Desmoplastic reaction of gastric carcinoma: a light- and electron-microscopic immunohistochemical analysis using collagen type-specific antibodies

The desmoplastic reaction in ten cases of gastric carcinoma was investigated light and electron immunohistochemically by using monospecific antibodies to collagen types. In addition to type I and III collagens, type V collagen was constantly recognized in the fibrous stroma, increasingly of the scirrhous carcinoma. Type IV collagen delineated the basement membranes of carcinoma nests linearly with occasional discontinuity, whereas in the scirrhous carcinoma, it was present along the thick bundles of collagenous fibers. Immunoelectron microscopic studies revealed that type I and III collagens were distributed on the collagen fibers, and type V collagen was stained in the margin of these fibers. These antibodies also reacted in the rough endoplasmic reticulum of fibroblasts or myofibroblasts in a few cases. Type IV collagen was localized in the periphery of smooth muscle cells, endothelial cells of collapsed capillaries, and myofibroblasts scattered in the stroma of scirrhous carcinoma. Carcinoma cells were not reactive with any antibodies examined. These findings suggest that type V collagen, as well as type I and III collagens, is involved in the formation of desmoplastic stroma, and that these collagens are reactively synthesized by fibroblasts and myofibroblasts in some interaction with invading carcinoma cells.     

Aberrant type I and type III collagen gene expression in human breast cancer in vivo

Increased synthesis and degradation of extracellular matrix components are associated with breast cancer development. This study evaluated type I and type III procollagen mRNA expression and the corresponding protein synthesis and maturation, as well as the tissue distribution of these collagens, in benign breast lesions, infiltrating ductal carcinomas, and their metastases by in situ hybridization and immunohistochemistry. In the benign lesions, the type I and type III collagen bundles were regularly organized and the expression of the corresponding mRNA was weak, indicating a relatively slow collagen turnover. In the malignant tumours, increased expression of type I and type III procollagen mRNAs was observed in the fibroblastic cells of the stroma; the malignant epithelial cells did not participate. The staining of corresponding newly-synthesized pN-collagens showed aberrant bundles in the invasive front of the malignant tumours. Newly-synthesized type I and type III procollagens were occasionally observed in fibroblastic cells, particularly in grade 2 and grade 3 tumours. Metastases of breast carcinoma resembled poorly differentiated primary tumours with respect to their collagen synthesis and deposition. The increased synthesis of fibrillar type I and type III procollagens may serve as a pathway for tumour invasion. The enhanced synthesis is associated with the formation of aberrant collagen bundles, which may be more readily degradable and may thus facilitate breast tumour invasion.     

The myofibroblast in wound healing and fibrocontractive diseases

The demonstration that fibroblastic cells acquire contractile features during the healing of an open wound, thus modulating into myofibroblasts, has open a new perspective in the understanding of mechanisms leading to wound closure and fibrocontractive diseases. Myofibroblasts synthesize extracellular matrix components such as collagen types I and III and during normal wound healing disappear by apoptosis when epithelialization occurs. The transition from fibroblasts to myofibroblasts is influenced by mechanical stress, TGF-beta and cellular fibronectin (ED-A splice variant). These factors also play important roles in the development of fibrocontractive changes, such as those observed in liver cirrhosis, renal fibrosis, and stroma reaction to epithelial tumours.     

Collagen matrix in development and progression of experimentally induced respiratory neoplasms in the hamster.

