Expression of decorin, biglycan, and collagen type I in human renal fibrosing disease

BACKGROUND: The extracellular matrix proteoglycans decorin and biglycan may have a pathogenic role in renal fibrosing disease via regulation of the activity of growth factors, such as transforming growth factor-beta, and effects on collagen type I fibrillogenesis. The expression of decorin and biglycan in human glomerular diseases characterized by mesangial sclerosis is unknown. METHODS: Decorin, biglycan, and collagen type I were localized immunohistochemically in human renal biopsy cases of amyloidosis (N = 18), diabetic nephropathy (N = 11), fibrillary glomerulonephritis (N = 5), immunotactoid glomerulopathy (N = 5), light-chain deposition disease (N = 4), idiopathic mesangial sclerosis (N = 4), and nephrosclerosis (N = 6), and in morphologically normal tissues obtained from tumor nephrectomies (N = 8). Decorin and biglycan mRNA synthesis was evaluated by in situ hybridization. RESULTS: Decorin and biglycan protein were not identified in normal glomeruli. Decorin accumulated in amyloid deposits, but not in deposits of fibrillary glomerulonephritis or immunotactoid glomerulopathy. Biglycan weakly accumulated in amyloid deposits, and both decorin and biglycan weakly stained mesangial nodules in cases of morphologically advanced light-chain deposition disease and diabetic nephropathy. In all analyzed cases, irrespective of the underlying disease, decorin and biglycan accumulated in glomeruli in areas of fibrous organization of the urinary space and in areas of tubulointerstitial fibrosis. Biglycan, but not decorin, accumulated in the neointima of arteriosclerotic blood vessels. Decorin and biglycan mRNA synthesis was detected at sites of proteoglycan accumulation in glomeruli, interstitium, and neointima. Collagen type I colocalized with decorin and biglycan deposits. CONCLUSIONS: Differences in extracellular matrix proteoglycan composition may be diagnostically useful in distinguishing morphologically similar diseases. Distinct patterns of proteoglycan expression may be related to modulation of specific growth factor activity in different glomerular diseases.     

The localized expression of extracellular matrix components in healing tendon insertion sites: an in situ hybridization study.

The localized expression of a number of extracellular matrix genes was evaluated over time in a novel rat rotator cuff injury model. The supraspinatus tendons of rats were severed at the bony insertion and repaired surgically. The healing response was evaluated at 1, 2, 4, and 8 weeks post-injury using histologic and in situ hybridization techniques. Expression patterns of collagens (I, II, III, IX, X, XII), proteoglycans (decorin, aggrecan, versican, biglycan, fibromodulin), and other extracellular matrix proteins (elastin, osteocalcin, alkaline phosphatase) were evaluated at the healing tendon to bone insertion site. Histologic results indicate a poor healing response to the injury, with only partial recreation of the insertion site by 8 weeks. In situ hybridization results indicate a specific pattern of genes expressed in each zone of the insertion site (i.e., tendon, fibrocartilage, mineralized cartilage, bone). Overall, expression of collagen types I and XII, aggrecan, and biglycan was increased, while expression of collagen type X and decorin was decreased. Expression of collagen type I, collagen type XII, and biglycan decreased over time, but remained above normal at 8 weeks. Results indicate that the rat supraspinatus tendon is ineffective in recreating the original insertion site, even at 8 weeks post-injury, in the absence of biological or biomechanical enhancements.     

Glomerular expression of C-C chemokines in different types of human crescentic glomerulonephritis.

