IL-6 deficiency in mice neither impairs induction of metabolic genes in the liver nor affects blood glucose levels during fasting and moderately intense exercise.

AIMS/HYPOTHESIS: Fasting and exercise are strong physiological stimuli for hepatic glucose production. IL-6 has been implicated in the regulation of gluconeogenic genes, but the results are contradictory and the relevance of IL-6 for fasting- and exercise-induced hepatic glucose production is not clear. METHODS: Investigations were performed in rat hepatoma cells, and on C57Bl6 and Il6(-/-) mice under the following conditions: IL-6 stimulation/injection, non-exhaustive exercise (60 min run on a treadmill) and fasting for 16 h. Metabolite analysis, quantitative real-time PCR and immunoblotting were performed. RESULTS: IL-6 stimulation of rat hepatoma cells led to higher glucose production. Injection of IL-6 in mice slightly increased hepatic Pepck (also known as Pck1) expression. Fasting of Il6(-/-) mice for 16 h did not alter glucose production compared with wild-type mice, since plasma glucose concentrations were similar and upregulation of phosphoenolpyruvate carboxykinase (PEPCK) and Pgc-1alpha (also known as Ppargc1a) expression was comparable. In the non-fasting state, Il6(-/-) mice showed a mild metabolic alteration including higher plasma glucose and insulin levels, lower NEFA concentrations and slightly increased hepatic PEPCK content. Moderately intense exercise resulted in elevated IL-6 plasma levels in wild-type mice. Despite that, plasma glucose, insulin, NEFA levels and hepatic glycogen content were not different in Il6(-/-) mice immediately after running, while expression of hepatic G6pc, Pgc-1alpha, Irs2 and Igfbp1 mRNA was similarly increased. CONCLUSIONS/INTERPRETATION: These data suggest that in mice IL-6 is not essential for physiologically increased glucose production during fasting or non-exhaustive exercise.

Activation of the mitogen-activated protein kinase (MAPK) signalling pathway in the liver of mice is related to plasma glucose levels after acute exercise.

AIMS/HYPOTHESIS: We aimed to identify, in the liver of mice, signal transduction pathways that show a pronounced regulation by acute exercise. We also aimed to elucidate the role of metabolic stress in this response. METHODS: C57Bl6 mice performed a 60 min run on a treadmill under non-exhaustive conditions. Hepatic RNA and protein lysates were prepared immediately after running and used for whole-genome-expression analysis, quantitative real-time PCR and immunoblotting. A subset of mice recovered for 3 h after the treadmill run. A further group of mice performed the treadmill run after having received a vitamin C- and vitamin E-enriched diet over 4 weeks. RESULTS: The highest number of genes differentially regulated by exercise in the liver was found in the mitogen-activated protein kinase (MAPK) signalling pathway, with a pronounced and transient upregulation of the transcription factors encoded by c-Fos (also known as Fos), c-Jun (also known as Jun), FosB (also known as Fosb) and JunB (also known as Junb) and phosphorylation of hepatic MAPK. Acute exercise also activated the p53 signalling pathway. A major role for oxidative stress is unlikely since the antioxidant-enriched diet did not prevent the activation of the MAPK pathway. In contrast, lower plasma glucose levels after running were related to enhanced levels of MAPK signalling proteins, similar to the upregulation of Igfbp1 and Pgc-1alpha (also known as Ppargc1a). In the working muscle the activation of the MAPK pathway was weak and not related to plasma glucose concentrations. CONCLUSIONS/INTERPRETATION: Metabolic stress evidenced as low plasma glucose levels appears to be an important determinant for the activation of the MAPK signalling pathway and the transcriptional response of the liver to acute exercise.

Increased accumulation of the glycoxidation product N(epsilon)-(carboxymethyl)lysine in human tissues in diabetes and aging.