Intratracheal instillations of 7H-dibenzo(c, g)carbazole (DBC), a tobacco smoke component, into Syrian golden hamsters, resulted in preneoplastic lesions and benign and malignant respiratory neoplasms. Neoplastic progression was associated with specific changes in the extracellular matrix (ECM), dependent on the stage of tumor development. DBC-induced tracheobronchial squamous metaplasia was associated with an increase in collagen type I and type III deposition in the subepithelial ECM, as observed by computer-assisted image analysis of immunohistochemical staining for the aminoterminal propeptides of collagen type I (PINP) and collagen type III (PIIINP). Increased collagen matrix synthesis was detected in dysplasia by in situ hybridization of alpha1(I) mRNA for collagen I and alpha1(III) mRNA for collagen type III after continued exposure to DBC. In well-differentiated squamous cell carcinomas with an expansive growth pattern, collagen deposition increased, as did fiber size. In moderately differentiated neoplasms, basement membrane (BM) destruction and invasion was associated with a destructive growth pattern and decreases in collagen synthesis and the deposition of new collagen. Preserved deposition of mature collagen was detected by staining for the telopeptide of collagen type I propeptide. In less differentiated tumors, ECM development was minimal, with few and small fibers, possibly explaining the rapid development of these neoplasms. Transforming growth factor beta (TGFbeta1) immunoreactivity was increased in hyperplastic epithelium and well differentiated neoplasms and decreased in dysplasia and less differentiated squamous cell carcinomas, while TGFbeta2 and TGFbeta3 expression was also distinct in neoplastic cells. Collagen synthesis and epithelial differentiation were associated with an increased number of myofibroblasts in the ECM and with increased TGFbeta3 immunoreactivity in differentiated cells and in the matrix. The nature of the composition of the ECM was related to neoplastic growth and progression when analyzed by computer-associated image analysis, revealing alterations in collagen structure, size, and shape.     

Fibrosis in undifferentiated (anaplastic) thyroid carcinomas: evidence for a dual action of tumour cells in collagen type I synthesis

The mechanisms involved in stromal reactions and fibrosis in solid malignant tumours are incompletely understood. In the present study, collagen type I production was investigated in tissues and cell lines derived from human undifferentiated (anaplastic) thyroid carcinomas, a highly aggressive, often fibrotic malignancy with mesenchymal phenotype. In situ hybridization showed the expression of pro-alpha1(I) collagen mRNA throughout the stromal part of the tumours. However, immunofluorescence staining using an anti-pro-collagen type I antibody revealed the synthesis of pro-collagen type I protein mainly in stromal cells juxtaposed to nests of tumour cells. In one out of five tissue samples from human undifferentiated thyroid carcinomas, pro-alpha1(I) collagen mRNA expression was also found in a small number of tumour cells. Several well-characterized cell lines established from undifferentiated thyroid carcinomas, two from tumours included in the present study, expressed both pro-alpha1(I) collagen and prolyl 4-hydroxylase mRNA, and three of these cell lines also synthesized native triple-helical collagen type I. Taken together, these data suggest that stromal fibroblasts are the main producers of collagen type I in anaplastic thyroid tumours. The carcinoma cells seem to play a regulatory role, stimulating the synthesis of collagen type I protein in the surrounding stroma by increasing pro-alpha1(I) collagen mRNA translation. However, collagen type I production by the carcinoma cells might also contribute to the marked desmoplasia commonly seen in these tumours.     

The extracellular matrix in oral Kaposi sarcoma (AIDS): the immunohistochemical distribution of collagens type IV,V, VI,of procollagens type I and III,of laminin and of undulin

Summary Twelve oral AIDS-associated Kaposi sarcomas (KS) were studied for the distribution of extracellular matrix components using indirect immunofluorescence. Staining for basement membrane (BM) components revealed two distinct patterns of distribution: a delicate and partly fragmented lining of BMs around sinusoid-like vascular spaces or an occasional diffuse interstitial fluorescence in the tumour stroma; or an irregular broad rim of fluorescence in walls of larger blood vessels. These findings support a vascular cell origin of the endothelial- and spindle cell component in KS. The tumour stroma was almost completely negative for collagen type V and undulin, whereas an intensive fluorescence was noted for procollagens type I, III and collagen type VI. In areas adjacent to KS a loss of procollagens type I and III, collagens type V, VI and undulin was noted. An intimal sheath of collagen type V was usually absent from blood vessels of the tumour or the peritumourous connective tissue. Immunohistochemical findings indicate that the preexisting interstitial connective tissue matrix is destroyed during tumour invasion and that subsequently procollagens type I, III and collagen type VI are synthesized de novo by cells of the tumour stroma.This study was supported by the Deutsche Forschungsgemeinschaft (Be 1017/1-2)     

