Biomedical subjects

J van den Born

Publications and source records attributed to J van den Born.

At least 19 recordsLinked to original sources

PubMed1998

Differential expression of agrin in renal basement membranes as revealed by domain-specific antibodies.

We determined the specificity of two hamster monoclonal antibodies and a sheep polyclonal antiserum against heparan sulfate proteoglycan isolated from rat glomerular basement membrane. The antibodies were characterized by enzyme-linked immunosorbent assay on various basement membrane components and immunoprecipitation with heparan sulfate proteoglycan with or without heparitinase pre-treatment. These experiments showed that the antibodies specifically recognize approximately 150-, 105-, and 70-kDa core proteins of rat glomerular basement membrane heparan sulfate proteoglycan. Recently, we showed that agrin is a major heparan sulfate proteoglycan in the glomerular basement membrane (Groffen, A. J. A., Ruegg, M. A., Dijkman, H. B. P. M., Van der Velden, T. J., Buskens, C. A., van den Born, J., Assmann, K. J. M., Monnens, L. A. H., Veerkamp, J. H., and van den Heuvel, L. P. W. J. (1998) J. Histochem. Cytochem. 46, 19-27). Therefore, we tested whether our antibodies recognize agrin. To this end, we evaluated staining of Chinese hamster ovary cells transfected with constructs encoding full-length or the C-terminal half of rat agrin by analysis on a fluorescence-activated cell sorter. Both hamster monoclonals and the sheep antiserum clearly stained cells transfected with the construct encoding full-length agrin, whereas wild type cells and cells transfected with the construct encoding the C-terminal part of agrin were not recognized. A panel of previously characterized monoclonals, directed against C-terminal agrin, clearly stained cells transfected with either of the constructs but not wild type cells. This indicates that both hamster monoclonals and the sheep antiserum recognize epitopes on the N-terminal half of agrin. By immunohistochemistry on rat renal tissue, we compared distribution of N-terminal agrin with that of C-terminal agrin. The monoclonal antibodies against C-terminal agrin stained almost exclusively the glomerular basement membrane, whereas the anti-N-terminal agrin antibodies recognized all renal basement membranes, including tubular basement membranes. Based on these results, we hypothesize that full-length agrin is predominantly expressed in the glomerular basement membrane, whereas in most other renal basement membranes a truncated isoform of agrin is predominantly found that misses (part of) the C terminus, which might be due to alternative splicing and/or posttranslational processing. The possible significance of this finding is discussed.

Agrin↗

PubMed1998

Extracellular matrix in human diabetic nephropathy: reduced expression of heparan sulphate in skin basement membrane.

In diabetic nephropathy, expression of glycosaminoglycan side chains of heparan sulphate proteoglycan in the glomerular basement membrane is reduced proportionally to the degree of proteinuria. We performed a cross-sectional study to evaluate whether non-vascular basement membranes also show a decrease in heparan sulphate side chain staining in patients with diabetic nephropathy. We evaluated the skin basement membrane for extracellular matrix components in the following groups: control subjects (n = 16); patients with Type 1 diabetes and normoalbuminuria (n = 17), microalbuminuria (n = 7), and macroalbuminuria (n = 16); patients with Type 1 diabetes and diabetic nephropathy undergoing renal replacement therapy (n = 13); and non-diabetic patients undergoing renal replacement therapy (n = 21). The following antibodies were used for this immunohistochemical study: monoclonal antibodies against the heparan sulphate side chain (JM403) and core protein (JM72) of the glomerular heparan sulphate proteoglycan; polyclonal antibodies against the core protein (B31); polyclonal antibodies against collagen types I, III, and IV, fibronectin, and laminin; and monoclonal antibodies against the noncollagenous domain of alpha1(collagen IV) and alpha3(collagen IV), against transforming growth factor beta(2G7), and against advanced glycosylation end products (4G9). Expression of heparan sulphate side chains was reduced in the skin basement membrane of patients with overt diabetic nephropathy, of those with Type 1 diabetes undergoing renal replacement therapy, and those with non-diabetic renal failure. Increased intensity of staining was found for collagen type I and advanced glycosylation end products in patients with diabetic nephropathy. Changes in the extracellular matrix of the skin basement membrane seem to be similar to those in the glomerular basement membrane. These findings support the suggestion that patients with diabetic nephropathy also have altered heparan sulphate and collagen staining in extrarenal basement membranes. However, patients with non-diabetic renal failure also had reduced expression of heparan sulphate in the skin basement membrane, suggesting that this finding is not specific for diabetic nephropathy.