N(epsilon)-(Carboxymethyl)lysine (CML), a major product of oxidative modification of glycated proteins, has been suggested to represent a general marker of oxidative stress and long-term damage to proteins in aging, atherosclerosis, and diabetes. To investigate the occurrence and distribution of CML in humans an antiserum specifically recognizing protein-bound CML was generated. The oxidative formation of CML from glycated proteins was reduced by lipoic acid, aminoguanidine, superoxide dismutase, catalase, and particularly vitamin E and desferrioxamine. Immunolocalization of CML in skin, lung, heart, kidney, intestine, intervertebral discs, and particularly in arteries provided evidence for an age-dependent increase in CML accumulation in distinct locations, and acceleration of this process in diabetes. Intense staining of the arterial wall and particularly the elastic membrane was found. High levels of CML modification were observed within atherosclerotic plaques and in foam cells. The preferential location of CML immunoreactivity in lesions may indicate the contribution of glycoxidation to the processes occurring in diabetes and aging. Additionally, we found increased CML content in serum proteins in diabetic patients. The strong dependence of CML formation on oxidative conditions together with the increased occurrence of CML in diabetic serum and tissue proteins suggest a role for CML as endogenous biomarker for oxidative damage.

High glucose-induced transforming growth factor beta1 production is mediated by the hexosamine pathway in porcine glomerular mesangial cells.

Previous studies revealed that exposure of mesangial cells to high glucose concentration induces the production of matrix proteins mediated by TGF-beta1. We tested if structural analogues of D-glucose may mimic the high glucose effect and found that D-glucosamine was strikingly more potent than D-glucose itself in enhancing the production of TGF-beta protein and subsequent production of the matrix components heparan sulfate proteoglycan and fibronectin in a time- and dose-dependent manner. D-Glucosamine also promoted conversion of latent TGF-beta to the active form. Therefore, we suggested that the hexosamine biosynthetic pathway (the key enzyme of which is glutamine:fructose-6-phosphate amidotransferase [GFAT]) contributes to the high glucose-induced TGF-beta1 production. Inhibition of GFAT by the substrate analogue azaserine or by inhibition of GFAT protein synthesis with antisense oligonucleotide prevented the high glucose-induced increase in cellular glucosamine metabolites and TGF-beta1 expression and bioactivity and subsequent effects on mesangial cell proliferation and matrix production. Overall, our study indicates that the flux of glucose metabolism through the GFAT catalyzed hexosamine biosynthetic pathway is involved in the glucose-induced mesangial production of TGF-beta leading to increased matrix production.

Competitive inhibition by glucose of myo-inositol incorporation into cultured porcine aortic endothelial cells.

To explore the significance of hyperglycaemia as a causal factor for the appearance of diabetic angiopathies we investigated aspects of myo-inositol metabolism in porcine aortic endothelial cells. myo-Inositol was shown to be a long-living metabolite. Its uptake into the cells was mediated by a high-affinity, Na(+)-dependent uptake system inhibitable by ouabain with an apparent KM of 18.6 mumols/l, which was responsible for more than 80% of total uptake at physiological myo-inositol concentrations. Inhibition of inositol uptake by D-glucose was exclusively competitive with an apparent Ki of 24 mmol/l as shown by Lineweaver-Burk- and Dixon-plot analysis. The specificity of competitive inhibition was studied. L-Glucose which is stereochemically related to myo-inositol in the same way as the D-isomer proved to be an equally potent inhibitor. The hexoses D-galactose, D-mannose and D-fructose inhibited myo-inositol uptake to a minor extent. D-allose and 3-O-methyl-D-glucose had no inhibitory effect indicating that the OH-group of the carbon atom in 3 position is essential for the interaction with the carrier. The acyclic hexitol sorbitol also did not compete. As expected, the aldose reductase blocker sorbinil did not influence the carrier since there is no polyol pathway operating in porcine aortic endothelial cells. In accordance with the results of the uptake experiments, the incorporation of exogenous myo-inositol into membrane phosphatidylinositol was reduced at elevated extracellular glucose levels. The results raise the possibility that hyperglycaemia impairs endothelial inositol supply.

Upregulation of myo-inositol transport compensates for competitive inhibition by glucose. An explanation for the inositol paradox?

High glucose concentrations inhibit the uptake of myo-inositol into cells. However, whether this leads to a depletion of intracellular myo-inositol levels has been debated, because unchanged, decreased, and increased cellular myo-inositol levels all have been reported for diabetic tissues. To evaluate whether cells are capable of counterregulating impaired uptake, we have investigated myo-inositol uptake in porcine aortic endothelial cells under short- and long-term hyperglycemic conditions. Although increasing glucose concentrations inhibited acute myo-inositol uptake competitively, the uptake was increased markedly, when cells were already preincubated in a high glucose medium for > 6 h. The stimulation was maximal at 20 mM of glucose with no further increase at 40 mM glucose. The same stimulation of uptake could be induced by 5 mM of glucose plus 35 mM of raffinose, whereas 35 mM of sorbitol or mannitol, which do not compete for myo-inositol uptake, were ineffective. Lineweaver-Burk analysis revealed an increased Vmax for the induced myo-inositol transport activity, whereas the Km for myo-inositol remained constant (18 microM). The upregulated inositol transporter was still Na+ and ATP dependent, indicating that the same carrier system was operating. Uptake returned to control values when cells were again exposed to normoglycemic medium conditions for an additional 24 h. When endothelial cells were incubated with D-[U-14C]glucose and 10 microM myo-[2-3H]inositol of equal specific radioactivity for 24 h, no 14C radioactivity was found in intracellular myo-inositol, indicating that conversion of glucose to myo-inositol was rather low.