The extracellular matrix in pigmented skin lesions: an immunohistochemical study

In recent years the interaction between tumour cells and the surrounding extracellular matrix in the process of tumour development, invasion and metastasis has been a focus of interest. We studied frozen sections of nine naevocellular naevi (junctional, compound and intradermal), 40 dysplastic naevi, six pagetoid in situ melanomas and 12 superficial spreading melanomas in order to determine the expression of: the basement membrane proteins collagen type IV and laminin, the interstitial collagen types I, III and VI, and fibronectin and tenascin. An indirect immunoperoxidase technique was used. In the various stages of melanocytic tumour progression we observed: 1 loss of type IV collagen and laminin within dermal melanocytic cell nests; 2 de novo expression of basement membrane type IV collagen and increased expression of the interstitial collagen types I, III and VI, as well as tenascin and fibronectin in the dermal stroma surrounding dysplastic naevus cells and melanoma cells; 3 presence of extracellular matrix components in close association with intra-epidermally located invading atypical melanocytes. These data demonstrate the complex alterations of the composition of the extracellular matrix from bland naevi through lesions with progressive atypia to invasive melanoma. The changes described result in a molecular environment which melanocytes with an altered adhesion molecule profile are able to invade.     

The differentiation of the dermis in the laboratory mouse

The purpose of this study was to provide a comprehensive and sequential account of the differentiation of the dermis in one body region in a mammalian species. A histological, histochemical, and ultrastructural study was made of each cellular and matrix component of the dermis of the upper lip of the mouse during prenatal development. On the basis of these observations, the development of the dermis was divided into four phases: I) undifferentiated mesenchyme (12, 13 days), II) cell differentiation (14, 15 days), III) dynamic transition (16 days), and IV) matrix differentiation (beginning at 17 days). The first phase was marked by a decrease in the cell density but no change in the ultrastructure of the undifferentiated mesenchyme cells. The second phase began with the cytodifferentiation of the mesenchyme cells and was characterized by the appearance of new cell types in the dermis (immature fibroblasts, mast cells, myoblasts, and cells of indeterminate type). During phase III the dermis was undergoing rapid change. Fibroblasts became fully differentiated, mast cell density reached a sharp peak, there was a marked increase in the number of collagen fibrils in the dermal matrix and the first collagen fibers were observed, and changes occurred in the pattern of proteoglycan synthesis. Aggregations of vesicles appeared to be extruded from cytoplasmic blebs on the fibroblasts in large quantities at this time. Further differentiation of the dermal intercellular matrix occurred during the fourth phase, which continued after birth, as more collagen was laid down to form the connective tissue stroma.     

Triggering the induction of myofibroblast and fibrogenesis by airway epithelial shedding

Myofibroblasts have been thought to participate in subepithelial fibrosis in asthma, but the mechanism of myofibroblast induction has not been fully understood. In this study we investigated injury-related myofibroblast induction in a coculture system of guinea-pig epithelial cells and fibroblasts cocultured in a human amnion chamber. After pseudostratified epithelial cells were mechanically scraped, migrated flat epithelial cells differentiated into cuboidal appearances on Day 4 and then returned to their original shapes on Day 8. During the course of the epithelial redifferentiation, it was found by Northern blot analysis, immunohistochemistry for alpha-smooth muscle actin, and electron microscopic observation that the myofibroblasts were transiently induced on Day 4. The myofibroblast induction was inhibited by the blocking of transforming growth factor (TGF)-beta1 and thrombospondin (TSP)-1, indicating that the activation of TGF-beta1 by TSP-1 would induce myofibroblasts. This finding was also supported by a transient upregulation of TSP immunoreactivity and TSP-1 messenger RNA (mRNA) in fibroblasts. Interestingly, epithelial injury reduced TGF-beta1 immunoreactivity in the amnion membrane but did not affect TGF-beta1 mRNA in epithelial cells and fibroblasts, indicating that TGF-beta1 supplied from the extracellular matrix can participate in myofibroblast induction. Concurrently with myofibroblast induction, procollagen type I and III mRNAs were upregulated in fibroblasts, and obvious collagen deposition was observed ultrastructurally around the myofibroblasts compared with the fibroblasts. These results indicate that induced myofibroblasts can be functionally more active in producing collagen than are resting fibroblasts. The present study suggests that epithelial injury stimulates TGF-beta1 release from the extracellular matrix and its activation via TSP-1 production, causing collagen synthesis through myofibroblast induction.     