BACKGROUND: Crescentic glomerulonephritis (CGN) presents a rapidly progressive glomerulonephritis clinically, in which macrophages play a crucial role in the pathogenesis. However, the precise molecular mechanism of macrophage recruitment and activation has not been fully elucidated. C-C chemokines, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha and beta (MIP-1alpha and MIP-1beta), are major chemoattractants for macrophages. We attempted to study the expression of C-C chemokines and their correlation with CD68-positive macrophages in crescentic glomeruli to investigate further their possible roles in crescent formation and progression to fibrosis in different types of human CGN. METHODS: The expression of MCP-1, MIP-1alpha, MIP-1beta and CD68 was detected in glomeruli with different forms of crescents (cellular, fibrocellular and fibrous crescents) by immunohistochemistry in serial sections of renal biopsies taken from 32 patients with biopsy-proven CGN including eight patients with anti-glomerular basement membrane (GBM) disease (type I CGN), 12 patients with immune complex-mediated CGN (type II CGN) and another 12 patients with pauci-immune CGN (type III CGN) enrolled in this study. Eight normal human kidneys were obtained from cadaveric renal transplant donors whose kidneys were technically unsuitable for transplantation, serving as controls. RESULTS: MCP-1, MIP-1alpha, MIP-1beta and CD68 were undetectable in glomeruli of normal kidney. In crescentic biopsies, MCP-1, MIP-1alpha, MIP-1beta and CD68 were detected in fibrocellular crescents and were even more prominent in cellular crescents, but were undetectable in fibrous crescents. Using consecutive sections for staining, it was demonstrated that a high proportion of infiltrating CD68-positive macrophages, mainly localized to the area of the expression of chemokines, were MCP-1, MIP-1alpha and MIP-1beta positive in crescents. Chemokines were expressed mainly by CD68-positive macrophages and parietal epithelial cells in crescents. The number of MCP-1- and MIP-1alpha-positive cells in glomeruli with cellular crescents was positively correlated with the number of CD68-positive cells (r = 0.568 and 0.749, respectively, both P < 0.01). The number of MCP-1- and MIP-1alpha-positive cells and the incidence of Bowman's capsule rupture in glomeruli of patients with type I CGN were higher than those of type II and type III CGN. CONCLUSIONS: These observations suggest that the expressed C-C chemokines, MCP-1, MIP-1alpha and MIP-1beta, may mediate the inflammatory process of crescent formation and progression to fibrosis. The strong correlation of MCP-1 and MIP-1alpha with infiltrating macrophages within glomeruli with cellular crescents suggested that these chemokines might be of particular importance for macrophage recruitment to this site. MCP-1 and MIP-1alpha were correlated to type I CGN with its more severe inflammatory course and worse prognosis. The variance of glomerular expression of C-C chemokines may contribute to the difference in histopathological features and prognosis in these three types of CGN.     

Increased Turnover of Small Proteoglycans Synthesized by Human Osteoblasts During Cultivation with Ascorbate and β-Glycerophosphate

The small proteoglycan decorin had been localized previously at the d-band in the gap zone of collagen fibrils in nonmineralizing tissues. In bone matrix this zone is proteoglycan free and is at least in some species the place where mineralization along collagen fibrils starts. To study the metabolism of the small proteoglycans decorin and biglycan under mineralizing conditions, osteoblasts from human nasal bone were cultured for several weeks in the presence or absence of β-glycerophosphate and ascorbate. An immediate consequence of the treatment was a reduced expression of decorin, as judged by immune precipitation, whereas the biosynthesis of biglycan was not affected. Pulse-chase experiments were performed with osteoblasts embedded in floating type I collagen gels. In the presence of β-glycerophosphate and ascorbate, a more rapid turnover of both proteoglycans was noted; the one of biglycan reached statistical significance. Indirect evidence for an enhanced rate of proteoglycan endocytosis was obtained. This effect was not seen in cultured skin fibroblasts. Thus, osteoblasts respond rapidly to mineralizing conditions with alterations of small proteoglycan biosynthesis and turnover. Received: 30 October 1995 / Accepted: 3 January 1997     

Proteoglycans and glycosaminoglycan fine structure in the mouse tail tendon fascicle.

The isolated mouse tail tendon fascicle, a functional and homogenous volume of tendon extracellular matrix, was utilized as an experimental system to examine the structure function relationships in tendon. Our previous work using this model system demonstrated relationships between mean collagen fibril diameter and fascicle mechanical properties in isolated tail tendon fascicles from three different groups of mice (3-week and 8-week control and 8-week Mov13 transgenic) K.A. Derwin, L.J. Soslowsky, J. Biomech. Eng. 121 (1999) 598-604. These groups of mice were chosen to obtain tendon tissues with varying collagen fibril structure and/or biochemistry, such that relationships with material properties could be investigated. To further investigate the molecular details of matrix composition and organization underlying tendon function, we report now on the preparation, characterization, and quantitation of fascicle PGs (proteoglycans) from these three groups. The chondroitin sulfate/dermatan sulfate (CS/DS)-substituted PGs, biglycan and decorin, which are the abundant proteoglycans of whole tendons, were also shown to be the predominant PGs in isolated fascicles. Furthermore, similar to the postnatal maturation changes in matrix composition previously reported for whole tendons, isolated fascicles from 8-week mice had lower CS/DS PG contents (both decorin and biglycan) and a higher collagen content than 3-week mice. In addition, CS/DS chains substituted on PGs from 8-week fascicles were shorter (based on a number average) and richer in disulfated disaccharide residues than chains from 3-week mice. Fascicles from 8-week Mov13 transgenic mice were found to contain similar amounts of total collagen and total CS/DS PG as age-matched controls, and CS/DS chain lengths and sulfation also appeared normal. However, both decorin and biglycan in Mov13 tissue migrated slightly faster on sodium dodecyl sulfate polyacrylamide gel electorphoresis (SDS-PAGE) than the corresponding species from 8-week control, and biglycan from the 8-week Mov 13 fascicles appeared to migrate as a more polydisperse band, suggesting the presence of a unique PG population in the transgenic tissue. These observations, together with our biomechanical data [Derwin and Soslowsky, 1999] suggest that compensatory pathways of extracellular matrix assembly and maturation may exist, and that tissue mechanical properties may not be simply determined by the contents of individual matrix components or collagen fibril size.     