Adult↗

PubMed1998

Decreased staining of heparan sulfate in non-lesional skin of a subgroup of patients with systemic lupus erythematosus.

Heparan sulfate proteoglycans (HSPGs) are components of the basement membrane of various tissues. They are composed of a core protein and of the negatively charged glycosaminoglycan side chain heparan sulfate, which is covalently bound to the core protein. We previously found that in both human and murine lupus nephritis, heparan sulfate staining in the basement membrane of the glomerulus is almost completely absent, and that there was an inverse correlation between heparan sulfate staining and glomerular immunoglobulin deposits. As immunoglobulin deposits are also present in the skin of systemic lupus erythematosus patients, we investigated the heparan sulfate staining pattern in the basement membrane of the dermal-epidermal junction. furthermore, we studied whether there was a correlation between heparan sulfate staining and deposition of immunoglobulin in the basement membrane of this junction, and between heparan sulfate staining and the presence of anti-DNA antibodies in the serum. Biopsies of non-lesional skin of 21 systemic lupus erythematosus patients (15 anti-DNA positive and 6 anti-DNA negative patients at the time of biopsy) were stained for the HSPG-core protein (mAb JM-72), highly sulfated stretches within heparan sulfate (JM-13), the low sulfated regions of heparan sulfate (mAb JM-403) and for immunoglobulin depositions. Abnormal and discontinuous staining of the low sulfated parts of heparan sulfate using mAb JM-403 in the basement membrane was found in skin biopsies of 4 out of 15 systemic lupus erythematosus patients with anti-DNA antibodies. In contrast, all specimens of anti-DNA negative patients showed normal continuous staining. Staining with JM-13 and JM-72 showed normal linear staining in both groups. The decreased heparan sulfate staining was correlated significantly with the presence of immunoglobulin deposits in the basement membrane. The subgroup could not be identified by its clinical picture. Our results suggest similar but not identical pathways in systemic lupus erythematosus nephritis and skin of systemic lupus erythematosus patients.

Adolescent↗

PubMed1998

Agrin is a major heparan sulfate proteoglycan in the human glomerular basement membrane.

Agrin is a heparan sulfate proteoglycan (HSPG) that is highly concentrated in the synaptic basal lamina at the neuromuscular junction (NMJ). Agrin-like immunoreactivity is also detected outside the NMJ. Here we show that agrin is a major HSPG component of the human glomerular basement membrane (GBM). This is in addition to perlecan, a previously characterized HSPG of basement membranes. Antibodies against agrin and against an unidentified GBM HSPG produced a strong staining of the GBM and the NMJ, different from that observed with anti-perlecan antibodies. In addition, anti-agrin antisera recognized purified GBM HSPG and competed with an anti-GBM HSPG monoclonal antibody in ELISA. Furthermore, both antibodies recognized a molecule that migrated in SDS-PAGE as a smear and had a molecular mass of approximately 200-210 kD after deglycosylation. In immunoelectron microscopy, agrin showed a linear distribution along the GBM and was present throughout the width of the GBM. This was again different from perlecan, which was exclusively present on the endothelial side of the GBM and was distributed in a nonlinear manner. Quantitative ELISA showed that, compared with perlecan, the agrin-like GBM HSPG showed a sixfold higher molarity in crude glomerular extract. These results show that agrin is a major component of the GBM, indicating that it may play a role in renal ultrafiltration and cell matrix interaction. (J Histochem Cytochem 46:19-27, 1998)

Adult↗

PubMed1997

Hydroxyl radicals depolymerize glomerular heparan sulfate in vitro and in experimental nephrotic syndrome.

