Interaction of transforming growth factor β1 with human glomerular epithelial cells in culture: opposite effects on synthesis of matrix proteins and on urokinase plasminogen activator

The effect of transforming growth factor- (TGF-) was analyzed on the synthesis of fibronectin, collagen type IV, and urokinase plasminogen activator in human glomerular epithelial cells in culture. An increase in the abundance of specific mRNA was found for collagen type IV and fibronectin. Fibronectin protein synthesis was also increased in TGF- treated cells; most of the de novo synthesized fibronectin was found as an unsoluble protein associated with extracellular matrix. In the same cells the amount of plasminogen activator mRNA was found leading also to a decreased surface expression of urokinase plasminogen activator. The data support the concept that by upregulating matrix protein synthesis and downregulating the plasminogen activator system, TGF- favors the development of sclerosis.Abbreviations FN Fibronectin - GEC Glomerular epithelial cells - TGF- Transforming growth factor - uPA Urokinase-type plasminogen activator     

Significance of fibrils in the formation of the Kimmelstiel-Wilson nodule

Summary The pathogenesis of the nodular lesion in diabetic glomerulosclerosis is described in association with fibrils. Thirteen diabetic patients with glomerular nodular lesions and 9 diabetics without the nodules were examined by electron microscopy using periodic acid-thio-carbohydrazide-silver proteinate staining. In cases of nodular glomerulosclerosis, abundant fibrillar structures mixed with electron-dense material were detected within the nodule and the mesangial matrix. They were also occasionally observed along the subendothelial space of the glomerular capillary walls. On the cross-section, these fibrils, including the lucent periphery, were 34 nm wide. Immunohistologically, collagen V and collagen VI were detected in nodular lesions. In contrast, in cases of the diffuse type of glomerulosclerosis, the widened mesangium was composed of dense material, which resembled the original mesangial matrix. The above fibrils were not detected in the mesangium. These findings suggest that the accumulation of the peculiar fibrils in the glomerular mesangium is a major pathogenic factor in the formation of Kimmelstiel-Wilson nodules.     

人α2（I）型胶原基因启动子活性研究

目的 探索器官纤维化形成中调控I型胶原基因高水平转录的启动片段及TGF-β、PDGF－BB、IGF－1等细胞因子对其活性的影响。方法 从人α2（I）胶原基因转录起始点上游－2.4kb至＋58bp的片段中，取长度不等的片段作为启动子与含氯霉素乙酰基转移酶（CAT）报告基因的质粒组成5个重组体，转染上述重组体至正常人原代培养皮肤成纤维细胞，测定细胞CAT表达水平以比较各重组体的启动子活性，同时加入细胞因子，以测定其对I型胶原启动序列的影响。结果 除－129～＋58bp序列外，其余4个重组体CAT表达水平均较高，其中－2292～＋58bp、－1476～ 58bp序列具较强启动CAT表达活性，－339～ 58bp、－616～ 58bp片段次之。TGF－β、IGF－1均能在一定程度上调人α2（I）胶原基因启动活性。结论 人α2（I）胶原基因片段－2292～ 58bp、－1476～ 58bp、－339～ 58bp有高启动活性，是进一步研究纤维化相关DNA结合蛋白的重要调控靶序列。TGF－β、IGF－1促进胶原表达，与其上调胶原基因启动活性有关。     

Intramembranous fine deposit disease associated with collagen disorders: a new morphological entity?

Summary A distinct, hitherto unknown renal histopathological appearance, consisting of diffuse thickening of the glomerular basement membrane (GBM) with fine intramembranous electron-dense deposits, was observed in the renal biopsies from three patients with collagen diseases. In each case, proteinuria was mild with normal urinary sediment. On light microscopy there were no particular abnormalities but a mild thickening of the glomerular capillary wall. Immunofluorescence studies revealed faint linear or extremely fine granular IgG deposition along the capillary wall. On electron microscopy, the GBM was diffusely thickened with fine intramembranous electron-dense deposits without spike formation. No other deposits were seen in the glomerulus. These histological features resembled those of membranous glomerulonephritis (MGN), although the possibility of the early change of MGN is excluded by specific findings in these cases. Other GBM-thickening diseases such as diabetic glomerulosclerosis were ruled out clinically and histologically. Our cases have a singular renal histopathology which differs from any of the previously established classifications of glomerular lesions. It may be a specific change associated with some type of collagen disease.     

Fibroblasts from recurrent fibrotic overgrowths reveal high rate of proliferation in vitro - findings from the study of hereditary and idiopathic gingival fibromatosis

ABSTRACT

Purpose: Investigate the content of fibrotic fibrils in gingival tissue and the proliferation of fibroblasts collected from recurrent and non-recurrent hereditary gingival fibromatosis (HGF) and idiopathic gingival fibromatosis (IGF).

