Titration of non-replicating adenovirus as a vector for transducing active TGF-β1 gene expression causing inflammation and fibrogenesis in the lungs of C57BL/6 mice

Investigators have shown that interstitial pulmonary fibrosis (IPF) can be induced in rats by overexpressing transforming growth factor beta₁ (TGF‐β₁) through a replication‐deficient recombinant adenovirus vector instilled into the lungs ( Sime et al. 1997 ). We have shown that this vector induces IPF in fibrogenic‐resistant tumour necrosis factor alpha‐receptor knockout (TNF‐αRKO) mice ( Liu et al. 2001 ). The object of our studies is to understand how peptide growth factors, such as TGF‐β₁, mediate interstitial lung disease (ILD). To do so, we must be able to manipulate the dose of the factor and sort out its effects on multiple other mediators in the lung parenchyma. As a step in this complex process, in the studies reported here, we have determined the concentrations of the recombinant adenovirus vector carrying the gene for porcine active TGF‐β₁ (AVTGFβ₁) that have little apparent effect, cause clear induction of disease, or severe disease. The disease largely resolves by 28 days in all cases, thus providing a valuable model to understand the mechanisms of the IPF that is mediated, at least in part, by TGF‐β₁. The findings here show that 10⁶ plaque‐forming units (pfu) of AVTGFβ₁, provide essentially a ‘no‐effect’ dose, but even this amount of TGF‐β₁ causes a significant increase in whole‐lung collagen by day 28 after treatment. In contrast, 10⁸ and 10⁹ pfu cause severe IPF in 4 days, whereas 10⁷ and 5 × 10⁷ are intermediate for all parameters studied, i.e. TGF‐β protein, inflammatory cells, cell proliferation, pro‐α 1(I) collagen gene expression and whole‐lung collagen accumulation, and expression of growth factors such as TGF‐β₁, TNF‐α and PDGF‐A and ‐B. Interestingly enough, TGF‐β₁, as a potent blocker of epithelial cell proliferation, appears to suppress airway epithelial cell growth that would be expected during the inflammatory phase of IPF. Thus, this model system helps us to understand some quantitative aspects of TGF‐β₁ biological activity and allows us to manipulate this potent factor as a mediator of interstitial fibrogenesis.     

Overexpression of transforming growth factor-beta 1 in the valvular fibrosis of chronic rheumatic heart disease

For the purpose of determining the pathogenic role of transforming growth factor-beta1 (TGF-beta 1) in the mechanism of chronic rheumatic heart disease, we evaluated the expression of TGF-beta 1, proliferation of myofibroblasts, and changes in extracellular matrix components including collagen and proteoglycan in 30 rheumatic mitral valves and in 15 control valves. High TGF-beta 1 expression was identified in 21 cases (70%) of rheumatic mitral valves, whereas only 3 cases (20%) of the control group showed high TGF-beta 1 expression (p<0.001). Additionally, increased proliferation of myofibroblasts was observed in the rheumatic valves. High TGF-beta1 expression positively correlated with the proliferation of myofibroblasts (p=0.004), valvular fibrosis (p<0.001), inflammatory cell infiltration (p=0.004), neovascularization (p=0.007), and calcification (p<0.001) in the valvular leaflets. The ratio of proteoglycan to collagen deposition inversely correlated with TGF-beta 1 expression in mitral valves (p=0.040). In conclusion, an ongoing inflammatory process, the expression of TGF-beta 1, and proliferation of myofibroblasts within the valves have a potential role in the valvular fibrosis, calcification, and changes in the extracellular matrix that lead to the scarring sequelae of rheumatic heart disease.     

Renal fibrosis. Extracellular matrix microenvironment regulates migratory behavior of activated tubular epithelial cells

During progression of chronic renal disease, qualitative and quantitative changes in the composition of tubular basement membranes (TBMs) and interstitial matrix occur. Transforming growth factor (TGF)-beta(1)-mediated activation of tubular epithelial cells (TECs) is speculated to be a key contributor to the progression of tubulointerstitial fibrosis. To further understand the pathogenesis associated with renal fibrosis, we developed an in vitro Boyden chamber system using renal basement membranes that partially mimics in vivo conditions of TECs during health and disease. Direct stimulation of TECs with TGF-beta(1)/epithelial growth factor results in an increased migratory capacity across bovine TBM preparations. This is associated with increased matrix metalloproteinase (MMP) production, namely MMP-2 and MMP-9. Indirect chemotactic stimulation by TGF-beta(1)/EGF or collagen type I was insufficient in inducing migration of untreated TECs across bovine TBM preparation, suggesting that basement membrane integrity and composition play an important role in protecting TECs from interstitial fibrotic stimuli. Additionally, neutralization of MMPs by COL-3 inhibitor dramatically decreases the capacity of TGF-beta(1)-stimulated TECs to migrate through bovine TBM preparation. Collectively, these results demonstrate that basement membrane structure, integrity, and composition play an important role in determining interstitial influences on TECs and subsequent impact on potential aberrant cell-matrix interactions.     