Heparan sulfate, the polysaccharide side chain of heparan sulfate proteoglycan, is important for the permselective properties of the glomerular basement membrane. In this report, we show a role for hydroxyl radicals in heparan sulfate degradation and an enhanced glomerular basement membrane permeability. First, in enzyme-linked immunosorbent assay, exposure of coated heparan sulfate (proteoglycan) to reactive oxygen species resulted in a +/-50% decrease of binding of a monoclonal antibody against heparan sulfate, whereas binding of an antibody against the core protein remained unaltered. Second, on polyacrylamide gel electrophoresis, the molecular weight of heparan sulfate exposed to radicals was reduced which indicates depolymerization. Both in enzyme-linked immunosorbent assay and gel electrophoresis, hydroxyl radicals are instrumental for heparan sulfate degradation as shown by the addition of various radical scavengers. Third, in an experimental model for human nephrotic syndrome (Adriamycin nephropathy in rats), glomerular basement membrane staining of two recently described anti-heparan sulfate antibodies (JM403 and KJ865) was reduced by 24 and 43%. Treatment of Adriamycin-exposed rats with the hydroxyl radical scavenger dimethylthiourea both reduced albuminuria by 37% (p < 0.01) and partly prevented loss of heparan sulfate staining by 53% (JM403) and 39% (KJ865) (p < 0.03). In contrast to the heparan sulfate side chains, the core protein expression and the extent of glycanation did not change in Adriamycin nephropathy. We conclude that glomerular basement membrane heparan sulfate is susceptible to depolymerization by hydroxyl radicals leading to loss of glomerular basement membrane integrity and albuminuria.

Animals↗

PubMed1997

Basal membrane heparan sulphate proteoglycan expression during wound healing in human skin.

Heparan sulphate proteoglycans (HSPGs) are integral components of the basement membrane (BM) in various tissues. HSPGs are important in the assembly and structure of the BM, and their putative functions include regulation of basement membrane permeability, binding of growth factors, and a role in cellular adhesion. In this study the expression of HSPG was examined during wound healing in human skin, using monoclonal antibodies (MAbs) that recognize the HSPG core protein and two different heparan sulphate (HS) epitopes, and the dynamics of HSPG expression were investigated in relation to epidermal cellular proliferation and permeability of the BM. Healing of excisional wounds in healthy volunteers was studied from day 0 up to 1 year. Intact human skin showed strong continuous staining of the dermo-epidermal BM and the vascular BM with all MAbs. Up to day 4 after wounding, staining for HSPG was absent under the ingrowing epidermis, with any of the MAbs, indicating that no complete BM was present. From day 7 onwards, the BM of the neo-epidermis showed positive staining for the HSPG core protein and a low sulphated HS epitope, and after day 14, the staining intensity was similar to normal skin. The staining patterns of these HSPG epitopes were similar to that of laminin. The staining pattern with a MAb against an epitope in the highly sulphated part of HS was found to be distinct from the other BM markers studied. This epitope was absent under the neo-epidermis up to 2 months after wounding. One year after wounding, the epitope was found to be present again. We observed that only in the time period between 2 months and 1 year had the epidermis normalized with respect to the number of cycling cells and the absence of high molecular weight plasma proteins. These findings suggest a correlation between normalization of epidermal proliferation, BM permeability, and regeneration of BM HS. It is proposed that complete BM maturation following skin wounding is a slow process and may account for the epidermal abnormalities that persist for a considerable period of time after wound healing.

Adult↗

PubMed1997

Epicutaneous application of leukotriene B4 induces patterns of tenascin and a heparan sulfate proteoglycan epitope that are typical for psoriatic lesions.

