori rats, an experimental model of spontaneous chronic pancreatitis, for ten weeks. Ellagic acid (100 mg/kg body weight/day) attenuated pancreatic inflammation and fibrosis. The protective effects were confirmed by an increase in pancreatic weight and decreases in myeloperoxidase activity (an index of neutrophil infiltration), collagen content, transforming growth factor-β1 expression, and the number of α-smooth muscle actin-positive cells (activated pancreatic stellate cells) and ED-1-positive cells (macrophages/monocytes). Ellagic acid inhibited the production of reactive oxygen species in pancreatic stellate cells in response to transforming growth factor-β1 or platelet-derived growth factor. Our results suggest that ellagic acid might be a candidate for treatment of chronic pancreatitis. N. Suzuki and K. Kikuta contributed equally to this work.     

rAAV2-TGF-β<Subscript>3</Subscript> Decreases Collagen Synthesis and Deposition in the Liver of Experimental Hepatic Fibrosis Rat

Background/Aims  

Hepatic fibrosis is one kind of common wound-healing response to chronic liver injury. Transforming growth factor (TGF)-β₃ performs an anti-fibrosis function under certain conditions such as pancreatic fibrosis and wound healing. This study aimed at investigating the effect of TGF-β₃ on the histology in the liver of rat with liver fibrosis.     

Intra- and extracellular enzymes of collagen biosynthesis as biological and chemical targets in the control of fibrosis

The several steps in the pathway for the biosynthesis of fibrillar collagen are reviewed to illustrate potential sites for the chemotherapeutic control of fibrosis. Particular emphasis is placed upon the properties and inhibition of lysyl oxidase, the enzyme which initiates the covalent crosslinking of extracellular collagen molecules converting these to insoluble fibers, and upon the properties and inhibition of prolyl hydroxylase, the intracellular enzyme which hyroxylates proline residues within collagen.     

Suppression of Collagen Production in Norepinephrine Stimulated Cardiac Fibroblasts Culture: Differential Effect of α and β-Adrenoreceptor Antagonism

Objectives  Prolonged sympathetic activation is damaging to the heart and experimental norepinephrine (NE) infusion induces the deposition of myocardial collagen. This study investigated the effects of NE on collagen and transforming growth factor-β1 (TGF-β1) gene expression in rat cardiac fibroblasts (CF) culture, and compared the anti-fibrotic effect of α and β (both selective and non-selective adrenergic receptor antagonists) receptors. Methods and Results  Rat CF were cultured in the presence of NE (0.01 to100 μM) for 24 hours. Procollagen types I and III as well as TGF-β1 gene expressions were measured by real-time quantitative PCR method. Collagen protein level was measured by Sirius red-based colorimetric method and Western blot analysis. The optimal dose of NE on fibrogenesis was 0.1 μM. Incubation for 24 hours increased procollagen I, III and TGF-β1 gene expression by 1.35 ± 0.23, 1.26 ± 0.16 and 1.35 ± 0.21 fold, respectively (all p < 0.05). The collagen protein was increased by both Sirius-red assay (0.120 ± 0.03 vs 0.093 ± 0.04 μg/total μg of protein, p < 0.05) and Western blot analysis (1.29 ± 0.26 fold, p < 0.05), when compared with the control group. Addition of carvedilol (a non-selective β-blocker with α-blockage activity) inhibited the effect of NE on procollagen I (0.64 ± 0.17 vs 1.28 ± 0.08 fold, p < 0.01), procollagen III gene (0.47 ± 0.16 vs 1.45 ± 0.29 fold, p < 0.01) and collagen protein expressions (0.99 ± 0.12 vs 1.26 ± 0.31 fold, p < 0.05, respectively). Doxazosin (an α-blocker) also inhibited the effect of NE on procollagen I (0.88 ± 0.30 vs 1.28 ± 0.08 fold, p < 0.01), procollagen III gene (0.64 ± 0.13 vs 1.45 ± 0.29 fold, p < 0.01) and collagen protein expression (0.90 ± 0.11 vs 1.26 ± 0.31 fold, p < 0.01 respectively). Such inhibitory effects were not seen in metoprolol (a β1-selective blocker) and propranolol (a non-selective β blocker). Furthermore, all the 4 drugs were unable to inhibit the NE induced TGF-β1 gene over-expression. Conclusions  In conclusion, NE increased collagen gene and protein expressions in CF culture. This effect is likely mediated through α-receptor as they were normalized by pretreatment with carvedilol and doxazosin, but not β-blockers such as propranolol and metoprolol. Also, TGF-β1 doesn’t seem to play a role in carvedilol inhibition of NE induced fibrogenesis.     