epsilon-Amino-lysine-bound glucose in human tissues obtained at autopsy. Increase in diabetes mellitus.

The level of nonenzymatically epsilon-lysine-bound glucose (NEBG) of tissue proteins obtained at autopsy has been determined with a specific method recently described. The mean values expressed as nmol lysine-bound glucose per mumol phenylalanine were 34 for tendon, 13 for aorta, 16 for coronary artery, 23.5 for femoral nerve, 10 for glomerular basement membrane, and 30 for lung parenchyma for tissues from nondiabetics and 86, 39.5, 34, 60, 29, and 57 for tissues form diabetics, respectively. NEBG was below detection limit in skeletal muscle from either nondiabetic or diabetic subjects. Tendon and aorta NEBG levels correlated well with those from other tissues (r = 0.8-0.94, except r = 0.68 for glomerular basement membrane). A fairly good correlation was also found when NEBG of aorta was compared with the mean blood sugar level determined during the last weeks of hospitalization. Finally, an arbitrary index of diabetic late complication was compared with NEBG of aorta. There appears to be a tendency of an increase of the index of complications along with an increase in tissue glucosylation. The possible role of nonenzymatic glucosylation in the long-term complication of diabetes is discussed.

Expression of glutamine:fructose-6-phosphate amidotransferase in human tissues: evidence for high variability and distinct regulation in diabetes.

Recent in vitro and in vivo studies suggested that the increased flux of glucose through the hexosamine biosynthetic pathway may contribute to glucose-induced insulin resistance and to the induction of the synthesis of growth factors. Because glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the first and rate-limiting step in the formation of hexosamine products, this enzyme is the key regulator in this pathway and is therefore possibly also involved in the alterations occurring in preclinical or manifest diabetic patients. To study the expression of GFAT in human tissues, we produced and characterized a peptic antiserum specifically recognizing GFAT protein and a riboprobe for the detection of GFAT mRNA. Immunohistochemical and nonradioactive in situ hybridization analysis revealed high levels of expression of GFAT protein and mRNA in adipocytes and skeletal muscle. Furthermore, a marked GFAT expression was found in vascular smooth muscle cells with unexpectedly high variability and lower levels in other cells, e.g., peripheral nerve sheath cells or endocrine-active cells, including the pancreatic islet cell. GFAT protein expression was below detection level in endothelium, osteocytes, lymphocytes, granulocytes, and in most quiescent fibroblasts. In renal tissue, GFAT was expressed in tubular epithelial cells, while glomerular cells remained essentially unstained. Renal sections obtained from patients with diabetic nephropathy showed significant GFAT expression in some glomerular epithelial and mesangial cells, indicating that GFAT expression may be induced by manifest diabetes. Our data indicate that GFAT is expressed in most tissues involved in the development of diabetic late complications. Furthermore, the results suggest that GFAT gene expression is highly regulated.

Clinical utility of nonenzymatically glycosylated blood proteins as an index of glucose control.

This study compares the utility of nonenzymatically glycosylated serum proteins (lys-GSP) to glycosylated hemoglobins (HbA1a-c) as control indices of glucose homeostasis in patients with IDDM. The diagnostic value of lys-GSP was also examined in patients with non-insulin-dependent diabetes mellitus, in subjects with impaired glucose tolerance, and in two patients with insulinoma. The intraindividual fluctuation of lys-GSP in normoglycemic subjects is very small, resulting in an interindividual range of 3.0 +/- 0.3 lysine-bound glucose/mg protein (means +/- SD, N = 52). HbA1a-c with a normal range of 6.4 +/- 0.9% (N = 52) shows greater variability. In IDDM there is no overlap of lys-GSP levels between the normal and the diabetic range at the 95% confidence level. In patients treated with an open-loop insulin delivery system failure of normalization of the glucose balance was clearly discernible by an elevation of GSP. In contrast, in about 40% of the patients with incomplete glycemic control the HbA1a-c levels fell within the normal range. The utility of lys-GSP for diagnosis of diabetes is compared with the results of 60 oral glucose tolerance tests. Two patients suffering from insulinoma displayed decreased lys-GSP values. From these results it appears that determination of lys-GSP represents a more sensitive parameter for long-term control than HbA1a-c and is suitable for monitoring even small fluctuations of blood glucose.