Characterization of proteoglycan production and processing by chondrocytes and BMSCs in tissue engineered constructs

OBJECTIVE: The goal of this study was to characterize the proteoglycan (PG) production and processing by bone marrow stromal cells (BMSCs) within a tissue engineered construct. METHODS: Bovine BMSCs and articular chondrocytes (ACs) were isolated from an immature calf, seeded into agarose gels, and cultured up to 32 days in the presence of TGF-beta1. The localization of various PGs was examined by immunofluorescence and histological staining. The role of proteolytic enzymes in construct development was further investigated by examining the effects of aggrecanase and MMP inhibitors on PG accumulation, aggrecan processing, and construct mechanics. RESULTS: BMSCs developed a matrix rich in sulfated-glycosaminoglycans (sGAG) and full-length aggrecan, but had low levels of versican. The BMSC constructs had less collagen II and aggrecan compared to the AC constructs cultured under identical conditions. AC constructs also had high levels of pericellular collagen VI, while BMSCs had a pericellular matrix containing little collagen VI and greater levels of decorin, biglycan, and fibronectin. Treatment with the aggrecanase inhibitor did not affect sGAG accumulation or the dynamic moduli of the BMSC constructs. The MMP inhibitor slightly but significantly inhibited sGAG accumulation and lowered the dynamic moduli of BMSC constructs. CONCLUSIONS: The results of this preliminary study indicate that long-term culture of BMSCs with TGF-beta1 promotes the development of an aggrecan-rich matrix characteristic of native articular cartilage; however, BMSCs accumulate significantly lower levels of sGAG and assemble distinct pericellular microenvironments compared to ACs. PG turnover does not appear to play a major role in the development of tissue engineered cartilage constructs by BMSCs.     

Transforming growth factor-beta regulates production of proteoglycans by mesangial cells

Accumulation of glomerular extracellular matrix is a prominent feature of most forms of progressive glomerular disease. Since some growth factors may play a role in extracellular matrix production, we examined the effects of transforming growth factor-beta (TGF-beta), interleukin 1, platelet derived growth factor, and tumor necrosis factor on the production of extracellular matrix components by cultured rat mesangial cells. In control experiments we found that mesangial cells produced two distinct proteoglycans identified as the small chondroitin/dermatan sulfate proteoglycans biglycan (PG I) and decorin (PG II) by showing that their mobility on SDS-PAGE changed upon digestion by chondroitinase ABC, and that they reacted with antibodies raised against synthetic peptides from the core protein sequence of human biglycan and decorin. Exposure to TGF-beta for 48 hours stimulated an 8- to 10-fold increase in the biglycan and decorin bands, and induced a structural change detected as a shift in electrophoretic mobility. TGF-beta did not demonstrably affect the production of other matrix proteins by the mesangial cells. The other growth factors tested had no comparable effect on the production of proteoglycans or other extracellular matrix components by these cells. Our results show that TGF-beta is unique among growth factors in its regulatory effects on mesangial cell proteoglycan production. The release or activation of TGF-beta during glomerular injury could mediate the accumulation of proteoglycans in the extracellular matrix and predispose the kidney to development of glomerulosclerosis.     