The extracellular matrix in "sclerosing" follicular center cell lymphomas: an immunohistochemical and ultrastructural study

"Sclerosis" is frequently seen in follicular center cell (FCC) lymphomas. The mechanism of its deposition, as well as its composition and significance, are unknown. Several clinical studies have suggested that the course of these lymphomas is more indolent than that of lymphomas of the same histologic type without sclerosis. Nine immunologically characterized cleaved FCC lymphomas with sclerosis and 14 reactive lymph nodes with follicular hyperplasia were investigated by special staining methods, electron microscopy, and immunohistochemical studies with antibodies to types I, III, IV, and V collagen, laminin, and fibronectin. The sclerotic tissue in FCC lymphomas stained uniformly with periodic acid-Schiff (PAS) and Masson's stain, with the patterns ranging from delicate filamentous strands to dense doubly refractile bands. Ultrastructurally, the bands of connective tissue were continuous with the adventitia of vessels and composed of varying amounts of banded collagen (types I and III) admixed with filamentous and flocculent material. In all cases the neoplastic lymphocytes were separated from the extra-cellular matrix by fibroblasts and myofibroblasts with long cell processes. Immunohistochemical studies demonstrated intense staining of sclerotic bands with antibodies to fibronectin and type I collagen and, usually, weaker marking with antibodies to types III and V collagen. No significant staining of sclerotic bands was found with antibodies to type IV collagen or with laminin. Weak pericellular staining for type V collagen was present in eight of nine lymphomas and half of the control lymph nodes. These studies suggest that the increased amounts of extracellular matrix in cleaved FCC lymphomas are produced primarily by fibroblasts and myofibroblasts and represent predominantly fibronectin and types I, III, and V collagen. The composition of the sclerotic areas of FCC lymphomas is similar immunohistochemically to that of the capsule and trabeculae of reactive lymph nodes, which are also intimately associated with fibroblasts and myofibroblasts.     

Immunoelectron microscopy shows an atypical pattern and a quantitative shift of collagens type I,III and VI in oral Kaposi's sarcoma of AIDS

Summary The localization of collagen types I, III and VI in normal human alveolar and palatal mucosa and in oral Kaposi's sarcoma (KS) was studied by light microscopy and cryo-immunoelectron microscopy. Normal oral mucosa revealed two different types of organization. The upper connective tissue stroma contained a loose reticular network mainly composed of collagen types III and VI, while collagen type I immunostaining predominated in the deeper stroma. Ultrastructurally, in the KS tumour stroma, a loose pattern of individual thin collagen fibrils was noted. These often fanned out at their ends showing a filamentous substructure. The fibrils consisted predominantly of collagen type I similar to individual fibrils of normal oral mucosa. However, there was a marked loss of thick fibre bundles of collagen types I and III in KS compared with normal oral mucosa, whereas collagen type VI was markedly increased and found preferentially in clusters and strands around cross-striated fibrils that often spanned the distance between single collagen fibres. The abundance of collagen type VI in a pattern similar to early stages of wound healing suggests that the KS stroma resembles an early organizational stage of the interstitial and vascular extracellular matrix subject to a high rate of collagen turnover. This character of the KS stroma appears to result from a continuous auto- and paracrine stimulation of cell growth and collagen synthesis and provides an excellent model to study the structural arrangement of collagen type VI in relation to the fibrillar collagen types I and III.     

