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Biomedical subjects

E Schleicher

Publications and source records attributed to E Schleicher.

At least 37 records · Page 2Linked to original sources
PubMed1999

N(epsilon)(carboxymethyl)lysin and the AGE receptor RAGE colocalize in age-related macular degeneration.

PURPOSE: To investigate whether glycoxidation products and the receptor for advanced glycation end products (RAGE) are present and colocalize in subfoveal membranes of patients with age-related macular degeneration (ARMD). METHODS: Surgically removed subfoveal fibrovascular membranes from 12 patients, 11 related to ARMD and 1 to an idiopathic membrane, were analyzed for the presence of the glycoxidation product N(epsilon)-(carboxymethyl)lysin (CML), one of the receptors for advanced glycation end products, RAGE, and the activation of NFkB, using immunohistochemistry. RESULTS: CML-like immunoreactivity was found in all ARMD specimens examined adjacent or colocalized with RAGE, but not in the idiopathic membrane. RAGE immunoreactive material was found in CD68-positive cells and in the fibrous matrix. CD68-positive cells and surrounding areas stained for p50, the activated form of NFkB. CONCLUSIONS: These results indicate that glycoxidation products are present in subretinal membranes of patients with ARMD. The concomitant expression of RAGE in these membranes and the finding of activated NFkB is suggestive of an implication of glycoxidation product formation in the pathogenesis of the disease.

Adolescent↗
PubMed1998

Glucosamine induces translocation of protein kinase C isoenzymes in mesangial cells.

Activation of protein kinase C (PKC) has been implicated in the high glucose-induced stimulation of matrix protein production in mesangial cells. Since we have found (Kolm-Litty et al., 1998) that glucosamine, similar to the PKC activator phorbol myristate acetate (PMA), mimicks high glucose-induced TGF-beta1 overexpression and subsequent matrix overproduction, the action of these agents on the translocation of PKC isoenzymes was studied in cultured mesangial cells. Exposure to 12 mM glucosamine resulted in rapid and specific translocation of PKC-isoenzymes in mesangial cells i.e. glucosamine caused an increased and sustained translocation of PKC-alpha, -beta and -epsilon while PKC-zeta was essentially unaffected. Comparison with PMA-induced translocation exhibited distinct differences. Exposure to high glucose concentrations of mesangial cells induced translocation of PKC-beta and down-regulation of PKC-epsilon while PKC-alpha and -zeta were essentially unaltered. Presence of azaserine an inhibitor of glutamine: fructose-6-phosphate amidotransferase, the key enzyme of the hexosamine pathway, attenuated the high glucose-induced effects on the membrane fraction of PKC-beta. Our results indicate that i) glucosamine is a potent stimulator of PKC-translocation exhibiting an isoenzyme specific translocation kinetic which is different from PMA-induced PKC-isoenzyme translocation ii) the hexosamine pathway may be possibly involved in the high glucose-induced activation of PKC.

Animals↗
PubMed1998

Intracellular processing of transforming growth factor-beta in mesangial cells.

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional regulator of cell-growth, differentiation and extracellular matrix formation in several physiological conditions. It plays a crucial role in the process of glomerulosclerosis. Mature TGF-beta 1 is secreted as a latent form associated with the latency associated peptide (LAP), and its activation occurs through the LAP cleavage. The intracellular localization and the mechanisms of activation of TGF-beta 1 protein have not been elucidated in the mesangial cell. In the present report we examined the intracellular processing from TGF-beta 1 precursor to the latent-TGF-beta 1 in cultured mesangial cells by immunocytochemistry, using three rabbit polyclonal antibodies directed against different epitopes of human TGF-beta 1. The anti-LAP-TGF-beta 1 precursor Ab stained mesangial cells in the perinuclear region and in the cytoplasm in the area corresponding to the rough endoplasmic reticulum; the anti-COOH-terminal fragment of TGF-beta 1 Ab reacted in the same area, in vesicular structures located in the cytoplasm and furthermore, in the mesangial cell clusters, so-called hillocks, with an extracellular pattern; the anti-NH2-terminal fragment of TGF-beta 1 Ab stained only large exocytotic vesicles at the periphery of the cytoplasma. Our investigations suggest a conformational rearrangement of pro-TGF-beta 1 molecule occurring between the rough endoplasmic reticulum and the TGF-beta 1 secretion and support the idea that in mesangial cells the activation of TGF-beta 1 occurs during the secretion process. In conclusion, the processing of TGF-beta 1 in mesangial cells seems to be similar to that one observed in other mesenchymal cells.