Immunohistochemical Identification of Decorin and Biglycan in Human Dentin: A Correlative Field Emission Scanning Electron Microscopy/Transmission Electron Microscopy Study

Decorin and biglycan, two small leucine-rich proteoglycans, have been proposed to play important roles in matrix-mediated formation of mineralized tissues, and their three-dimensional arrangement in human dentin is still not completely understood. The aim of this study was to immunohistochemically analyze the distribution of decorin and biglycan in human predentin/dentin organic matrix under a high-resolution field emission in-lens scanning electron microscope (FEI-SEM) and a transmission electron microscope (TEM). Tooth dentin specimens were submitted to either a preembedding or a postembedding immunolabeling technique using primary antibodies antidecorin and antibiglycan and gold-conjugated secondary antibodies. Correlative FEI-SEM/TEM observations showed that the two antibodies yielded a similar labeling pattern over the processes of odontoblasts and the predentin. Decorin and biglycan were mainly associated with the collagen fibers within the predentin layer, revealing a moderate immunoreaction that was significantly higher compared to the one observed on dentin. Thus, a generally weak labeling for decorin was found in dentin, which, however, was significantly higher on odontoblast processes within dentinal tubules than in intertubular dentin. On the other hand, biglycan immunolocalization on dentin revealed few gold particles rather uniformly distributed, without showing significant differences between tubular and intertubular regions. In conclusion, this study reveals distinct distribution patterns of decorin and biglycan and their relation with collagen. Decorin’s and biglycan’s precise roles within prematrix and mineralized matrix in human teeth should be further clarified.     

TGF-β/Extracellular Matrix Interactions in Dentin Matrix: A Role in Regulating Sequestration and Protection of Bioactivity

TGF-β isoforms sequestrated in dentin matrix potentially provide a reservoir of bioactive molecules that may influence cell behavior in the dentin–pulp complex following tissue injury. The association of these growth factors with dentin matrix and the influence of such associations on the bioactivity of growth factors are still unclear. We used surface plasmon resonance technology in the BIAcore^TM 3000 system to investigate the binding of TGF-β isoforms 1 and 3 to purified decorin, biglycan, and EDTA soluble dentin matrix components. TGF-β isoforms 1 and 3 were immobilized on sensorchips CM4 through amine coupling. For kinetic studies of protein binding, purified decorin and biglycan, isolated EDTA soluble dentin matrix, and dentin matrix immunodepleted of decorin and/or biglycan were injected over TGF-β isoforms and allowed to interact. Programmed kinetic analysis software provided sensorgrams for each concentration of proteoglycan or dentin matrix extract injected. Purified decorin and biglycan and dentin matrix extract bound to the TGF-β isoforms. However, the association with TGF-β3 was much weaker than that with TGF-β1. After immunoaffinity depletion of the dentin matrix extract, the level of interaction between the dentin matrix extract and TGF-β was significantly reduced. These results suggest isoform-specific interactions between decorin/biglycan and TGF-β isoforms 1 and 3, which may explain why TGF-β3 is not detected in the dentin matrix despite being expressed at higher levels than TGF-β1 in odontoblasts. These proteoglycans appear to play a significant role in TGF-β/extracellular matrix interactions and may be important in the sequestration of these growth factors in the dentin matrix.     

Joint immobilization reduces the expression of sensory neuropeptide receptors and impairs healing after tendon rupture in a rat model

Healing after mobilization versus immobilization was assessed in a model of rat Achilles tendon rupture, by RT‐PCR at 8 and 17 days and by histological analyses at 14 and 28 days postrupture. The expression of mRNA for extracellular matrix (ECM) molecules (collagen type I and type III, versican, decorin, and biglycan), and the subjective histological maturation of the healing area were analyzed. Effects of immobilization on healing were related to changes in the peripheral expression of substance P (NK₁)‐ and calcitonin gene‐related peptide (CRLR and RAMP‐1)‐ receptors. At 8 days postinjury, mRNA levels for ECM molecules were equal in both groups. However, by day 17, the ECM mRNA expression in the mobilized group had increased up to ～14× that of the immobilized group, which were comparable to intact tendon values. Histological analysis confirmed a higher regenerating activity in the mobilized group, with an increased amount of blood vessels, fibroblasts, and new collagen. The expression of sensory neuropeptide receptors in the mobilized group exhibited a significant increase from 8 to 17 days postinjury similar to the increased ECM mRNA expression, whereas the immobilized group at 17 days exhibited levels comparable to the intact tendon values. Therefore, immobilization postrupture appears to hamper tendon healing, a process which may prove to be directly linked to a downregulated peripheral sensitivity to sensory neuropeptide stimulation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:274–280, 2009     

Roles of and correlation between alpha-smooth muscle actin, CD44, hyaluronic acid and osteopontin in crescent formation in human glomerulonephritis