Myofibroblastic sarcomas: a brief review of sarcomas showing a myofibroblastic line of differentiation and discussion of the differential diagnosis

Although myofibroblasts have been intensively studied during the last three decades there is still a lack of consensus regarding their exact role and position in the spectrum of proliferative and neoplastic spindle cell lesions. This especially concerns the question of whether myofibroblastic neoplasms, or in other words a myofibroblastic line of differentiation in mesenchymal neoplasms, exist or not. Some authors question the existence of benign and particularly malignant myofibroblastic neoplasms, and suggest rather that myofibroblasts represent a functional stage in neoplastic processes arising from fibroblasts, smooth muscle cells or pericytes. It has been claimed that the ultrastructural evidence of so-called fibronexus (microtendons) and stress fibres plays a crucial role in the diagnosis of myofibroblastic neoplasms, and that myofibroblasts are defined essentially by electron microscopy. However, in diagnostic surgical pathology mesenchymal neoplasms are classified according to the cell type they most closely resemble, and it is not surprising that, in neoplastic processes, variable genetic and morphological changes occur. Therefore it seems impractical that a single specialised organelle such as the enigmatic fibronexus should be the limitation for the delineation of a line of differentiation. For practical purposes, myofibroblastic neoplasms are composed of spindle-shaped tumour cells set in a more or less collagenous matrix. Myofibroblastic tumour cells contain ill-defined eosinophilic cytoplasm and fusiform nuclei that are either elongated and wavy or plumper and vesicular. Neoplastic myofibroblasts stain positively for muscle actin, alpha-smooth muscle actin, and/or desmin and are characterised by a variable immunophenotype. Ultrastructurally, myofibroblasts share features of fibroblasts and smooth muscle cells but differ from both cell types. Given these diagnostic criteria it is most likely that, in addition to well-known reactive and benign myofibroblastic proliferations, a variety of clinicopathological forms of myofibroblastic sarcomas (myofibrosarcomas) occur. In this spectrum congenital and infantile fibrosarcoma, inflammatory myofibroblastic tumour, low-grade myofibroblastic sarcoma, and high-grade (‘MFH’-like) myofibroblastic sarcoma are briefly reviewed and their differential diagnosis is discussed.     

Gadodiamide contrast agent 'activates' fibroblasts: a possible cause of nephrogenic systemic fibrosis

Nephrogenic systemic fibrosis (NSF) is a fibrotic disease generating intense interest due to its recent discovery, and unknown cause. It appears confined to patients with renal disease and presents as grossly thickened, indurated, tight skin that is woody to palpation. Histologically, the dermis contains thickened collagen bundles, numerous plump fibroblast-like cells, and elevated hyaluronan expression. Recent data suggest a link between the use of gadolinium chelate as an MRI contrast agent and the onset of the disease. Fibroblasts from the lesions of six NSF patients, all of whom were exposed to gadodiamide, were compared with control fibroblasts for hyaluronan and collagen synthesis. Serum from NSF patients was assessed for fibroblast hyaluronan-stimulating activity, collagen synthesis, and gadodiamide for its effect on fibroblast proliferation and matrix synthesis. NSF fibroblasts synthesized excess levels of hyaluronan and collagen compared with control fibroblasts, with up to 2.8-fold and 3.3-fold increases, respectively. NSF patient serum stimulated control fibroblast hyaluronan synthesis by up to 7-fold, and collagen synthesis by up to 2.4-fold. 1 mM gadodiamide added to culture medium stimulated fibroblast growth in a dose-dependent manner, decreasing their doubling time from 28 h to 22 h, and increasing the maximum cell density. Even a short exposure to gadodiamide stimulated cell growth, suggesting that the cells were activated by the gadodiamide. The growth of fibroblasts within contracted collagen lattices was also significantly stimulated by gadodiamide, while fibroblasts exposed to gadodiamide synthesized increased levels of hyaluronan. Control fibroblasts exposed to gadodiamide, and NSF fibroblasts exhibited an extensive pericellular coat of hyaluronan, and expressed alpha-smooth muscle actin. Gadolinium chloride did not affect fibroblast growth. This report demonstrates that NSF fibroblasts synthesize excess levels of hyaluronan and collagen, and that gadodiamide stimulates control fibroblast growth, matrix synthesis, and differentiation into myofibroblasts, suggesting a possible role for gadodiamide in the pathophysiology of NSF.     

