Collagen in the cellular and fibrotic stages of scleroderma

The collagen in localized and systemic scleroderma skin was studied by light microscopy with silver impregnation (50 patients), electron microscopy (14 patients), and immunofluorescence microscopy using specific antibodies against Type I and Type III collagens (12 patients). In the cellular stage, the dermis and adipose tissue revealed perivascular or diffuse cellular infiltrates (mostly lymphocytes, plasma cells, and macrophages), accompanied by deposition of Type III collagen. The lower dermis also showed an increase in Type III collagen. In the fibrotic stage, the papillary layer showed a reduction and/or clumping of Type III collagen as compared to normal skin. The lower dermis and the adipose tissue revealed compact collagen consisting exclusively of Type I collagen or a mixture of Type I and Type III collagen. The pattern of Type III collagen distribution was similar to that of reticulin, thus suggesting that at least some reticulin fibrils may represent Type III collagen.     

Collagen in the cellular and fibrotic stages of scleroderma.

The collagen in localized and systemic scleroderma skin was studied by light microscopy with silver impregnation (50 patients), electron microscopy (14 patients), and immunofluorescence microscopy using specific antibodies against Type I and Type III collagens (12 patients). In the cellular stage, the dermis and adipose tissue revealed perivascular or diffuse cellular infiltrates (mostly lymphocytes, plasma cells, and macrophages), accompanied by deposition of Type III collagen. The lower dermis also showed an increase in Type III collagen. In the fibrotic stage, the papillary layer showed a reduction and/or clumping of Type III collagen as compared to normal skin. The lower dermis and the adipose tissue revealed compact collagen consisting exclusively of Type I collagen or a mixture of Type I and Type III collagen. The pattern of Type III collagen distribution was similar to that of reticulin, thus suggesting that at least some reticulin fibrils may represent Type III collagen.     

Increased expression of dermatopontin mRNA in the infarct zone of experimentally induced myocardial infarction in rats: comparison with decorin and type I collagen mRNAs

Objectives: Dermatopontin, a 22 kDa extracellular matrix (ECM) protein, has been shown to interact with other ECM components, especially decorin, and to regulate ECM formation. We examined dermatopontin mRNA expression in the myocardial infarct zone. Methods: The cDNA encoding the rat dermatopontin was cloned by RT-PCR based on screening results from the Expressed Sequence Tag™ database. The dermatopontin mRNA expression was examined in the infarct zone after experimentally induced myocardial infarction in rats by the methods of Northern blotting and in situ hybridization. The expression of dermatopontin mRNA was compared to that of decorin and type I collagen mRNAs. Results: The isolated clone contained a 609 bp cDNA insert containing a complete open reading frame encoding 202 amino acids. The rat dermatopontin cDNA showed high homology to human and mouse counterparts (>96 %). Northern blotting demonstrated that dermatopontin mRNA expression did not markedly increase on day 2, but was increased on days 7, 14 and 28 by 2.4-, 4.1- and 4.2-fold, respectively, compared to that in preligation hearts. Dermatopontin mRNA expression was regulated almost in parallel with decorin mRNA expression. In situ hybridization demonstrated mRNA signals for dermatopontin in macrophages and spindle-shaped mesenchymal cells (fibroblasts and myofibroblasts) located in the infarct interior zone around infarcted necrotic tissue on day 7. Coexpression of dermatopontin mRNA with decorin and type I collagen mRNAs was observed in spindle-shaped mesenchymal cells. Conclusions: The present results demonstrated the time-dependent increase in the expression of dermatopontin mRNA in parallel with that of decorin mRNA in the infarct zone. Coexpression of dermatopontin mRNA with decorin and type I collagen mRNAs suggests that dermatopontin plays a role in ECM (fibrillar collagen matrix) reformation in the infarct along with decorin and type I collagen. Received: 7 March 2002, Returned for revision: 8 April 2002, Revision received: 16 May 2002, Accepted: 3 June 2002 Correspondence to: S. Kusachi, MD     

Dermal extracellular matrix in cutaneous leprosy lesions.