Animals↗
PubMed1997

A detailed protocol for the measurement of TGF-beta1 in human blood samples.

Quantification of the multifunctional cytokine Transforming Growth Factor-beta1 (TGF-beta1) in blood samples has aroused increasing interest in recent years, since an abnormal regulation of this cytokine appears to play a key role in the pathogenesis of different diseases, such as autoimmundiseases or malignant tumors. The measurement of TGF-beta1 is complicated by a lot of problems concerning the collection, preparation and handling of blood samples, the platelet contamination, and the TGF-beta1 activation procedure. Here, we recommend detailed instructions for measurement of TGF-beta1 in blood plasma samples which should be followed to exclude the determination of false positive or negative results.

Humans↗
PubMed1996

Structural and functional changes in diabetic glomerulopathy.

Diabetic nephropathy is characterized by glomerular basement membrane thickening and mesangial expansion. Immunohistochemical studies of diabetic kidneys showed an increased collagen type IV synthesis and deposition in the mesangial matrix, while the glomerular heparan sulfate proteoglycan content was decreased. In nodular glomerulosclerosis massive deposition of collagens III and VI appears, possibly indicating irreversibility of the pathological process. These structural changes seem to be the underlying cause for the alterations of renal functions like persistent albuminuria and proteinura. In a recent study significant glomerular infiltration by macrophages at all stages of glomerulosclerosis was observed. The pathogenesis of the multitude of cellular, structural, and functional abnormalities in diabetic nephropathy is likely to be multifactorial, involving chronic hyperglycemia as well as genetic determinants. In vitro studies with cultured glomerular cells have indicated that hyperglycemia induces transforming growth factor beta, a matrix-producing cytokine. The hyperglycemia-induced cytokine production may involve protein kinase C activation and/or the formation of advanced glucosylation end products. The elucidation of the pathogenesis of diabetic nephropathy may suggest new ways for therapeutic interventions.

Animals↗
PubMed1996

Distribution of basement membrane components in normal adipose tissue and in benign and malignant tumors of lipomatous origin.

Normal adipose tissue as well as 13 benign and 17 malignant lipomatous tumors (lipomas, hibernomas, lipoblastomas, and liposarcomas) were immunohistochemically analyzed for their expression of the basement membrane components collagen IV, laminin, heparan sulfate proteoglycan, and fibronectin. Monovacuolar cells in normal white fat tissue and in lipomas generally exhibited a distinctive pericellular basement membrane composed of collagen IV and laminin, whereas heparan sulfate proteoglycan and fibronectin were almost completely missing. In brown fat tissue and hibernomas the characteristic multivacuolated cells differed from the monovacuolated white fat cells by the additional content of heparan sulfate proteoglycan and fibronectin and the more intensive staining for the other components tested. In contrast, multi-/monovacuolated cells in lipoblastomas exhibited no characteristic immunohistochemical feature because they reacted irregularly and only faintly for collagen IV, laminin, and heparan sulfate proteoglycan. Spindle cell areas in benign lipomatous tumors displayed more fibronectin than laminin and heparan sulfate proteoglycan indicating a "preadipose" fibroblast-like cellular differentiation. In liposarcomas, only well-differentiated lipoma-like neoplasms revealed a basement membrane pattern resembling that of white fat tissue. Otherwise, in nonlipoma-like liposarcomas, a marked decrease particularly of collagen IV staining was evident. Poorly differentiated liposarcomas mostly failed to express any of the basement membrane components, but showed a relative increase of fibronectin. Our results provide evidence that the staining pattern of basement membrane components parallels the histogenetic derivation of benign lipomatous tumors from either brown or white adipose tissue and, additionally, may reflect such a derivation in liposarcomas.