Methods: Gingival biopsies were collected from HGF (n = 3) and IGF (n = 3) donors with recurrent and non-recurrent gingival overgrowths and from a control group (Ctrl, n = 3). Hematoxylin staining was performed to evaluate the histomorphology of gingival tissue. Heidenhain’s AZAN trichrome staining served for visualization of fibrotic fibrils in gingiva. Quantitative analysis of the content of fibrotic fibrils in gingival tissue was performed using a polarized light microscope. Proliferation was evaluated at 24 h, 48 h, and 72 h in fibroblast cultures using a cell proliferation ELISA assay based on 5-bromo-2?-deoxyuridine (BrdU).

Results: Numerous blood vessels and fibroblasts were observed in recurrent overgrowths, whereas moderate blood vessels and moderate to scanty fibroblasts were detected in non-recurrent overgrowths. Heidenhain’s staining revealed numerous collagen fibers in both recurrent and non-recurrent overgrowths. Quantitative analysis in a polarizing microscope showed significant accumulation of fibrotic fibrils exclusively in the overgrowths with the recurrence. In all time-points, increased proliferation of cells from all recurrent overgrowths was observed, but not from overgrowths which do not reoccur.

Conclusions: The study revealed that recurrent gingival overgrowths consist of highly fibrotic and dense connective tissue with numerous blood vessels and abundant fibroblasts. We also demonstrated that unlike fibroblasts derived from overgrowths, which did not present recurrence, fibroblasts derived from highly fibrotic and recurrent overgrowths maintain high rate of proliferation in vitro.     

Age-related changes in the articular cartilage of human sacroiliac joint

 Iliac and sacral articular cartilage of 25 human sacroiliac joints (1–93 years) are examined by light microscopy and immunohistochemistry in order to gain further insight into the nature and progress of degenerative changes appearing during aging. These changes can already be seen in younger adults as compared to cartilage degeneration known in other diarthrodial joints. Structural differences between sacral and iliac cartilage can already be observed in the infant: the sacral auricular facet is covered with a hyaline articular cartilage, reaching 4 mm in thickness in the adult and staining intensely blue with alcian blue at pH1. Iliac cartilage of the newborn is composed of a dense fibrillar network of thick collagen bundles, crossing each other at approximately right angles. A faint staining with alcian blue suggests a low content of acidic glycosaminoglycans. In the adult, iliac cartilage becomes hyaline and its maximal thickness reaches 1–2 mm. Both articular facets exhibit morphological changes during aging that are more pronounced in the iliac cartilage and resemble osteoarthritic degeneration; the staining pattern of the extracellular matrix becomes inhomogenous, chondrocytes are arranged in clusters and the articular surface develops superficial irregularities and fissures. Sometimes fibrous tissue fills up these defects. Nevertheless, large areas of iliac cartilage remain hyaline in nature. Sacral articular cartilage often remains largely unaltered until old age. The sacral subchondral bone plate is usually thin and shows spongiosa trabeculae inserted at right angles, suggesting a perpendicular load on the articular facet. Iliac subchondral spongiosa shows no definite alignment and joins the thickened subchondral bone plate in an oblique direction. The iliac cartilage therefore seems to be stressed predominantly by shearing forces, arising from the changing monopodal support of the pelvis during locomotion. The subchondral bone plate on both the iliac and sacral auricular facet is penetrated by blood vessels that come into close contact with the overlying articular cartilage. These vessels may contribute to the high incidence of rheumatoid and inflammatory diseases in the human sacroiliac joint. Immunolabelling with an antibody against type II collagen reveals a diminished immunoreactivity in the upper half of adult sacral cartilage and only a faint and irregular labelling in the iliac cartilage. Type I collagen can be detected in a superficial layer on the sacral articular surface and around chondrocyte clusters in iliac cartilage, as in dedifferentiating chondrocytes during the development of osteoarthritis. Accepted: 22 April 1998     

Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells Cultured onto Three Different Polymers <Emphasis Type="Italic">In Vitro</Emphasis>