Thirty-eight biopsies of cutaneous lesions from leprosy patients [borderline tuberculoid (BT) 14, borderline lepromatous (BL) 18, lepromatous (LL) 6] were processed for staining of some extracellular matrix (ECM) components (collagen, proteoglycans, elastic fibers and fibronectin). Specific histological staining and the indirect immunofluorescence method with antibodies to collagen and fibronectin were utilized. The ECM of the normal dermis was strikingly modified in the inflammatory infiltrate. By Gomori's reticulin and anti-fibronectin immunostaining, replacement of the dense interlaced collagen fibers with a reticular mesh was observed in the infiltrate. The immunoreactivity obtained with anti-type I and anti-type III collagens showed positive fibrils and a lumpy pattern in the lepromatous and tuberculoid lesions with a higher amount in the lepromatous lesions. The lack of clear-cut boundaries between the normal dermis and the inflammatory infiltrate in the lepromatous (BL, LL) lesions was correlated with the blurred limits of the clinical lesions of this pole of the leprosy spectrum. Absence of elastic fibers in the infiltrate was a constant finding, and fuchsin-positive microfibrils were found in some infiltrates. The clear zone of lepromatous lesions was devoid of oxytalan fibers. Elaunin fiber rings around sweat gland acini were present even when the leprosy infiltrate was seen enveloping them. The original ECM is replaced by a newly assembled one, which is suited for the dynamic nature of the inflammatory process. The trophic effects of the ECM upon the cutaneous epithelial structures are modified so that atrophy and late degeneration ensues. These ECM modifications contribute, therefore, to the biological alterations of the skin functions in leprosy.     

Morphometric analysis of dermal collagen fibers in normal human skin as a function of age

A quantitative study of dermal collagen as a function of age was carried out by computerized digital image analysis. Fast Green-Syrius Red stained sections were obtained of skin biopsies taken from the upper inner arm of 33 healthy women and 38 healthy men. The Leitz texture Analysis System (Leitz-TAS) and mathematical morphology (Serra, 1982) were used for the evaluation of the data. Collagen was studied in the superficial dermis and also in the reticular dermis using the same program. There were significant correlations, firstly between the percentage of collagen measured by the morphometric method and the concentration of collagen analysed biochemically (microg/mm(2) of tissue section) (r = 0.79, p < 0.001) and secondly between the decreased concentration of collagen and age (r = 0.58, p < 0.05). The morphometrical measurements have shown that the relative percentage of collagen bundles (surface of collagen fibers as a function of the dermal area analyzed) was 93.35% in the superficial dermis and 89.2% in the reticular dermis. Although this value is higher than the chemically determined ratio of collagen to other proteins (over 70%), this may be due to the relatively uniform distribution pattern of (type I and III) collagen through the dermis covering most other components of the skin. As the collagen fiber density per unit dermal surface did not change with age, the decrease in collagen content of the skin may be ascribed to the loss of about 6% of dermal mass per decade (Branchet, 1990), although large individual variations exist. The histogram of the diameter distribution of collagen fiber bundles of the reticular dermis showed thinner diameters in persons between 20 and 40 years of age than in older persons. The histogram of the distribution of interfiber spaces did not show any variation with age in the superficial dermis, while in the reticular dermis there was a predominance of smaller interspaces in persons older than 50 years.     

Expression of fibrillar types I and III and basement membrane collagen type IV genes in myocardium of tight skin mouse

STUDY OBJECTIVE--The aim was to study the expression of fibrillar collagen types I and III and basement membrane type IV collagen in the heart of the tight skin mouse, a genetic mutant with collagen overproduction in various organs. DESIGN--Collagen gene expression was measured in the ventricular tissues of the heart of the tight skin mouse and the age matched homozygous (+/+) litter mates by the use of cDNA probes to alpha 2 (I), alpha 1 (III) and alpha 2 (IV) procollagen and northern and dot blot analysis. Collagen deposition was examined by immunofluorescent light microscopy using monospecific antibodies to types I, III and IV collagens. EXPERIMENTAL MATERIAL--Heterozygous male (TSK/+) and normal (+/+) mice, 1.5-2 months old of the C57BL/6 strain were used. The animals were anaesthetised and the hearts were rapidly excised and processed for RNA extraction and antibody staining. MEASUREMENTS AND RESULTS--The results of northern and dot blot analyses showed a 41% increase in mRNA level for collagen type I, a 63% increase in mRNA level for type III and a 33% increase in type IV collagen in the ventricular myocardium (right and left ventricles) of the tight skin mouse compared to its counterpart in age matched homozygous (+/+) litter mates. mRNA levels for beta actin showed no significant increase. Immunofluorescent light microscopy and monospecific antibodies to types I, III and IV collagens were used to examine collagen deposition. The results showed that collagen type I fibres are thicker and denser in perivascular areas of the tight skin mouse heart compared to normal heart. No abnormal accumulation of type III fibres was observed. CONCLUSIONS--The heart of the tight skin mouse may be an appropriate model for studying the up regulation of cardiac collagen gene expression and its potential contribution to myocardial diseases.     

Collagen degradation in rat skin but not in intestine during rapid growth: effect on collagen types I and III from skin.