TGF-β1介导的RhoA/ROCK通路在大鼠肺肌成纤维细胞分化中的调节作用

 目的：探讨转化生长因子β1 (TGF-β1)介导的RhoA/Rho相关卷曲螺旋形成蛋白激酶(ROCK)通路在调控肺成纤维细胞向肌成纤维细胞分化过程中的作用。方法：胰酶消化法获得原代培养的肺成纤维细胞,进行以下实验：(1) 观察TGF-β1诱导肺成纤维细胞不同时间后p-RhoA、ROCK、磷酸化肌球蛋白磷酸酶靶亚基(p-MBS)、血清应答因子(SRF)、α-平滑肌肌动蛋白(α-SMA)、I型和III型胶原蛋白表达变化;(2) 将细胞分为对照组、TGF-β1诱导分化组和Y-27632（ROCK通路阻滞剂）干预组,免疫细胞化学染色与Western blotting法分别观察与检测ROCK、p-MBS、SRF、α-SMA、I型和III型胶原蛋白在细胞内的定位分布与表达。结果：（1）经TGF-β1诱导24 h后,大鼠肺成纤维细胞胞体内出现大量平行或交叉排列的α-SMA抗体标记的肌丝。随着TGF-β1诱导刺激时间的延长,p-RhoA、ROCK、p-MBS、SRF、α-SMA、I型和III型胶原蛋白表达逐渐增高,其中RhoA/ROCK信号（p-RhoA、ROCK、p-MBS）、SRF和α-SMA蛋白分别在诱导后6 h、12 h和24 h达到高峰。I型胶原和III型胶原蛋白表达均在24 h达高峰,分别为诱导前的2.19和3.04倍（P<0.05）。 (2)与TGF-β1诱导分化组比较,当给予Y-27632干预后,在相应时点ROCK、p-MBS、SRF、α-SMA、I型和III型胶原蛋白表达均明显降低,差异有统计学意义（P<0.05）。结论：TGF-β1通过ROCK信号转导通路的活化,刺激了大鼠肺成纤维细胞向肌成纤维细胞分化,进而促进了胶原蛋白的合成,可能在（矽）肺纤维化形成过程中发挥着重要作用。     

Heterogeneous response of nasal and lung fibroblasts to transforming growth factor-β1

Background Nasal polyposis is characterized by marked oedema, sparse extracellular matrix (ECM) and proliferating blood vessels. Pulmonary fibrosis is characterized by inflammatory cells accumulation, considerable ECM deposition and vascular abnormalities. Although lung fibrosis is not only and necessarily an inflammatory disorder, we hypothesized that the difference between nasal polyposis and pulmonary fibrosis may, in part, be due to the heterogeneity between nasal and lung fibroblasts. Fibroblasts participate in the inflammatory response by releasing ECM proteins and cytokines. TGF‐β is thought to participate in chronic inflammation and fibrosis. Myofibroblasts are the activated form of fibroblasts. A phenotypic hallmark of myofibroblasts is the expression of smooth muscle α‐actin (SMA). Objective We examined whether there is any heterogeneity between nasal and lung fibroblasts upon stimulation with TGF‐β₁ with regard to the synthesis of SMA, pro‐collagen type I and vascular endothelial growth factor (VEGF) as well as translocation of Smad proteins. Methods Fibroblasts lines were established from human biopsy tissue. The expression of SMA, pro‐collagen type I, VEGF mRNA was evaluated by reverse transciptase RT‐PCR. The amount of pro‐collagen type I and VEGF was measured by ELISA. By immunocytochemistry, we analysed the expression of SMA and Smad2, 3, 4 in cultured fibroblasts. Results TGF‐β₁ induced SMA and pro‐collagen type I synthesis in lung, but not in nasal fibroblasts. By contrast, TGF‐β₁ induced VEGF synthesis in both lung and nasal fibroblasts. After stimulation with TGF‐β₁, Smad2, 3, 4 were translocated from the cytoplasm to the nucleus in lung fibroblasts, whereas only Smad3 was translocated in nasal fibroblasts. Conclusion These results establish the heterogeneous responsiveness of fibroblast populations in the airways to TGF‐β₁ and that such a heterogeneity may contribute, at least in part, to the different pathological outcomes of inflammation in the upper and lower airways.     