Transfer of the active form of transforming growth factor-beta 1 gene to newborn rat lung induces changes consistent with bronchopulmonary dysplasia

Bronchopulmonary dysplasia is a chronic lung disease of premature human infancy that shows pathological features comprising varying sized areas of interstitial fibrosis in association with distorted large alveolar spaces. We have previously shown that transfer of active transforming growth factor (TGF)-beta 1 (AdTGF beta 1(223/225)) genes by adenovirus vector to embryonic lungs results in inhibition of branching morphogenesis and primitive peripheral lung development, whereas transfer to adult lungs results in progressive interstitial fibrosis. Herein we show that transfer of TGF-beta1 to newborn rat pups results in patchy areas of interstitial fibrosis developing throughout a period of 28 days after transfer. These areas of fibrosis appear alongside areas of enlarged alveolar spaces similar to the prealveoli seen at birth, suggesting that postnatal lung development and alveolarization has been inhibited. In rats treated with AdTGF beta 1(223/225), enlarged alveolar spaces were evident by day 21, and by 28 days, the mean alveolar cord length was nearly twice that in control vector or untreated rats. Hydroxyproline measurements confirmed the presence of fibrosis. These data suggest that overexpression of TGF-beta 1 during the critical period of postnatal rat lung alveolarization gives rise to pathological, biochemical, and morphological changes consistent with those seen in human bronchopulmonary dysplasia, thus inferring a pathogenic role for TGF-beta in this disorder.     

Age-dependent decrease in serum transforming growth factor (TGF)-beta 1 in healthy Japanese individuals; population study of serum TGF-beta 1 level in Japanese

Transforming growth factor-beta1 (TGF-beta1), a multi-functional cytokine, is involved in regulating a variety of cellular activities and the serum/plasma TGF-beta1 level is altered with various diseases. However, most published reports have described adult patients, and so we investigated the clinical significance of serum TGF-beta1 level in pediatric patients. The diagnostic application of the measurement of serum TGF-beta1 level depends critically on the control value, however, there is no information on the control value of serum TGF-beta1 for children. In the present study, we determined the serum TGF-beta1 level of healthy Japanese children as a control value with enzyme-linked immunosorbent assay (ELISA). The serum TGF-beta1 level of children (0-14 years old) was significantly higher than that of adults (over 15 years old) (p < 0.01). Thus, it is recommended that when the serum TGF-beta1 levels of patients are evaluated, they should be compared with those of age-matched controls.     

Protective effect of decorin on acute ischaemia-reperfusion injury in the rat kidney

Introduction

Transforming growth factor-β1 (TGF-β1) has a crucial role in collagen synthesis and fibrosis. TGF-β1 can be antagonized and/or reduced by the action of certain agents. We propose to identify the role of decorin in treatment of tubular and interstitial fibrosis and in the inhibition of TGF-β1 in an acute ischaemic kidney.

Material and methods

We grouped 34 female Sprague Dawley type rats into 3 groups as 9 sham, 9 ischaemia-reperfusion (I/R) and 16 I/R + decorin respectively. The rats in the I/R + decorin group had decorin administered intraperitoneally at the dose of 0.1 mg/kg for 9 days after reperfusion. After 9 days, all the rats in the 3 groups were unilaterally nephrectomized. The TGF-β1 level was measured immunohistochemically in the nephrectomized material.

Results

The TGF-β1 level was lower in the I/R + decorin group. Evaluation of apoptotic activity level by caspase staining showed a statistically significant difference between the 3 groups. The number of caspase stained cells was lower in the I/R + decorin group. The amount of collagen in interstitial tissue was higher in the I/R group than in the I/R + decorin group, but this difference was not statistically significant.

Conclusions

We found that the TGF-β1 level – the so-called initiator of fibrotic activity – and apoptotic activity were low in the I/R + decorin group. Additional studies must be performed to understand the role of decorin in inhibition of TGF-β1 and to assess decorin’s routine use in acute renal ischaemia.     