Application of leukotriene B4 (LTB4) to normal human skin induces changes similar to those found in psoriatic skin, and it has proved to be a useful model for studying the pathogenesis and treatment of psoriasis. We studied the expression patterns of molecules that have recently been shown to be altered in lesional psoriatic skin, including the extracellular matrix protein tenascin (TN) and the basement membrane and cell surface-associated heparan sulfate proteoglycans (HSPGs). During 72-h the expression of these markers was studied immunohistochemically and the expression of TN was correlated with epidermal proliferation and influx of inflammatory cells. Following the peak influx of polymorphonuclear leukocytes, a marked increase in TN expression was noted in the papillary dermis 72 h after LTB4 application. The expression patterns of basal membrane-associated epitopes of HSPG remained unaltered, whereas the expression of cell surface-associated HSPG disappeared 72 h after LTB4 application. A significant correlation was found between dermal TN expression and epidermal hyperproliferation, and between TN expression and the presence of dermal T cells. These findings indicate that the model of LTB4-induced acute cutaneous inflammation displays many characteristics of early psoriatic lesions and could serve as a model to study some of the cell biological changes in this disease.

Administration, Cutaneous↗

PubMed1997

Nucleosomes and histones are present in glomerular deposits in human lupus nephritis.

BACKGROUND: Recently we showed that antinuclear autoantibodies complexed to nucleosomes can bind to heparan sulphate (HS) in the glomerular basement membrane (GBM) via the histone part of the nucleosome. Histones have been identified in glomerular deposits in human and murine lupus nephritis. In addition, a decreased HS staining in the GBM was found, most probably due to masking by deposition of antibodies complexed to nucleosomes. METHODS: In this study we first investigated whether histones or nucleosomes could be identified in glomerular deposits in human lupus nephritis, and secondly whether the presence of these nuclear components was correlated with absence of HS staining. Kidney biopsies of SLE patients (11 with diffuse proliferative glomerulonephritis (DPGN) and six with membranous glomerulonephritis (MGN)) and non-SLE glomerular diseases were stained for histones. DNA, nucleosomes, IgG and HS. RESULTS: Using a polyclonal anti-H3 1 21 antiserum, histones were detected in all patients with DPGN and in two of six patients with SLE-MGN (P < 0.01). Using a monoclonal antihistone antibody, histones were stained in three patients with DPGN, but in none of the biopsies with MGN. Using nucleosome specific monoclonal antibodies, nucleosomes were detected in five patients with DPGN, in two patients with MGN, but in none of the biopsies with non-SLE glomerulonephritis. HS staining was nearly absent in DPGN, whereas staining was only moderately reduced in patients with MGN and controls (P = 0.001). CONCLUSION: Using polyclonal and monoclonal antihistone antisera, histones were identified in all patients with DPGN and their presence was associated with a decrease of HS staining. Nucleosomes were identified in five of 11 patients with DPGN and in two of six patients with MGN. This is the first demonstration of nucleosomes in glomerular deposits in SLE nephritis.

Adolescent↗

PubMed1997

Antiproteinuric effect of ciclosporin A in adriamycin nephropathy in rats.

Ciclosporin A (CsA) can reduce proteinuria in various forms of human and experimental glomerulopathies. This antiproteinuric effect can be the result of a decrease of immunological damage, a decrease in the glomerular filtration rate (GFR), or a change in the permselective properties of the glomerular capillary wall. In this study we investigated the effect of CsA on Adriamycin-induced nephropathy in rats. A single intravenous injection of Adriamycin (5 mg/kg body weight) induced a severe nephrotic syndrome with a massive albuminuria (+/- 400 mg/24 h from 3 weeks onwards) and a hypoalbuminemia (+/- 7 mg/ml after 5 weeks). The IgG/albumin selectivity index was 0.16 +/- 0.05, indicating a preferential loss of albumin. A 5-day treatment with CsA reduced the albumin excretion by almost 50% (from 336 +/- 91 to 178 +/- 58 mg/24 h; p = 0.002) and induced an increase in the serum albumin level (from 7.1 +/- 4.1 to 12.8 +/- 3.2 mg/ml; p = 0.002) in contrast to the vehicle olive oil (OO). CsA also decreased the GFR by 40% (from 0.74 +/- 0.11 to 0.41 +/- 0.11 mg/ml/100 g body weight; p = 0.002). Albuminuria corrected for the GFR (fractional excretion of albumin, FE(alb)) was still significantly lower in CsA-treated than in OO-treated animals (FE(alb) CsA: 1.35 +/- 0.88, FE(alb) OO: 3.17 +/- 2.29%; p = 0.0005). This suggests that other factors are also involved in the reduction of albuminuria. To exclude that CsA has an effect on the tubular reabsorption of albumin, we evaluated the blockade of the tubular reabsorption by lysine and found no difference in albuminuria between the CsA- and OO-treated groups. These experiments suggest that the antiproteinuric effect of CsA is not (only) due to a decrease in the GFR, but also to a decrease of the enhanced permeability of the glomerular capillary wall for albumin.