Metabolic degradation of prelabeled collagen in whole body skin and whole intestine was compared to that of types I and III collagens from skin in young, rapidly growing rats. Pregnant rats were given [3H]proline during the last week of gestation; and after birth, littermates were compared. Between the second and sixth weeks of age, there was a 43% loss of radioactivity from dermal collagen but no significant loss of radioactivity from intestinal collagen. Pepsin treatment solubilized 90% of the dermal collagen but only 12% of intestinal collagen. Skin from 2- and 6-week-old rats yielded the same proportions of type I and type III collagens (type I, 82%; type III, 18%). The relative losses of total radioactivity from types I and III were similar to each other (50 and 44%, respectively) and to the loss from whole skin. Because types I and III collagens are known to be present in both skin and intestine, the marked degradation of both collagen types in skin but not in the intestine may be related to the amount and kind of intermolecular crosslinks present.     

Regulation of fibrillar collagen types I and III and basement membrane type IV collagen gene expression in pressure overloaded rat myocardium

Left ventricular hypertrophy is based on cardiac myocyte growth. The hypertrophic process can be considered heterogeneous based on whether it also includes a remodeling and accumulation of fibrillar types I and III collagens that are responsible for impaired myocardial stiffness. In the heart, the messenger RNA (mRNA) for fibrillar collagen types I and III has been detected only in cardiac fibroblasts, whereas mRNA for basement membrane collagen type IV is present in both fibroblasts and myocytes. We studied the early and long-term expression of these collagenous proteins in rat myocardium after abdominal aortic banding with renal ischemia. Complementary DNA probes for rat pro-alpha 2 (I), mouse type III and mouse type IV collagens, and chicken beta-actin were used. Northern and dot blot analysis on total RNA extracted from left ventricular tissue indicated a sixfold increase in steady-state levels of mRNA for collagen type I on day 3 of abdominal aortic banding, which had declined to control levels by day 7 where it remained rather constant at 4 and 8 weeks. Type III collagen showed a similar pattern of gene expression after banding. mRNA levels for type IV collagen, on the other hand, were elevated on day 1 after banding, returning to control at day 7 and remaining constant. Actin mRNA levels also increased on day 1 of banding, followed by a rapid return to control levels. Monospecific antibody to types I and III collagens and immunofluorescent light microscopy on frozen sections of the myocardium revealed that at 1 week after banding, the distribution and density of these collagens were similar to those of control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-dependent variations of the biosyntheses of fibronectin and fibrous collagens in mouse skin

Skin explant cultures from hairless mice of increasing age were incubated with radioactive precursors in order to determine the age-dependent variations of the biosyntheses of fibronectin and fibrous collagens (types I and III). Total collagen synthesis expressed as a percentage of total protein synthesis did not vary with age but, if expressed as micrograms hydroxyproline per mg wet weight of skin, decreased by about 30% between 2 and 22 months of age. Hydroxylation of collagen, expressed as the ratio of 3H-hypro over 3H (pro + hypro) incorporated in freshly synthesized collagen, decreased with age by about 40% between 2 and 22 months of age. The proportion of type III collagen expressed as % of type I + type III collagens increased progressively with age by about 25% at 12 months to 60% at 22 months of age. Fibronectin biosynthesis, determined by immunoprecipitation of 35S-methionine labeled peptides in SDS-extracts of skin increased progressively with age from about 2% of total incorporated radioactivity in fibronectin at 2 months to 4% at 22 months. Plasma fibronectin, of hepatic origin, was shown already to increase with age in humans. It appears thus that the expression of genes coding for extracellular matrix macromolecules is under age-dependent regulation. This regulation appears to be different for the investigated macromolecules.     

Increase in fibronectin in the deep dermis of involved skin in progressive systemic sclerosis

The distribution and amount of fibronectin in both involved and uninvolved skin from scleroderma patients and controls were compared by indirect immunofluorescence. A marked increase in fibronectin was found in the deep dermis of involved scleroderma skin, while the subepidermal papillary regions of all specimens revealed little variation in fibronectin content. The distribution of the accumulated fibronectin appeared to parallel that of the accumulated collagen in the involved reticular dermis.     

Increase in fibronectin in the deep dermis of involved skin in progressive systemic sclerosis.

The distribution and amount of fibronectin in both involved and uninvolved skin from scleroderma patients and controls were compared by indirect immunofluorescence. A marked increase in fibronectin was found in the deep dermis of involved scleroderma skin, while the subepidermal papillary regions of all specimens revealed little variation in fibronectin content. The districution of the accumulated fibronectin appeared to parallel that of the accumulated collagen in the involved reticular dermis.     

Changes in extracellular matrix and in transforming growth factor beta isoforms after coronary artery ligation in rats.

