Role of collagen-binding heat shock protein 47 and transforming growth factor-beta1 in conjunctival scarring in ocular cicatricial pemphigoid.
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PURPOSE: Submucosal fibrosis due to excessive accumulation of collagens is an important histologic feature in the pathogenesis of ocular cicatricial pemphigoid (OCP). Heat shock protein 47 (HSP47), a collagen-binding protein, plays an important role in the biosynthesis of procollagens. In the present study, we examined the role of HSP47 in conjunctival scarring in patients with OCP. METHODS: Biopsy specimens of the conjunctiva of 15 patients with OCP and 5 normal subjects were studied for the expression of HSP47, transforming growth factor (TGF)-beta1, type I collagen, and type III collagen. The role of TGF-beta1 on the induction of HSP47 and type I collagen by conjunctival fibroblasts was studied by immunostaining, Western blot analysis, and quantitative real-time PCR. RESULTS: Compared with the control, increased accumulations of type I and type III collagens were detected by immunohistochemistry in fibrotic conjunctiva of patients with OCP. Weak and sparse expression of HSP47 was detected in the epithelial cells and stromal fibroblasts in control conjunctival tissues. In contrast to the control, the expression of HSP47 was markedly increased in the stromal fibroblasts in conjunctival tissues obtained from patients with OCP, as detected by immunohistochemistry. By quantitative real-time PCR, compared with control conjunctival tissues, a 3.4-fold increase in the expression of HSP47 was noted in the conjunctival tissues obtained from patients with OCP. Similar to conjunctival tissues, fibroblasts isolated from conjunctiva of patients with OCP exhibited 4.8-fold increase in the expression of HSP47, compared with control fibroblasts. When conjunctival fibroblasts were treated with various concentration of TGF-beta1, upregulation in the expression of HSP47 and type I collagen was detected. CONCLUSIONS: This study demonstrated increased expression of HSP47 and TGF-beta1 by conjunctival fibroblasts in biopsy specimens obtained from patients with OCP. TGF-beta1 induced the expression of HSP47 and type I collagen by conjunctival fibroblasts. Increased levels of TGF-beta1 and HSP47 may regulate increased synthesis, assembly, and production of collagens and thereby could significantly contribute to the process of conjunctival scarring in patients with OCP. (+info)
Progressive adult-onset emphysema in transgenic mice expressing human MMP-1 in the lung.
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Mice with lung-specific expression of human matrix metalloproteinase-1 (MMP-1) develop emphysematous changes similar to those seen in smoking-induced emphysema in humans. Morphometric analyses of three transgenic lines [homozygous colony (Col) 34, Col 50, and Col 64] with varying temporal expression of MMP-1 were undertaken to determine the validity of this animal as a model of adult-onset emphysema. Line 50 mice, which have early expression of MMP-1 (14 days postconception), exhibited morphometric changes by 5 days of age. In contrast, homozygous line 34 and 64 with delayed expression (birth and 2 wk of age) were normal up until 4 wk of age when progressive changes in their mean linear intercept were first noted. In contrast, heterozygous mice from line 34 with lower transgene expression did not develop emphysema until 1 yr of age. The changes in mean linear intercept coincided with an increase in lung compliance. Emphysema in these mice was associated with decreased immunostaining for type III collagen within the alveolar septa. This study provides evidence that MMP-1 induces progressive adult-onset emphysema by the selective degradation of type III collagen within the alveolar wall. (+info)
Morphology of aging lung in F344/N rat: alveolar size, connective tissue, and smooth muscle cell markers.
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This study investigated the morphological changes of lungs in F344/N rats (9-36 months old). We initially examined general and quantitative morphological changes, and then we used immunohistochemistry to detect distributional changes in collagen subtypes (types I, III, and IV) and smooth muscle cell (SMC) markers (alpha-smooth muscle actin (ASMA), gamma-smooth muscle actin (GSMA), desmin, and vimentin) in the lungs. In 24-month-old rats, alveolar ducts and alveolar sacs were enlarged, and alveoli were wider and shallower than in younger animals. In old rats (>/=27 months), terminal and respiratory bronchioles and alveolar ducts were dilated and alveoli were more extended than in 24-month-old rats. No age-related distributional changes were observed for collagen types I, III, and IV as revealed by immunohistochemistry, or elastin as revealed by resorsin fuchsin. SMCs in the extra- and intrapulmonary bronchi were immunoreactive for ASMA, GSMA, and desmin, but not for vimentin at all ages. In old rats (>/=27 months), SMCs were loosely arranged in comparison with younger animals, and stainability for GSMA and desmin was decreased. In the respiratory bronchioles and alveolar ducts, a few cells immunoreactive for ASMA and vimentin were observed in the smooth muscle aggregations of the alveolar orifice in rats younger than 12 months. In older rats (>20 months), cells immunoreactive for ASMA and vimentin were increased in septal tips. In conclusion, extension of distal airways and immunohistochemical changes of SMC markers in F344/N rat lungs were evident by approximately 24 months of age, but there was no apparent change in connective tissue morphology. (+info)
Chymase inhibition prevents cardiac fibrosis and improves diastolic dysfunction in the progression of heart failure.