Albuminuria↗

PubMed1997

Immunohistochemical quantification of heparan sulfate proteoglycan and collagen IV in skeletal muscle capillary basement membranes of patients with diabetic nephropathy.

In IDDM patients, an increased permeability of the glomerular capillaries has been associated with a general loss of negatively charged heparan sulfate proteoglycans (HSPGs) within basement membranes (BMs). In the present study, we used immunohistochemical staining to quantify heparan sulfate (HS), HSPG core protein, and collagen IV in capillary basement membranes of skeletal muscle biopsies taken from 9 healthy control subjects (C) and 20 IDDM patients: 7 with normal albumin excretion rate (<30 mg/24 h) (D0), 5 with incipient nephropathy (albumin excretion rate 30-300 mg/24 h) (D1), and 8 with clinical nephropathy (albumin excretion rate >300 mg/24 h) (D2). In the capillaries, staining was measured by a scanning and integrating microspectrophotometer. A significant difference in the absorbance of HS was found among the four subgroups (means +/- SD): 0.477 +/- 0.082 (C), 0.627 +/- 0.031 (D0), 0.542 +/- 0.098 (D1), and 0.371 +/- 0.118 (D2) (P = 0.006). Similarly, an overall significant difference in the absorbance of collagen IV was demonstrated (means +/- SD): 0.836 +/- 0.111 (C), 0.838 +/- 0.300 (D0), 0.970 +/- 0.173 (D1), and 0.512 +/- 0.248 (D2) (P = 0.02). No statistical difference in the absorbance of core protein was demonstrated among the groups. Within the diabetic groups, HS was inversely correlated to albuminuria (r = -0.76, P = 0.003) and albuminuria corrected for creatinine clearance (r = -0.69, P = 0.008). Because, in IDDM patients with albuminuria, alterations of the content of HS and collagen IV within the capillary BM have been demonstrated immunohistochemically, not only in the glomerular filtration barrier, but also in the skeletal muscle capillary BM, we suggest that these changes reflect universal quantitative or qualitative alterations within the capillary filtration barrier.

Adult↗

PubMed1997

Altered distribution of heparan sulfate proteoglycans in psoriasis.

Heparan sulfate proteoglycans (HSPGs) are components of the basement membrane (BM) of various tissues. They consist of a core protein and negatively charged glycosaminoglycan side chains: the heparan sulfate (HS) moieties. In psoriasis, uninvolved skin of psoriasis patients and in normal skin, the distribution of HSPGs was studied immunohistochemically by means of three different monoclonal antibodies: JM-72, directed against the HSPG-core protein, JM-13 against the sulfated domains of HS and JM-403 against the unmodified/low sulfated parts of HS. In psoriasis JM-13 staining was consistently absent in the tips of the dermal papillae, whereas JM-13 showed a continuous staining in the BM of uninvolved and normal skin. JM-403 staining was present in BM of all specimens. In addition, a honeycomb-like staining was found in epidermis of normal skin and to a lesser extent in uninvolved skin, due to binding with plasma membrane-associated HS. In psoriasis this JM-403 staining of the epidermis was invariably absent. JM-72 showed a continuous staining of BM in all biopsies. In conclusion, normal human skin and involved psoriatic skin show clear differences in expression of HS. These data may provide insight into the role of HSPGs in psoriasis, which remains further to be elucidated.

Adult↗

PubMed1997

Vessel wall heparan sulfate and transcapillary passage of albumin in experimental diabetes in the rat.