In this study, the osteoinductive and cell-binding properties of three different resorbable polymers were evaluated by human mesenchymal stem cells (MSCs). MSCs were isolated, expanded, and cultivated onto resorbable D,D,L,L-polylactide (PLLA), collagen I/III, and polygalactin-910/polydioxanone (PGPD) scaffolds in vitro. To evaluate the influence of dexamethasone, ascorbic acid, and beta-glycerolphosphate (DAG) on osteoblast differentiation, MSCs were incubated in a DAG-enriched medium. After a 28-day period in vitro, the cellular loaded polymers were digested enzymatically by papain and HCl. The Ca(2+) content of the biomembranes was evaluated by an o-kresolphthalein-complexon reaction via photometer. A PicoGreen assay was performed for dsDNA quantification. Significant differences between the number of adherent MSCs were documented (collagen > PLLA > PGPD). Compared to the initial number of adherent cells, all biomaterials induced a significant decrease in cellular adherence after 28 days in vitro. The presence of DAG-enriched culture medium stimulated the cellular proliferation for PLLA and slightly for PGPD, whereas cell proliferation was inhibited when MSCs were cultivated onto collagen I/III. In comparison with the control groups, all biomaterials (PLLA, PGPD, and collagen I/III) showed a significant increase in local Ca(2+) accumulation under DAG stimulation after 28 days in vitro. Furthermore, collagen I/III and PLLA scaffolds showed osteoinductive properties without DAG stimulation. These results were verified by immunocytochemical stainings against osteoblast-typical markers (osteopontin and alkaline phosphatase) and completed by calcified matrix detection (von Kossa staining). MSCs were identified by CD105 and CD13 antigen expression. Corresponding to an absence of CD34, CD45, and collagen II expression, we found no chondrogenic or hematopoietic cell differentiation. The results indicate significant differences for the proliferation, differentiation, adherence, and Ca(2+) accumulation between the tested polymers in a MSC culture.     

FT-IR study for hydroxyapatite/collagen nanocomposite cross-linked by glutaraldehyde

FT-IR analysis was performed for the hydroxyapatite (HAp)/collagen (COL) nanocomposite cross-linked by glutaraldehyde (GA). The amide bands I, II and III from COL matrix, and phosphate and carbonate bands from HAp were identified. The amide B band arising from C–H stretching mode showed a sensitive conformation by the degree of cross-linking. The amide I band showed a complicate conformational change by the degree of cross-linking. The characteristic amide I band at 1685 cm⁻¹, which is known as an aging parameter in the biological bone, did not show a monotonous tendency by the degree of cross-linking. The relative contents of the organics in the cross-linked HAp/COL nanocomposite were evaluated as an integration ratio between the amide I band at 1600–1700 cm⁻¹ and PO₄³⁻ band at 900–1200 cm⁻¹. The increase of the organics content by the cross-linking is enabled by the further organization of Ca²⁺ ions of HAp crystals in HAp/COL nanocomposite. The complicate conformational behavior in the amide I, II and III bands seems to be affected by the cross-linking induced directional arrangement of HAp/COL nanocomposite fibrils.     

Scanning electron microscopic studies of reticular framework in the rat mesenteric lymph node

Background: The reticular framework in the lymph node has in the past been studied mainly by light microscopy of silver-impregnated specimens. The aim of the present study is to understand three-dimensionally the ultrastructure and organization of the reticular framework better than before. Methods: The mesenteric lymph nodes of the rat were prepared either an alkali-water maceration method or a conventional method and were observed in a scanning electron microscope (SEM). Results: The SEM study of alkali-water macerated tissues visualized directly the reticular fiber network in the lymph node. The reticular fibers consisted of thin bundles of collagem fibrils. They were continuous with the collagen fibriliar sheaths of blood vessels and lymphatic sinuses as well as with the fibrous capusule, thus acting as a skeleton of the lymph node. The arrangement of the reticulum was variable, depending on individual compartments. The SEM study of conventionally treated tissues, on the other hand, clarified the shape of reticular cells and their relationship with the reticular fibers. The sinus reticular cells connected with the sinus lining cells but separated from the parenchymal reticular cells, indicating that the former two originate from lymphatic endothelial cells. The parenchymal reticular cells varied in shape depending on their locations but essentially shared features with fibroblasts. Conclusions: The arrangements of the reticular fibers in the parenchyma were closely related to the associated reticular cells, showing the possibility that the reticular cells maintain the shape of the reticular framework suitable for each compartment of the lymph node. © 1995 Wiley-Liss, Inc.     

Age- and training-related changes in the collagen metabolism of rat skeletal muscle

Summary The effects of ageing and life-long endurance training on the collagen metabolism of skeletal muscle were evaluated in a longitudinal study. Wistar rats performed treadmill running 5 days a week for 2 years. The activities of collagen biosynthesis enzymes, prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase, were highest in the muscles of the youngest animals, decreased up to the age of 2 months and from then on remained virtually unchanged. The enzyme activity in young animals was higher in the slow collagenous soleus muscle than in the rectus femoris muscle. The enzyme activity in the soleus muscle was higher for older trained rats than older untrained rats. The relative proportion of type I collagen increased and that of type III collagen decreased with age, suggesting a more marked contribution by type I collagen to the agerelated accumulation of total muscular collagen. The results show that collagen biosynthesis decreases with maturation and that life-long endurance training maintains a higher level of biosynthesis in slow muscles.