Morphological aspects of early and late collagen degradation in granulation tissue.

A sequential histologic, ultrastructural and immuno pathologic study was carried out in the Selye's inflammatory pouch model to observe extracellular matrix and cellular changes during granulation tissue formation. Besides changes involving different components of the connective tissue, it was observed that collagen resorption occurred under a biphasic process. At an early phase (3rd to 15th day), in which exudative inflammatory changes predominated, signs of collagen synthesis and degradation were seen simultaneously. Extracellular breakdown and internalization of collagen fragments within fibroblasts and myofibroblasts were observed. Later on (30th to 60th day), changes affecting collagen had a different ultrastructural appearance. Collagen fragmentation, focal "lytic" and "electron dense" changes occurred in the extracellular space specially at the periphery of fibroblasts, myofibroblasts and smooth muscle cells. Collagen degradation, thus seems to be a continuous process in granulation tissue, occurring with different morphologies at different times.     

Lysyl oxidase gene expression in the stromal reaction to in situ and invasive ductal breast carcinoma.

Lysyl oxidase is involved in the main pathway of collagen and elastin cross-linking: it has a role in the maturation of fibrillar matrix proteins in fibrosing processes and dictates their stability against metalloproteases. The stromal reaction patterns in ductal breast carcinoma are known to be morphologically varied. This has raised the hypothesis that there might be a differential expression of the lysyl oxidase gene as a function of stromal reaction pattern. The present study investigates this potential correlation and the role of matrix protein cross-linking in stromal differentiation. Lysyl oxidase was detected by immunohistochemistry and lysyl oxidase gene expression by in situ hybridization. Maximal expression was observed in myofibroblasts and myoepithelial cells around in situ tumors and in the reactive fibrosis facing the invasion front of infiltrating tumors. The lysyl oxidase substrates were observed in parallel, resulting in the stabilization of a scar-like peritumor barrier. In contrast, a lack of lysyl oxidase was associated with the loose or scirrhous stroma accompanying invading tumors; here, in situ hybridization revealed type I collagen synthesis, resulting in the deposition of non-cross-linked matrix proteins susceptible to degradation. The early development of a cross-linked matrix around ductal breast carcinoma suggests a possible bost defense mechanism, whereas the synchronous or late stromal reaction lacking lysyl oxidase favors tumor dispersion.     

Activation of adventitial fibroblasts contributes to the early development of atherosclerosis: a novel hypothesis that complements the "Response-to-Injury Hypothesis" and the "Inflammation Hypothesis"

The role of the adventitia in vascular function and vascular lesion formation has been largely ignored. This article introduces the hypothesis that the activation of the adventitia, specifically the fibroblasts, contributes to the formation of intimal atherosclerotic lesions. The evidence for this hypothesis includes: (a) the early proliferative changes seen in fibroblasts found in the adventitia; (b) the increase and the alteration of extracellular matrix deposition in the adventitia; (c) fibroblast differentiation into myofibroblasts and migration into the intima; and (d) fibroblast synthesis and release of cytokines that have potent effects on neighboring smooth muscle and endothelial cells prior to intimal lesion formation. In conclusion, the activation of adventitial fibroblasts is a key regulator of vascular function and structure from the "outside-in" and contributes to the development of atherosclerotic lesions. The outer location of the adventitia makes it a suitable location for drug delivery and gene therapy aimed at preventing and treating atherosclerosis.     