BACKGROUND: To investigate whether albuminuria in streptozotocin-induced diabetes mellitus in the rat is a symptom of a more generalized vessel wall permeability, and whether changes in vessel wall heparan sulfate (HS) are associated with alterations in vessel wall permeability. METHODS: The transcapillary escape rate of albumin (TERalb) was calculated from the disappearance rate from the circulation of i.v. injected radiolabeled albumin, together with the regional clearance of albumin (RCalb) in several tissues using a double isotope technique. These measurements were performed in seven rats one year after diabetes induction and in seven sex- and age-matched control rats. To evaluate the association between vessel wall HS and the transcapillary passage of albumin, we determined the content of basement membrane HS in tissue homogenates of heart, liver, kidneys, and lungs with a sensitive inhibition-ELISA using a monoclonal antibody (JM-403), which specifically recognizes basement membrane HS. RESULTS: Diabetic rats developed albuminuria (31.7 +/- 10.8 mg/24 h) in contrast to control animals (2.2 +/- 1.5 mg/24 h; P = 0.0006). TERalb was increased from 13.3 +/- 1.7 in control rats to 15.6 +/- 2.6%/h in diabetic rats, P = 0.02. RCalb was significantly increased in heart, liver, skeletal muscle and aorta, unchanged in kidneys and skin, and significantly decreased in lung tissue. We found a decrease in HS content in heart tissue of diabetic rats, and a correlation between HS content and RCalb (r = -0.72, P = 0.004), in contrast with an increase in lung HS content that correlated with a decrease in RCalb (r = -0.64, P = 0.014). No changes in HS content were found in kidney and liver tissue. CONCLUSIONS: These data indicate that in one-year diabetic rats albuminuria coincides with an increased TERalb and RCalb in most, but not all tissues, and that alterations in basement membrane HS content correlate with changes in the RCalb, which suggests a functional relationship.

Animals↗

PubMed1996

Identification of the endothelial cell binding site for factor IX.

We previously demonstrated that the primary region of factor IX and IXa responsible for saturable specific binding to bovine aortic endothelial cells resides in residues 3-11 at the amino terminus of factor IX. We also demonstrated that mutations of lysine to alanine at residue 5, factor IX K5A, or valine to lysine at residue 10, factor IX V10K, resulted in a molecule unable to bind to endothelial cells. Moreover, a mutation with lysine to arginine at residue 5, factor IX K5R, resulted in a factor IX molecule with increased affinity for the endothelial cell binding site. In this paper we report that collagen IV is a strong candidate for the factor IX binding site on endothelial cells. Factor IX and factor IX K5R compete with 125I-labeled factor IX for binding to tetrameric collagen IV immobilized on microtiter plates, while factor X, factor VII, and factor IX K5A or V10K fail to compete. The Kd for wild-type factor IX binding to collagen IV in the presence of heparin was 6.8 +/- 2 nM, and the Kd for factor IX K5R was 1.1 +/- 0.2 nM, which agrees well with our previously published Kd values of 7.4 and 2.4 nM for binding of the same proteins to endothelial cells. Our working assumption is that we have identified the endothelial cell binding site and that it is collagen IV. Its physiological relevance remains to be determined.

Animals↗

PubMed1996

Effects of high glucose on the production of heparan sulfate proteoglycan by mesangial and epithelial cells.