Adipose Tissue↗
PubMed1996

Increased glomerular alpha 1 (IV) collagen expression and deposition in long-term diabetic rats is prevented by chronic glycosaminoglycan treatment.

Diabetic nephropathy is associated with thickening of the glomerular basement membrane and, in particular, with mesangial matrix expansion. Previous studies have indicated that administration of chemically modified, low-anticoagulant heparins prevents some of the morphologic and physiologic alterations occurring in experimental diabetic nephropathy. The effect of prolonged heparin treatment on the glomerular expression and deposition of alpha 1 (IV) collagen, which is a major component of the mesangial matrix, has not been reported previously. Four groups of rats were studied for 12 months: two control groups and two groups of streptozotocin-diabetic rats. One group in each branch received modified heparin continuously from the induction of diabetes. After 12 months synthesis and deposition of alpha 1 (IV) collagglomerula and adjacent tubuli were determined by nonradioactive in situ hybridization and immunohistochemistry. Mesangial cells were localized by Thy 1.1 staining. Long-term diabetes caused a significant increase in alpha 1 (IV) collagen deposition in the mesangial matrix and a more than 2-fold enhancement of glomerular cell alpha 1 (IV) collagen transcript levels, mainly in mesangial cells. The alpha 1 (IV) collagen mRNA levels of proximal tubular cells and visceral epithelial cells were similarly increased. Chronic treatment of diabetic rats with modified heparin completely prevented the increased alpha 1 (IV) collagen deposition and expression. The increased glomerular deposition of alpha 1 (IV) collagen observed in long-term streptozotocin diabetic rats appears to depend on an increased alpha 1 (IV) collagen synthesis. Because chronic application of low-anticoagulant heparin completely prevents the increased alpha 1 (IV) collagen synthesis by mesangial cells, this result suggests a new therapeutic option for the prevention of diabetic nephropathy in humans.

Animals↗
PubMed1996

Glomerular basement membrane-derived perlecan inhibits mesangial cell adhesion to fibronectin.

To explore whether the heparan sulfate proteoglycan, periecan, may act as a regulator of glomerular cell behavior, we examined the effects of perlecan on adhesive properties of rat and human mesangial cells in culture. We observed that mesangial cells did not adhere to purified perlecan. Mesangial cell adhesion to fibronectin, but not to laminin, was inhibited when perlecan was present in the substratum, reaching complete inhibition at 20 micrograms/ml perlecan. Similarly, perlecan reduced mesangial cell adhesion to the 120 kDa fragment of fibronectin and to bovine serum albumin-coupled RGD peptides both lacking the fibronectin heparin-binding domains, indicating that the inhibitory effect of perlecan does not require interaction of its heparan sulfate chains with fibronectin. The core protein of perlecan seems to be relevant, since perlecan retained most of its anti-adhesive potency after total deglycosylation. It is unlikely that perlecan causes steric inhibition of the cell binding sites of fibronectin by direct binding, since the 120 kDa fragment of fibronectin which includes the cell binding site, did not bind to intact perlecan. In the presence of submaximal inhibitory concentrations of perlecan, soluble RGD peptides (100 micrograms/ml), by themselves without effect, completely inhibited cell adhesion to fibronectin. Using immunocytochemistry, we examined the effects of perlecan on the organization of fibronectin-specific, RGD-dependent beta 1 integrins in focal contacts. When perlecan was added to the substratum, human mesangial cells adhered to fibronectin only in the presence of collagen I. Under these conditions, perlecan did not significantly alter the organization of alpha 5 beta 1 integrin into focal contacts of mesangial cells which adhered to a substratum composed of collagen I plus fibronectin. These data suggest that perlecan exerts its anti-adhesive effects on mesangial cells via a core protein-dependent mechanism which reduces the avidity of the fibronectin receptor. The resulting avidity level is too low for mediating cell binding but sufficient to induce integrin organization into focal contacts.