Oxidant-antioxidant imbalance as a potential contributor to the progression of human pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia. IPF is a disease with poor prognosis and an aggressive nature, and poses major challenges to clinicians. Thus, a large part of research in the area has focused on the pathogenesis on IPF. Characteristic features in IPF include fibrotic lesions devoid of inflammatory cell infiltrates. There are experimental models of lung fibrosis (e.g., bleomycin-induced fibrosis), but they typically contain a prominent inflammatory pattern in the lung, which leads to relatively diffuse lung fibrosis. Nonetheless, experimental models have provided important information about the progression and pathways contributing to the lung fibrosis, including activation of transforming growth factor beta (TGF-beta). Both patient material and experimental models of lung fibrosis have displayed marked elevation of several markers of oxidant burden and signs for disturbed antioxidant/oxidant balance. Several studies also suggest that reactive oxygen species can cause activation of growth-regulatory cytokines, including TGF-beta. In addition, there are indications that endogenous and exogenous antioxidants/redox modulators can influence fibrogenesis, protect the lung against fibrosis, and prevent its progression. Factors that restore the antioxidant capacity and prevent sustained activation of growth-regulatory cytokines may have a therapeutic role in IPF.     

TNF-alpha, PDGF, and TGF-beta(1) expression by primary mouse bronchiolar-alveolar epithelial and mesenchymal cells: tnf-alpha induces TGF-beta(1)

The bronchiolar-alveolar epithelium (BAE) is a primary target site for inhaled agents that cause lung injury. These cells, consequently, release a broad range of mediators that influence other cell populations, including interstitial lung fibroblasts that are central to the development of interstitial pulmonary fibrosis (IPF). A number of peptide growth factors (GF) have been postulated to be essential in the pathogenesis of IPF. We demonstrate here that primary populations of mouse BAE and mesenchymal cells, maintained in culture, synthesize four potent GF. These are platelet-derived growth factor isoforms (PDGF) A and B, transforming growth factor beta-1 (TGF-beta(1)), and tumor necrosis factor alpha (TNF-alpha). A mouse lung epithelial cell isolation technique pioneered in this laboratory has been used to purify the BAE cells to greater than 85% (80 +/- 5.6% alveolar type II and 9 +/- 2.3% Clara cells) in culture. Northern analysis, RNase protection assay, and immunocytochemistry (ICC) were used to establish mRNA and protein expression of the GF over time in the cultured BAE and mesenchymal cells. We show for the first time in these primary mouse lung cells that treatment of both cell types with TNF-alpha upregulates expression of TGF-beta(1). The four GF are produced by both epithelial and mesenchymal cells but with different temporal patterns. TGF-beta(1) is expressed constitutively by BAE and mesenchymal cells, whereas TNF-alpha expression wanes over time. The findings by ICC were consistent with levels of mRNA expression in both cell types. As genetically defined and altered mouse strains are becoming increasingly valuable for modeling lung disease, studying the gene expression patterns of target cells from these animals in vitro would be useful in sorting out the complex responses by individual cell types of the lung and the interactions among the multitude of mediators that are released during lung cell injury.     

The +869T/C polymorphism in the transforming growth factor-beta1 gene is associated with the severity and intractability of autoimmune thyroid disease

The severity of Hashimoto's disease (HD) and the intractability of Graves' disease (GD) vary among patients. To clarify whether the +869T/C polymorphism in the transforming growth factor-beta1 (TGF-beta1) gene, which is associated with TGF-beta1 expression, is involved in the intractability of GD and severity of HD, we genotyped the TGF-beta1 +869T/C polymorphism by polymerase chain reaction-restriction fragment length polymorphism method in genomic DNA samples from 33 patients with HD who developed hypothyroidism before they were 50 years old (severe HD) and 30 untreated, euthyroid patients with HD who were older than 50 years (mild HD). We also examined 48 euthyroid patients with GD who had been under treatment and were still positive for anti-thyrotropin receptor antibodies (intractable GD), 20 euthyroid patients with GD in remission and 45 healthy controls. The frequency of the T allele and the TT genotype were higher in patients with severe HD than in those with in mild HD. In contrast, the frequency of the CC genotype was higher in patients with intractable GD than in patients with GD in remission. In conclusion, the +869T/C polymorphism in the TGF-beta1 gene is associated with the severity and intractability of autoimmune thyroid disease.     