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BACKGROUND: Angiotensin (Ang) II, which plays a crucial role in the cardiac remodeling process, is generated via angiotensin-converting enzyme (ACE); however, an alternative generation pathway, chymase, which is stored in the mast cells, also exists in the heart. Cardiac chymase is insensitive to ACE inhibitors (ACEIs), and heart chymase promotes interstitial fibrosis by affecting collagen metabolism via transforming growth factor-beta in vitro. Therefore, selective chymase blockade seems to be an important strategy in the prevention of cardiac remodeling METHODS AND RESULTS: We evaluated the effects of a specific chymase inhibitor, SUNC8257 (Chy I; 10 mg/kg twice a day; n=7), on changes in cardiac structures, Ang II levels, and gene expressions, which are characterized as molecular markers for fibrosis, in dogs with tachycardia induced heart failure (HF). In HF, the number of chymase enzyme-positive mast cells increased in the left ventricle (LV) compared with the normal group; however, Chy I significantly decreased the mast cell density and cardiac Ang II levels. Despite no significant differences in LV systolic function compared with the vehicle group, Chy I decreased LV end-diastolic pressure and shortened the prolongation of tau. Chy I suppressed collagen-type I and III and transforming growth factor-beta mRNA levels and decreased fibrosis in the LV compared with the vehicle. CONCLUSIONS: The chymase pathway may be critical for cardiac diastolic dysfunction accompanied with fibrosis. Chronic chymase inhibition may therefore become an important strategy in the prevention of cardiac remodeling in HF. (+info)
Determination of serum fibrosis indexes in patients with chronic hepatitis and its significance.
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OBJECTIVES: To study the relationship between serum levels of hyaluronic acid (HA), type III procollagen (PCIII), laminin (LN), type IV collagen (IV-C) and hepatic fibrosis and to determine their value in clinical practice. METHODS: 2600 serum samples from chronic hepatitis patients were assayed for fibrosis indexes including HA, PCIII, LN and IV-C with RIA. Liver biopsy was performed in 280 of those patients and the biopsy material was examined histopathologically. The inflammation grade of the liver, stage of fibrosis and degree of chronic hepatitis were recorded and were compared with fibrotic indexes. RESULTS: Among 2600 chronic hepatitis patients, every fibrotic index had a significant correlation with the inflammation grade, fibrosis staging and the degree of chronic hepatitis (P < 0.01). The coefficient correlation of the results of histopathological examinations to HA was 0.544, 0.548 and 0.468 respectively, that to PCIII, 0.495, 0.424 and 0.335, that to LN, 0.214, 0.204 and 0.184, and that to IV-C, 0.406, 0.404 and 0.412, respectively. CONCLUSIONS: Serum fibrosis indexes are fairly well correlated with the inflammation grade of the liver, fibrosis staging and the degree of chronic hepatitis. However, as diagnostic markers, they should be considered in combination with liver function tests, ultrasonography and clinical manifestations. (+info)
Should increasing the dose or adding an AT1 receptor blocker follow a relatively low dose of ACE inhibitor initiated in acute myocardial infarction?