Extensive myocardial remodeling occurs after transmural myocardial infarction (MI). The infarcted myocardium is being replaced by scar tissue after gradual resorption of the necrotic tissue. The remodeling process involves both synthesis and degradation of collagens as major components of the extracellular matrix (ECM). In the present study we have analyzed the time-dependent changes of the processes related to this fibrosis in the infarct area and in the non-infarcted left ventricle (LV) six hours to 82 days after occlusion of the left anterior descending coronary artery (LAD) in rats. We also examined whether changes occurred in the expression pattern of the transforming growth factor (TGF) beta isoforms, since this cytokine is known as powerful inductor of fibrosis. Elevation in colligin expression preceded the pronounced increase in mRNA expression of both type I and type III collagen after MI from day three onwards. The maximal increase in colligin protein in the infarct area coincided with the most pronounced expression of collagen I and collagen III mRNA expression. Also, the expression and activity of matrix metalloproteinases (MMPs) and of tissue inhibitor of matrix metalloproteinase (TIMP)-2 mRNA were increased predominantly in the infarct area. TGF beta(1)and TGF-beta(2)expression increased within the first days after MI, whereas TGF-beta(3)expression was elevated predominantly in the infarct area. This pronounced increase in TGF-beta(3)persisted up to 82 days and correlated positively with the parameters of ECM metabolism. Thus, the scar formation is an ongoing dynamic process in which TGF-beta(3)seems to play an active role in the complex ventricular remodeling.     

Type II Collagen Is a Major Component of Bovine Retinal Microvessel Extracellular Matrix

Objective: To determine which interstitial collagen types may be present in bovine retinal microvessel extracellular matrix (ECM). Methods: Dissociated bovine microvessels were treated with detergents and the resultant purified ECM monitored by transmission electron microscopy (TEM). Pepsin-extracted soluble ECM collagens were identified by Western blots. Collagens were further purified by neutral salt precipitation and carboxymethyl cellulose (CMC) chromatography before cyanogen bromide (CNBr) peptide mapping and two-dimensional peptide mapping of CMC-generated fractions. Interstitial collagens were localized by immunofluorescence on frozen sections. Results: Transmission electron microscopy of detergent-purified microvessel ECM demonstrated numerous 10–50-nm collagen fibrils associated with basal laminae regardless of vessel diameter. Western blots showed that soluble ECM collagens were strongly positive for type II, moderate for type III, and weak for type I. CNBr peptide maps and two-dimensional maps of neutral salt and CMC-purified fractions confirmed the presence of type II collagen. Immunofluorescence localized type II collagen in large and small vessels of the retina. Conclusions: Type II collagen is an unexpected major component of bovine microvessel ECM, whereas types I and III are present in minor amounts. Type V collagen is also a substantial ECM component. Accordingly, all four types may contribute to a heterogeneous population of collagenous fibrils identified by TEM in intact isolated retinal microvessel ECM.     

Connective tissue metabolism including cytokines in scleroderma.

Scleroderma fibrotic lesions demonstrate vascular disease, mononuclear cell infiltrates, and increased collagen. Fibroblasts in these lesions are activated to synthesize increased extracellular matrix substances, a phenotype that continues when these cells are removed and grown in tissue culture. Levels of messenger RNA for connective-tissue substances, measured directly in biopsies of scleroderma skin, show increased message for type I collagen, but not type III collagen or fibronectin. Increased procollagen type I in scleroderma skin occurs in the papillary dermis, perivascular areas, and deep interstitium, even in skin areas that are not yet fibrotic. Scleroderma fibroblasts express more intercellular adhesion molecule 1 on their surfaces than do normal cells, and this molecule is increased in endothelial cells, mononuclear cells, and fibroblasts. In vitro scleroderma fibroblasts adhere more frequently to extracellular matrix substances and retract collagen lattices to a greater extent. Peripheral blood lymphocytes from scleroderma patients produce excessive amounts of interleukin-2 when incubated with type I collagen, and circulating basophils release more histamine than do normal cells. There is evidence for activated eosinophils both in the dermis and pulmonary lesions in scleroderma, which may play a role in fibrosis. Transforming growth factor-beta is overexpressed by alveolar macrophages from patients with fibrotic pulmonary disease. Scleroderma fibroblasts, when exposed to transforming growth factor-beta, overexpress the alpha-type receptor for platelet-derived growth factor. Scleroderma sera more frequently contain measurable quantities of interleukin-4, interleukin-6, and interleukin-2. Interleukin-4 causes adult dermal fibroblasts to proliferate and to make interleukin-6. Interleukin-6 has been shown to stimulate fibroblast synthesis of collagen and glycosaminoglycans.(ABSTRACT TRUNCATED AT 250 WORDS)