Serotonin mechanisms in heart valve disease I: serotonin-induced up-regulation of transforming growth factor-beta1 via G-protein signal transduction in aortic valve interstitial cells

Clinical disorders associated with increased serotonin [5-hydroxytryptamine (5-HT)] levels, such as carcinoid syndrome, and the use of serotonin agonists, such as fenfluoramine have been associated with a valvulopathy characterized by hyperplastic valvular and endocardial lesions with increased extracellular matrix. Furthermore, 5-HT has been demonstrated to up-regulate transforming growth factor (TGF)-beta in mesangial cells via G-protein signal transduction. We investigated the hypothesis that increased exposure of heart valve interstitial cells to 5-HT may result in increased TGF-beta1 expression and activity because of serotonin receptor-mediated signal transduction with activation of Galphaq, and subsequently up-regulation of phospholipase C. Thus, in the present study we performed a clinical-pathological investigation of retrieved carcinoid and normal valve cusps using immunohistochemical techniques to detect the presence of TGF-beta1 and other proteins associated with TGF-beta expression, including TGF-beta receptors I and II, latent TGF-beta-associated peptide (LAP), and alpha-smooth muscle actin. Carcinoid valve cusps demonstrated the unusual finding of widespread smooth muscle actin involving the interstitial cells in the periphery of carcinoid nodules; these same cells were also positive for LAP. Normal valve cusps were only focally positive for smooth muscle actin and LAP. In sheep aortic valve interstitial cell cultures 5-HT induced TGF-beta1 mRNA production and increased TGF-beta1 activity. 5-HT also increased collagen biosynthesis at the dosages studied. Furthermore, TGF-beta1 added to SAVIC cultures increased the production of sulfated glycan and hyaluronic acid. In addition, overexpression of Galphaq using an adenoviral expression vector for a constitutively active Galphaq mutant (Q209L-Galphaq) resulted in increased phospholipase C activity as well as up-regulation of TGF-beta expression and activity. These results strongly support the view that G-protein-related signal transduction is involved in 5-HT up-regulation of TGF-beta1. In conclusion, 5-HT-associated valve disease may be, in part, because of TGF-beta1 mechanisms.     

Increased iron deposition in rat liver fibrosis induced by a high-dose injection of dimethylnitrosamine

Using a developed rat model of hepatic necrosis and subsequent fibrosis induced by a high-dose intraperitoneal injection of dimethylnitrosamine (DMN), we studied iron deposition and expression of transforming growth factor-beta(1) (TGF-beta(1)) during the development of persistent liver fibrosis. Rats were sacrificed at several timepoints from 6 h to 10 months post-injection and the livers were examined for iron content and distribution, and for expression of alpha-smooth muscle actin, ED-1, TGF-beta(1), and collagen (alpha(2))I. Morphologic evidence of acute submassive hemorrhagic necrosis peaked at 36 h; on day 3 the residual parenchyma contained activated hepatic stellate cells (HSCs) and necrotic areas contained numerous macrophages; and on day 5, necrotic tissues and erythrocytes had been phagocytosed and macrophages contained abundant iron deposits. From days 7 to 10, iron-laden macrophages and activated HSCs (myofibroblasts) populated the fibrous septa in parallel. From week 2 to month 10, closely arranged macrophages and myofibroblasts were found in central-to-central bridging fibrotic tissue. TGF-beta(1) was strongly detected in both macrophages and HSCs during development of liver fibrosis. Our data suggest that increased iron deposition may be involved in the initiation and perpetuation of rat liver fibrosis. Iron-laden macrophages may influence HSCs through the action of TGF-beta(1) in DMN-induced liver fibrosis.     

HIF-1α and VEGF expression correlates with thrombus remodeling in cases of intravascular papillary endothelial hyperplasia

Intravascular papillary endothelial hyperplasia (IPEH) is histopathologically characterized by endothelium-lined papillary structures encircling an acellular fibrin core. The process of IPEH pathogenesis is unclear. The purpose of our study was to identify histopathological and immunohistochemical characteristics of IPEH to better understand the pathogenesis of this disease. After reviewing microscopic and medical records from Kyungpook National University Hospital, we selected 16 cases of IPEH. Masson’s trichrome and immunohistochemical staining as well as hematoxylin-eosin staining for 16 cases of IPH were performed. Immunohistochemical studies included CD31, CD68, mast cell tryptase, hypoxia-inducible factor-1 (HIF-1α), and vascular endothelial growth factor (VEGF). Sections from all our cases showed three distinct histological regions including a papillary portion with hyalinized fibrous or fibroblastic cores, an area containing an unorganized thrombus, and organization area with an ingrowth of endothelial cells, myofibroblasts, and fibroblasts. In the organization area, HIF-1α-positive cells were identified in the loose connective tissue. Endothelial cells forming vascular channels were negative for HIF-1α while VEGF was highly expressed in both interstitial mononuclear and endothelial cells. In the papillary portion, the cellular cores were strongly positive for both HIF-1α and VEGF, but the acellular cores were negative. Our investigation confirmed that IPEH is a reactive lesion that incidentally arises during the organization process of older thrombi. It was also found that HIF-1α and VEGF expression was dependent on the thrombus remodeling stage in cases of IPEH.     