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OBJECTIVE: Based on currently available clinical evidence, we should use high-dose angiotensin converting enzyme inhibitor (ACE-I) for patients with acute myocardial infarction (MI), initiating it at incremental doses to avoid excessive hypotension. Recent animal studies with acute MI models failed to demonstrate the superiority of the combination therapy of ACE-I and angiotensin receptor blocker (ARB) to high-dose ACE-I treatment with comparable blood pressure reductions, which however might be attributed to the initiation of the targeted doses from the beginning. The aim of this study was to compare the effect of increasing the dose of ACE-I with that of adding ARB following a relatively low dose of ACE-I on the survival and left ventricular (LV) remodeling after MI. METHODS: Rats underwent left coronary artery ligation and were treated with either ACE-I temocapril (5 mg/kg/day) or vehicle for 2 weeks, which was initiated 3 days after the surgery. The rats treated with temocapril were further randomly assigned to receive either high-dose temocapril (10 mg/kg/day) or combination therapy (temocapril 5 mg/kg/day+olmesartan 2.5 mg/kg/day), which was continued for another 6 weeks. RESULTS: Both treatments similarly reduced the blood pressure, improved survival and ameliorated LV enlargement. In contrast, several parameters of LV function were significantly ameliorated only by the high-dose ACE-I but not by the combination therapy. CONCLUSIONS: After the initiation of a relatively low dose of ACE-I in acute MI, increasing the dose of ACE-I or adding ARB may equally improve survival and LV remodeling in the setting of an equal hypotensive effect. Further study with a longer treatment protocol is required to determine whether the several favorable effects on LV function elicited only by the high-dose ACE-I treatment provide further beneficial effects on survival and LV remodeling compared with the combination therapy. (+info)
Assembly of stable human type I and III collagen molecules from hydroxylated recombinant chains in the yeast Pichia pastoris. Effect of an engineered C-terminal oligomerization domain foldon.
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The C-propeptides of the pro alpha chains of type I and type III procollagens are believed to be essential for correct chain recognition and chain assembly in these molecules. We studied here whether the 30-kDa C-propeptides of the human pC alpha 1(I), pC alpha 2(I), and pC alpha 1(III) chains, i.e. pro alpha chains lacking their N-propeptides, can be replaced by foldon, a 29-amino acid sequence normally located at the C terminus of the polypeptide chains in the bacteriophage T4 fibritin. The alpha foldon chains were expressed in Pichia pastoris cells that also expressed the two types of subunit of human prolyl 4-hydroxylase; the foldon domain was subsequently removed by pepsin treatment, which also digests non-triple helical collagen chains, whereas triple helical collagen molecules are resistant to it. The foldon domain was found to be very effective in chain assembly, as expression of the alpha 1(I)foldon or alpha 1(III)foldon chains gave about 2.5-3-fold the amount of pepsin-resistant type I or type III collagen homotrimers relative to those obtained using the authentic C-propeptides. In contrast, expression of chains with no oligomerization domain led to very low levels of pepsin-resistant molecules. Expression of alpha 2(I)foldon chains gave no pepsin-resistant molecules at all, indicating that in addition to control at the level of the C-propeptide other restrictions at the level of the collagen domain exist that prevent the formation of stable [alpha 2(I)]3 molecules. Co-expression of alpha 1(I)foldon and alpha 2(I)foldon chains led to an efficient assembly of heterotrimeric molecules, their amounts being about 2-fold those obtained with the authentic C-propeptides and the alpha 1(I) to alpha 2(I) ratio being 1.91 +/- 0.31 (S.D.). As the foldon sequence contains no information for chain recognition, our data indicate that chain assembly is influenced not only by the C-terminal oligomerization domain but also by determinants present in the alpha chain domains. (+info)
Autologous chondrocyte implantation using a bilayer collagen membrane: a preliminary report.
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OBJECTIVE: To present preliminary clinical experience with Matrix-induced autologous chondrocyte implantation, a new tissue-engineering technique for treatment of deep cartilage defects, in which autologous chondrocytes are seeded on a tridimensional scaffold provided by a bilayer type I-III collagen membrane. METHODS: From December 1999 to January 2001, 13 patients underwent implantation procedure for deep cartilage defects. Age of patients ranged from 18 to 49 years (mean age, 35 years). The mean defect size was 3.5 cm(2) (range, 2.0-4.5 cm(2)). Clinical and functional evaluation were performed using various score systems for the ankle and the knee, and magnetic resonance imaging was performed at 6 and 12 months postoperatively. Membrane structure and cellular population were investigated by light microscopy, scanning electron microscopy, and electrophoresis before implantation. RESULTS: The mean follow-up was 6.5 months (range, 2-15 months). No complications were observed in the postoperative period. The 6 patients with a minimum follow-up of 6 months showed an improvement in clinical and functional status after surgery. Magnetic resonance images showed the presence of hyaline-like cartilage at the site of implantation; there was evidence of chondroblasts and type II collagen inside the seeded membrane. CONCLUSION: Matrix-induced autologous chondrocyte implantation offers several advantages with respect to the traditional cultured cell procedure. These include technical simplicity, short operating time, minimal invasiveness, and easier access to difficult sites. It appears to be a reliable method for the repair of deep cartilage defects. (+info)