Animals↗
PubMed1994

Atherogenic levels of low density lipoprotein alter the permeability and composition of the endothelial barrier.

In the present study we investigated the influence of elevated low density lipoprotein (LDL) concentration on endothelial permeability. Endothelial cells were cultured on microporous membranes until confluence and albumin, dextran and LDL transfer across endothelial monolayers was determined to assess macromolecular permeability. Exposure of proliferating aortic endothelial cells to LDL levels of more than 1 mg/ml LDL-cholesterol induced a concentration-dependent exponential increase in the permeability of confluent endothelial monolayers. Acute addition of high LDL concentration did not alter macromolecular permeability. Once elevated permeability was induced, it persisted. It was not readily reversible after addition of normal LDL levels. Change in permeability was accompanied by a selective decrease in basement membrane associated heparan sulfate proteoglycan (HSPG) content. The apparent parallel between the loss in endothelial barrier function and HSPG decrease implicates a connection between the two events. Prolonged, but not acute, incubation with antiserum directed against the core-protein of HSPG also led to increased permeability, suggesting a causal role of HSPG for the proper function of endothelium. The fact that non-atherogenic LDL-cholesterol levels had no effect indicates that a 'threshold' concentration for LDL-cholesterol may exist, leading to nondenuding injury in the endothelial barrier as an early event in development of atherosclerosis.

Animals↗
PubMed1994

[Significance of the basement membrane for growth of tumors--immunohistochemical aspects with special reference to tumors of the head/neck area].

This report presents the state of current knowledge regarding the importance of basement membrane (BM) for the growth of benign and malignant tumours with special emphasis on tumours of the head and neck. The BM forms a barrier that has to be destroyed by infiltrative tumour cells. Recent immunohistochemical analyses have revealed both quantitative and qualitative changes in the BM composition following malignant transformation of the epithelial cells, thereby allowing malignant ingrowth into the stroma. This BM pattern, however, may also allow an early diagnosis of tumour invasion. Additionally, carcinomas of the larynx demonstrate a correlation between the amount of preserved BM deposition and the grade of tumour differentiation. This fact may also be important in tumour prognosis.

Basement Membrane↗
PubMed1994

Heparin induces endothelial extracellular matrix alterations and barrier dysfunction.

We investigated the influence of heparin on the composition of the subendothelial matrix and on endothelial permeability to elucidate the structure-function relationship of matrix composition and permeability. Albumin flux across the confluent endothelial monolayers was used to assess the macromolecular permeability. Lowest values were obtained when 100% serum was used as medium for permeability studies. The endothelial matrix components, fibronectin and basement membrane-associated heparan sulfate proteoglycan (HSPG), were measured by enzyme immunoassay. Treatment of proliferating endothelial cells with heparin (0-900 micrograms/ml) induced a dose-dependent decrease in endothelial HSPG content, whereas the fibronectin content was unaltered. This structural change was accompanied by an increase in albumin permeability. Both heparin effects exhibited similar dose-response curves with half-maximal effects at approximately 5 micrograms/ml heparin. Acute addition of 300 micrograms/ml heparin had no effect on permeability or HSPG content. When endothelial cells were preincubated with an HSPG antiserum, the endothelial permeability increased nearly threefold. Our results indicate that heparin-induced loss of HSPG may cause the increase in endothelial permeability. The data underline the importance of HSPG for the integrity of the endothelial barrier.

Animals↗
PubMed1993

Alterations of glomerular matrix proteins in the pathogenesis of diabetic nephropathy.