Serum Markers for Hepatic Fibrosis in Alcoholic Liver Disease: Which Is the Best Marker,Type III Procollagen,Type IV Collagen,Laminin, Tissue Inhibitor of Metalloproteinase,or Prolyl Hydroxylase?

Although various serum markers for the evaluation of hepatic fibrosis have been introduced, it remains unclear which is the best marker to evaluate the hepatic fibrosis observed in alcoholic liver disease (ALD). In this study, we measured serum concentrations of the immunoreactive β-subunit of prolyl hydroxylase, procollagen type III peptide, the 7s domain (7s-IV) and triple-helix domain (TH-IV) of type IV collagen, laminin, and tissue inhibitor of metalloproteinase (TIMP) in patients with and without ALD (non-ALD), and controls to evaluate the best serum marker reflecting the characteristic histologic features of ALD. Alter Azan-Mallory and silver-impregnated reticulin staining, histologic specimens were examined; and the degree of hepatic fibrosis was classified as mild, moderate, or severe. Although serum concentrations of all markers, except for TIMP, in patients with each type and stage of liver disease were higher than cut-off values and these concentrations increase with the progression of liver disease, statistical analyses indicate that serum TH-IV concentration is the best marker to distinguish ALD from non-ALD. A good correlation was also found between the hepatic type IV collagen content and serum TH-IV, but not serum 7s-IV concentration. Moreover, after abstinence from alcohol, serum concentrations of TH-IV decreased more quickly than other serum markers. These results clearly suggest that, compared with other markers, serum concentration of TH-IV may more strongly reflect the histologic features of ALD. However, other serum markers, except for TIMP, may be useful in evaluating the degree of hepatic fibrosis.     

TGF-β1 Induces Human Bronchial Epithelial Cell-to-Mesenchymal Transition in Vitro

The subepithelial fibrosis component of airway remodeling in asthma is mediated through induction of transforming growth factor-β1 (TGF-β1) expression with consequent activation of myofibroblasts to produce extracellular matrix proteins. The number of myofibroblasts is increased in the asthmatic airway and is significantly correlated with the thickness of lamina reticularis. However, much is still unknown regarding the origin of bronchial myofibroblasts. Emerging evidence suggests that myofibroblasts can derive from epithelial cells by an epithelial-to-mesenchymal transition (EMT). In this study we investigated whether TGF-β1 could induce bronchial epithelial EMT in the human bronchial epithelial cell. Cultured human bronchial epithelial cells, 16HBE-14o, were stimulated with 10 ng/ml TGF-β1. Morphologic changes were observed and stress fiber by actin reorganization was detected by indirect immunostaining. The expression of α-SMA (α-smooth muscle actin) and the epithelial cell marker E-cadherin were detected in those 16HBE-14o cells after TGF-β1 stimulation for 72 h, using immunostaining and RT-PCR. The contents of collagen I were determined by radioimmunoassay, and the levels of endogenous TGF-β1 were measured with ELISA. Human bronchial epithelial cells stimulated with TGF-β1 were converted from a “cobblestone” epithelial structure into an elongated fibroblast-like shape. Incubation of human bronchial epithelial cells with TGF-β1 induced de novo expression of α-SMA, increased formation of stress fiber by F-actin reorganization, and loss of epithelial marker E-cadherin. Moreover, a significant increase in the levels of collagen I and endogenous TGF-β1 released from bronchial epithelial cells stimulated with TGF-β1 were observed. These results suggested that human bronchial epithelial cells, under stimulation of TGF-β1, underwent transdifferentiation into myofibroblasts.