Changes in heparan sulfate metabolism may be important in the pathogenesis of diabetic nephropathy. Recent studies performed on renal biopsies from patients with diabetic nephropathy revealed a decrease in heparan sulfate glycosaminoglycan staining in the glomerular basement membrane without changes in staining for heparan sulfate proteoglycan-core protein. To understand this phenomenon at the cellular level, we investigated the effect of high glucose conditions on the synthesis of heparan sulfate proteoglycan by glomerular cells in vitro. Human adult mesangial and glomerular visceral epithelial cells were cultured under normal (5 mM) and high glucose (25 mM) conditions. Immunofluorescence performed on cells cultured in 25 mM glucose confirmed and extended the in vivo histological observations. Using metabolic labeling we observed an altered proteoglycan production under high glucose conditions, with predominantly a decrease in heparan sulfate compared to dermatan sulfate or chondroitin sulfate proteoglycan. N-sulfation analysis of heparan sulfate proteoglycan produced under high glucose conditions revealed less di- and tetrasaccharides compared to larger oligosaccharides, indicating an altered sulfation pattern. Furthermore, with quantification of glomerular basement membrane heparan sulfate by ELISA, a significant decrease was observed when mesangial and visceral epithelial cells were cultured in high glucose conditions. We conclude that high glucose concentration induces a significant alteration of heparan sulfate production by mesangial cells and visceral epithelial cells. Changes in sulfation and changes in absolute quantities are both observed and may explain the earlier in vivo observations. These changes may be of importance for the altered integrity of the glomerular charge-dependent filtration barrier and growth-factor matrix interactions in diabetic nephropathy.

Adult↗

PubMed1996

Altered composition of urinary heparan sulfate in patients with COPD.

In patients with emphysema the integrity of the extracellular matrix (connective tissue skeleton) is compromised. In this study we analyzed glycosaminoglycans, which are main constituents of this matrix, in urines from patients with chronic obstructive pulmonary disease (COPD)/emphysema. Glycosaminoglycans (GAGs) were purified by anion exchange chromatography and quantified using the 1,9-dimethylmethylene blue assay. Heparan sulfate (HS) was assayed using three different chemical methods: digestion with heparitinase or with nitrous acid and by use of an adapted 1,9-dimethylmethylene blue assay. A specific epitope on the HS molecule, defined by the monoclonal antibody JM403, was determined using an inhibition enzyme immunoassay. In patients with COPD total urinary glycosaminoglycan and HS content were not altered. The JM403 epitope of HS, however, was greatly decreased in patients (0.6 versus 4.1 units/mg creatinine for control subjects, p < 0.0001). A similar pattern was observed when patients with bronchial carcinoma with and without emphysema were compared (0.4 versus 2.4 units/mg creatinine respectively, p < 0.0005). Patients with sarcoidosis did not show a decreased epitope content. These results indicate a structural change or an altered processing of the HS molecule in patients with emphysema. Taking into consideration the importance of HS for the stability of the alveolar extracellular matrix, this change may be associated with the pathogenesis of emphysema.

Adult↗

PubMed1996

Differential effects of angiotensin II and transforming growth factor beta on the production of heparan sulfate proteoglycan by mesangial cells in vitro.

This study approaches the question of whether angiotensin II (AngII) and transforming growth factor beta (TGF-beta) are important mediators for mesangial heparan sulfate proteoglycan (HSPG) production. This might explain the beneficial effects of angiotensin-converting enzyme inhibitors observed in several kidney diseases independent from their hemodynamic effects. Metabolic-labeling studies revealed that AngII induced a decrease of HSPG synthesis with decreases in N-sulfation of the glycosaminoglycan side chains. ELISA measurements with a heparan sulfate (HS)-specific monoclonal antibody confirmed that AngII decreased HS production. AngII increased TGF-beta production in a dose-dependent fashion. Specific mRNA for the large basement membrane HSPG (perlecan) decreased, whereas mRNA for TGF-beta increased after incubation with AngII. Blockade of the Subtype 1 Ang-II receptor (ATR1) reversed both the effects of AngII on HSPG and TGF-beta production. Coincubation of the mesangial cells with neutralizing antibodies against TGF-beta significantly reduced the production of HS as compared with control and AngII. These results indicate that the decrease in HS synthesis induced by AngII is not mediated by an increase in TGF-beta, but on the contrary, the increase in TGF-beta partially counteracts the inhibition of HS production by AngII. Considering the important role of HSPG in maintaining the glomerular charge barrier, cell proliferation, and matrix interaction, downregulation of the production of this molecule by increased local AngII concentrations could have important consequences, such as albuminuria and matrix expansion.

Adult↗

PubMed1996

Autoantibodies to myeloperoxidase aggravate mild anti-glomerular-basement-membrane-mediated glomerular injury in the rat.