Enhanced myofibroblastic differentiation and survival in Thy-1(-) lung fibroblasts

Thy-1 is a glycosylphosphatidyl-inositol-linked cell surface glycoprotein whose exact biological role remains unclear. Differential expression of Thy-1 affects fibroblast proliferation and fibrogenic signaling. In idiopathic pulmonary fibrosis, the proliferating myofibroblasts within the fibroblastic foci are Thy-1(-), whereas normal lung fibroblasts are predominantly Thy-1(+). In this study, we used rat lung fibroblasts sorted for Thy-1 expression to examine myofibroblastic differentiation in response to fibrogenic stimuli. We examined the effects of transforming growth factor-beta, endothelin-1, and connective tissue growth factor on the expression of myofibroblast proteins and myogenic regulatory factors by real-time RT-PCR and immunoblotting. Thy-1(-) cells have significantly higher myofibroblast and myogenic regulatory factor gene and protein expression compared with Thy-1(+) cells, confirmed by immunofluorescence. We also used floating collagen matrix contraction assays to assess the functional differentiation of the fibroblasts. At baseline and after stimulation with transforming growth factor-beta and endothelin-1, Thy-1(-) cells caused significantly greater collagen contraction than did Thy-1(+) cells, supporting the hypothesis that Thy-1(-) cells are more fully differentiated myofibroblasts. Because apoptosis has been implicated in the regression of myofibroblasts, we examined the percentage of apoptotic cells in the contracted collagen matrices at baseline and after stimulation with fibrogenic agents. A significantly greater proportion of Thy-1(+) cells underwent apoptosis in all conditions compared with Thy-1(-) fibroblasts. Transfection of Thy-1 into Thy-1(-) cells inhibits collagen matrix contraction and reduces cell survival. Our data indicate that Thy-1 regulates myogenic gene expression, myofibroblastic differentiation, and survival in lung fibroblasts.     

Activation of Toll-like receptor 3 augments myofibroblast differentiation

Airway remodeling is observed in the airways of patients with asthma, and differentiation of fibroblasts to myofibroblasts plays a critical role in the progress of airway remodeling. Viral infection induces not only the disease development and exacerbations but also airway remodeling. The aim of this study was to evaluate whether the activation of Toll-like receptor 3 (TLR3) can affect the differentiation of fibroblasts to myofibroblasts and the extracellular matrix (ECM) protein production. Human fetal lung fibroblasts (HFL-1) and adult lung fibroblasts were treated with a synthetic double-stranded RNA, polyinosine-polycytidylic acid (poly[I:C]) and the expression of alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblast differentiation, was evaluated. The release of transforming growth factor-beta(1) (TGF-beta(1)) and ECM protein production were assessed. The effect of anti-TGF-beta antibody on the alpha-SMA and ECM production was also assessed. Poly(I:C) significantly augmented the alpha-SMA expression (P < 0.01) and release of TGF-beta(1) (P < 0.01) compared with control. Bafilomycin, an inhibitor of TLR3 signaling, diminished poly(I:C)-augmented TGF-beta(1) release. Anti-TGF-beta(1) antibody inhibited the poly(I:C)-augmented alpha-SMA expression. Poly(I:C) enhanced translocation of nuclear factor-kB (NF-kappaB) and interferon regulatory factor-3 (IRF-3) into the nucleus. Poly(I:C)-augmented TGF-beta(1) release was almost completely blocked by NF-kappaB inhibitors, but not by silencing IRF-3. The production of fibronectin and collagen I expression were significantly increased by poly(I:C) (P < 0.01) and they were inhibited by anti-TGF-beta antibody. These results suggest that activation of TLR3 can affect the differentiation to myofibroblasts and enhance ECM production via the NF-kappaB-TGF-beta(1)-dependent pathway.