Idiopathic pulmonary fibrosis fibroblasts become resistant to Fas ligand‐dependent apoptosis via the alteration of decoy receptor 3

Idiopathic pulmonary fibrosis (IPF) is an irreversible lethal lung disease with an unknown etiology. IPF patients' lung fibroblasts express inappropriately high Akt activity, protecting them in response to an apoptosis‐inducing type I collagen matrix. FasL, a ligand for Fas, is known to be increased in the lung tissues of patients with IPF, implicated with the progression of IPF. Expression of Decoy Receptor3 (DcR3), which binds to FasL, thereby subsequently suppressing the FasL–Fas‐dependent apoptotic pathway, is frequently altered in various human disease. However, the role of DcR3 in IPF fibroblasts in regulating their viability has not been examined. We found that enhanced DcR3 expression exists in the majority of IPF fibroblasts on collagen matrices, resulting in the protection of IPF fibroblasts from FasL‐induced apoptosis. Abnormally high Akt activity suppresses GSK‐3β function, thereby accumulating the nuclear factor of activated T‐cells cytoplasmic 1 (NFATc1) in the nucleus, increasing DcR3 expression in IPF fibroblasts. This alteration protects IPF cells from FasL‐induced apoptosis on collagen. However, the inhibition of Akt or NFATc1 decreases DcR3 mRNA and protein levels, which sensitizes IPF fibroblasts to FasL‐mediated apoptosis. Furthermore, enhanced DcR3 and NFATc1 expression is mainly present in myofibroblasts in the fibroblastic foci of lung tissues derived from IPF patients. Our results showed that when IPF cells interact with collagen matrix, aberrantly activated Akt increases DcR3 expression via GSK‐3β–NFATc1 and protects IPF cells from the FasL‐dependent apoptotic pathway. These findings suggest that the inhibition of DcR3 function may be an effective approach for sensitizing IPF fibroblasts in response to FasL, limiting the progression of lung fibrosis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Correlative insights into the immunoexpression of transforming growth factor beta-1 in acutely rejected renal allografts

Recent data suggest that early changes in the process of acute renal transplant rejection (ARTR) occurring during the first 3 months after transplantation include interstitial fibrosis. Transforming growth factor beta (TGF-beta) has been recognized as a key mediator of renal fibrogenesis; therefore, the present study was conducted to ascertain immunoexpression of TGF-beta in ARTR. Another purpose of our study was to determine whether TGF-beta could correlate with the interstitial area and to examine a possible relationship between TGF-beta and interstitial alpha-smooth muscle actin (alpha-SMA), endothelin-1 (ET-1) expression, interstitial T lymphocytes, and monocytes/macrophages. Twenty-four renal allograft biopsy specimens obtained from patients with ARTR were examined using percutaneous renal biopsy. As a control, we used 11 allograft biopsy specimens obtained from patients without any sign of rejection. Staining intensities of TGF-beta-1 in tubuli and of ET-1 in the endothelium of peritubular capillaries, in arterioles, and in the renal tubular epithelial cells were recorded semiquantitatively, whereas interstitial CD3+ cells, CD68+ cells, alpha-SMA expression, and the interstitial area were assessed quantitatively using computer image analysis system. Our study revealed that in the ARTR group, the mean values of the immunoexpression of TGF-beta-1, ET-1, interstitial CD3+ cells, CD68+ cells, alpha-SMA expression, and the interstitial area were significantly increased as compared with controls. In the ARTR group, there were significant positive correlations between immunostaining of TGF-beta-1 and ET-1, immunostaining of TGF-beta-1 and alpha-SMA, as well as immunostaining of TGF-beta-1 and interstitial volume. The correlation between immunostaining of TGF-beta-1 and CD 3+ cells tended to be negative; however, this did not reach statistical significance. We did not find any significant relationship between TGF-beta-1 and interstitial monocytes/macrophages. In controls, all these correlations were not significant. In conclusion, our correlative study suggests a role of TGF-beta-1 in early interstitial fibrotic changes in acutely rejected renal allografts, and we hypothesize that endothelin-1 and myofibroblasts pathways may play an important role in this process.