Diabetic late complications are characterized by morphological and biochemical alterations of the extracellular matrix. In particular, longstanding diabetes causes quantitative and qualitative changes in basement membrane structure of retinal and renal capillaries. Immunohistochemical investigations of diabetic kidneys with diffuse glomerulosclerosis show increased collagen type IV deposition in the mesangial matrix and decreased heparan sulfate proteoglycan content in the mesangial matrix and glomerular basement membrane as well. In nodular glomerulosclerosis normal basement membrane components are decreased or absent while the occurrence of collagen type III in this stage has been interpreted as an irreversible alteration of the glomerular structure. These changes seem to be the underlying cause for the alterations in renal functions like persistent albuminuria and proteinuria. Increased intra- and extracellular levels of glucose and its derivatives are thought to be responsible for diabetic tissue dysfunction although there are reports on possible genetic defects causing increased susceptibility to develop diabetic nephropathy. Recent results, however, focus on the role of glucose-induced cytokine secretion as mediator for altered metabolism of glomerular matrix proteins. In vitro studies with cultured kidney cells have shown that the glucose-induced dysregulation of the basement membrane synthesis may be mediated by a glucose dependent activation of protein kinase C. Alternatively or synergistically, the formation of AGE products formed after prolonged exposure of matrix proteins to elevated glucose may also lead to cytokine secretion subsequently inducing synthesis of extracellular matrix proteins. Studies in experimental animals confirm the diabetes induced dysregulation of the synthesis of extracellular matrix components on the molecular level.

Animals↗
PubMed1992

Comparison of various basement membrane components in benign and malignant peripheral nerve tumours.

Immunohistochemical methods were used to analyse benign and malignant tumours of peripheral nerve tissue. We tested for the distribution of basement membrane (BM) components collagen IV, laminin, heparan sulphate proteoglycan, fibronectin, for S100 protein and for the presence of interstitial collagens III and V. Laminin was generally noted in association with Schwann cells, but collagen IV occurred with perineural cells. When tested for BM components, fibroblasts were notably non-reactive except for fibronectin. Three specific area-dependent BM patterns were observed in the benign tumours: (a) Schwann cell-like, in fascicular areas (Antoni A areas of schwannoma, central fibrous bundles of plexiform neurofibromas and central areas of cutaneous neurofibroma), (b) perineural cell-like (capsular structures of schwannoma) and (c) fibroblast-like (myxoid and fibrously transformed areas). Most malignant tissues showed a variably fragmentary focal deposition of laminin. Other BM components were present only in well-differentiated areas. Poorly differentiated tumours demonstrated fibronectin reactivity alone. Our results provide evidence that the specific staining pattern for BM components helps to differentiate the various cellular proliferations in neurogenic tumours. Schwann cells are not only distinguishable from perineural cells by S100 protein staining, but also by their specific BM staining. In addition, perineural cells can be separated from fibroblasts, which do not express BM material. The "tropism" of laminin in normal nerves and benign neural tumours--which persists in neurogenic sarcomas--indicates preferential Schwann cell differentiation in these cells.

Basement Membrane↗
PubMed1992

Renal sorbitol, myo-inositol and glycerophosphorylcholine in streptozotocin-diabetic rats.

The polyols, sorbitol and myo-inositol, seem to be involved in the development of diabetic complications of different organs. High concentrations of both polyols were found in kidney medulla in addition to trimethylamines. To investigate the influence of diabetes mellitus on the regulation of both polyols and glycerophosphorylcholine in kidney, these osmolytes were quantitated enzymatically along the corticopapillary axis in untreated, streptozotocin-diabetic and insulin-treated streptozotocin-diabetic rats. In control animals three individual osmolyte patterns were found: a steep gradient of sorbitol in the papilla, increasing amounts of glycerophosphorylcholine from the outer medulla to the papilla, and nearly equal amounts of myo-inositol in the renal medulla, decreasing towards the cortex. Diabetic rats exhibit an up to fourfold increase of inner medullary sorbitol, whereas myo-inositol was only elevated in the outer medulla. Glycerophosphorylcholine was lowered in the papillary tip and elevated in the outer medulla and cortex. Insulin treatment reduced sorbitol to a concentration between those of diabetic and control rats, caused a restoration of glycerophosphorylcholine in the papillary tip and outer medulla to control values, and increased cortical myo-inositol. These data confirm previous in vitro data, which show that papillary sorbitol specifically increases in hyperglycaemic states, thereby counteracting the increased extracellular tonicity due to elevated tissue glucose concentrations. Imbalance of extra- vs intracellular osmolality during insulin treatment may be involved in the pathomechanism of renal papillary necrosis.

Animals↗