BACKGROUND: alpha-Smooth muscle actin (SMA), CD44, hyaluronic acid (HA) and osteopontin (OPN) are involved in crescent formation; however, the correlation between these molecules during the formation and progression of the crescents in human glomerulonephritis (GN) has not been fully evaluated. METHODS: To investigate the expression of alpha-SMA, CD44, HA, OPN and CD68 renal biopsy specimens from 14 patients with crescentic GN were examined by immunohistochemistry. All crescents were separated into cellular, fibrocellular and fibrous. The extent of staining in each crescent was scored semiquantitatively. The change in the expression of each molecule and its correlation with other molecules during the formation and progression of the crescents were estimated statistically. RESULTS: The expression of alpha-SMA was significantly up-regulated in the fibrocellular crescents compared with that in the cellular and fibrous crescents. The expression of CD44, OPN and CD68 was significant in the cellular crescents compared with that in the fibrocellular and fibrous crescents. The deposition of HA in the three groups of crescents was high level. However, that of HA was not significant among three groups of crescent. The expression of CD44 in the cellular crescents correlated significantly with the expression of OPN and CD68, and the deposition of HA in the cellular crescents. The expression of OPN in the cellular crescents correlated with the deposition of HA and the expression of CD68 in the cellular crescents. The expression of alpha-SMA in the cellular and fibrocellular crescents correlated with the deposition of HA in the cellular and fibrocellular crescents. CONCLUSION: The expression of CD44, HA, OPN and CD68 was up-regulated at the early stage of the crescent formation in human crescentic GN. Moreover, myofibroblasts and cell-matrix interactions mediated by the CD44-OPN and CD44-HA receptor-ligand pairs may play important roles in the formation and progression of the crescents.     

Production of extracellular matrix by glomerular epithelial cells is regulated by transforming growth factor-beta 1.

Transforming growth factor-beta (TGF-beta) has widespread effects on extracellular matrix production by many cultured cell lines and appears to play a role in the pathological accumulation of extracellular matrix that accompanies inflammatory and fibrotic diseases such as glomerulonephritis. Earlier experiments have shown that mesangial cells respond to TGF-beta 1 with a marked increase in the production of two chondroitin/dermatan sulfate proteoglycans, decorin and biglycan, but their production of other matrix components elevated in glomerulonephritis is not substantially affected by TGF-beta 1. Since the glomerular epithelial cells are also thought to contribute to matrix production in the glomerulus, we examined the ability of these cells to produce some of the nonproteoglycan matrix components in response to TGF-beta 1. Exposure of glomerular epithelial cells to TGF-beta 1 increased the production of fibronectin and type IV collagen, in addition to biglycan. Enhancement of the cell layer accumulation of laminin was also observed. These results show that TGF-beta 1 has a differential effect on extracellular matrix production by epithelial and mesangial cells from glomeruli. TGF-beta 1 released in the glomerulus secondary to injury could thus affect both cell types and lead to increased intraglomerular production of proteoglycans, whereas the increased fibronectin, type IV collagen, and laminin may primarily originate from the epithelial cells.     

Immunoelectron Microscopic Visualization of Pro- and Secreted Forms of Decorin and Biglycan in the Predentin and During Dentin Formation in the Rat Incisor

Using antibodies raised against the proform and fully processed (secreted) forms of the proteoglycans decorin and biglycan, combined with gold electron immunohistochemistry, we observed in the incisors of five Sprague-Dawley rats that the proforms were mostly located in the cell bodies of odontoblasts, with a presence reduced to one-third or one-fourth in the processes. Proforms, also present in the extracellular matrix, were uniformly distributed throughout predentin, with higher labeling for probiglycan than pro-decorin. Both were present in lesser amounts in metadentin and dentin. With respect to the secreted form, grain density was at a constant level for biglycan in predentin and dentin, whereas a gradient was detected for decorin, the grain density being increased three times in the distal predentin. Although decorin labeling was diminished in metadentin, staining in circumpulpal dentin was similar to that found in distal predentin. We have previously reported a reverse gradient for chondroitin sulfate/dermatan sulfate distribution. To reconcile these diverging data, our hypothesis is that enzymatic proteolytic cleavage may remove the glycosylated N-terminal-containing region, resulting in a non- proteoglycan form of the molecule. Although substantial differences in distribution were apparent between the two proteoglycans, increasing interactions between proteoglycans and collagen, facilitated by the cleavage and loss of the N-terminal glycosaminoglycan chain region in the distal predentin, may be a prerequisite for dentin mineralization.