Autoantibodies to myeloperoxidase (MPO) are present in sera from patients with various forms of vasculitis-associated glomerulonephritis. Evidence for a pathogenic role of anti-MPO antibodies has been provided mainly by in vitro studies. We studied the pathogenic role of autoantibodies to MPO in a rat model of mild immune-mediated glomerular injury. Brown Norway rats were immunized with human MPO in complete Freund's adjuvant or with complete Freund's adjuvant alone. At 2 weeks after immunization, rats had developed antibodies to human and rat MPO as detected by indirect immunofluorescence, enzyme-linked immunosorbent assay, and immunoprecipitation. At this time point, rats were intravenously injected with a subnephritogenic dose of 150 micrograms of rabbit anti-rat GBM. Rats were sacrificed at 4 hours, 24 hours, 4 days, and 10 days after antibody administration. Control immunized rats developed mild glomerulonephritis characterized by slight proteinuria at day 10 (14.8 +/- 8.1 mg/24 hours) and moderate intraglomerular accumulation of ED1+ macrophages. Crescent formation, tuft necrosis, and tubular atrophy were not observed in those rats. In contrast, rats immunized with MPO developed severe glomerulonephritis characterized by the early occurrence of severe hematuria, marked proteinuria at day 10 (76.2 +/- 18.2 mg/24 hours), and massive glomerular deposition of fibrin. Complement and rat IgG were present in insudative lesions, but no linear pattern along the glomerular capillary wall was observed. By light microscopy, severe glomerular lesions were found at day 10 consisting of crescent formation and fibrinoid necrosis of capillary loops. In the interstitium, tubular necrosis and atrophy and marked interstitial mononuclear infiltration were found in conclusion, autoantibodies to MPO severely aggravate subclinical anti-GBM disease demonstrating their in vivo pathogenic potential.

Animals↗

PubMed1995

Presence of N-unsubstituted glucosamine units in native heparan sulfate revealed by a monoclonal antibody.

Immunohistochemical application of antibodies against heparan sulfate proteoglycan core protein and heparitinase-digested heparan sulfate stubs showed the presence of heparan sulfate proteoglycan in all basement membranes of the rat kidney. However, a monoclonal antibody (JM-403) against native heparan sulfate (van den Born, J., van den Heuvel, L. P. W. J., Bakker, M. A. H., Veerkamp, J. H., Assmann, K. J. M., and Berden, J. H. M. (1992) Kidney Int. 41, 115-123) largely failed to stain tubular basement membranes, suggesting the presence of heparan sulfate chains lacking the specific JM-403 epitope. Heparan sulfate preparations from various sources differed markedly with regard to JM-403 binding, as demonstrated by liquid phase inhibition in enzyme-linked immunosorbent assay, the interaction decreasing with increasing sulfate contents of the polysaccharide. Mapping of the JM-403 epitope indicated that it was dominated by one or more N-unsubstituted glucosamine unit(s), since treatments that destroyed or altered the structure of such units in heparan sulfate preparations (cleavage at N-unsubstituted glucosamine units with HNO2 at pH 3.9 and N-acetylation with acetic anhydride, respectively), abolished antibody binding. Conversely, immunoreactivity could be induced in a (D-glucuronyl-1,4-N-acetyl-D-glucosaminyl-1,4) polysaccharide by the generation of N-unsubstituted glucosamine N-unsubstituted glucosamine in a JM-403-binding heparan sulfate (preparation HS-II from human aorta) was demonstrated by an approximately 3-fold reduction in molecular size following HNO2 (pH 3.9) treatment. Further characterization of the epitope recognized by JM-403, based on enzyme-linked immunosorbent assay inhibition tests with chemically/enzymatically modified polysaccharides, indicated that one or more N-sulfated glucosamine units are invariable present, whereas L-iduronic acid and O-sulfate residues appear to inhibit JM-403 reactivity. It is concluded that the epitope contains one or more N-unsubstituted glucosamine and D-glucuronic acid units and is located in a region of the heparan sulfate chain composed of mixed N-sulfated and N-acetylated disaccharide units.

Animals↗