High concentrations of low density lipoprotein decrease basement membrane-associated heparan sulfate proteoglycan in cultured endothelial cells.

The effect of increasing low density lipoprotein (LDL) concentrations on the synthesis of basement membrane components was investigated in proliferating porcine aortic endothelial cells (PAEC) in culture. Basement membrane-associated heparan sulfate proteoglycan (HSPG) and fibronectin were determined by enzyme immunoassay. Low extracellular LDL-levels increase, high extracellular LDL-levels decrease the HSPG content of PAEC. Fibronectin synthesis was only slightly affected while proliferation and metabolic activity as assessed by lactate production were constant. Insulin or high extracellular glucose did not influence the effect of LDL on basement membrane components.

Overexpression of glutamine:fructose-6-phosphate-amidotransferase induces transforming growth factor-beta1 synthesis in NIH-3T3 fibroblasts.

Increased flux through the hexosamine biosynthetic pathway with glutamine:fructose-6-phosphate-amidotransferase (GFAT) as rate-limiting enzyme has been linked to the enhanced bioactivity of the prosclerotic cytokine transforming growth factor beta1 (TGF-beta1) in fibrotic complications, particularly in diabetic kidney disease. Here, we investigate in a stable transfection system the effect of overexpression of GFAT on TGF-beta1 synthesis in NIH-3T3 fibroblasts. We demonstrate a 1.8-fold stimulation of TGF-beta1 mRNA and a 1.9-fold increased protein expression, whereas TGF-beta2 production was not upregulated. The 1.5-fold enhanced TGF-beta1 promoter activity suggests a transcriptional regulation. The elevated TGF-beta1 protein is biologically active since GFAT-overexpressing cells exhibit a 2-fold fibronectin production. The results indicate a GFAT-dependent induction of TGF-beta1 synthesis.

N(epsilon)-(carboxymethyl)lysine in atherosclerotic vascular lesions as a marker for local oxidative stress.

Previous studies suggested that N(epsilon)-(carboxymethyl)lysine (CML), as the major product of oxidative degradation of glycated proteins and unsaturated fatty acids, represents an integrative biomarker for oxidative stress. In the present study, the level of CML in morphologically normal as well as atherosclerotic vessel walls are immunohistochemically analyzed and the in vitro formation of CML determined from glycoxidation and lipid peroxidation processes. The analysis revealed negative staining results in normal arterial walls of fetal, juvenile and young adult origin. A minor positive staining was seen in normal arteries from adults between 40 and 60 years of age with a rise in the CML-staining further increasing with rising individual age. This staining was mainly restricted to the intimal extracellular matrix and there was no intracellular staining. In arteriosclerotic vessels, in contrast, the extracellular CML-staining was significantly increased by approximately 3-fold also affecting the vascular media and adventitia. A strong intracellular staining was seen in macrophages. The degree of CML-staining correlated with the extent of the atherosclerotic changes. The in vitro studies showed a slow formation of CML of glycated proteins under aerobic conditions. No CML was formed under anaerobic conditions. Unsaturated fatty acids revealed a much faster formation of CML which reached high levels. The addition of vitamin E did not substantially suppress the CML-formation. The data suggest that the endogenous biomarker CML for oxidative stress accumulates slowly in normal arterial walls. This process is significantly increased in atherosclerosis. While the accumulation of CML in the extracellular matrix seemed to be the result of ongoing glycoxidation, the significant intracellular CML-formation in macrophages may have resulted from lipid peroxidation.

Divergence in distribution and prognostic significance of major basement components in laryngeal carcinomas.

In previous studies, we demonstrated a loss of major basement membrane (BM) components in laryngeal squamous cell carcinomas and provided initial evidence that this was of potential prognostic significance. In our current study, we extended the panel of BM antibodies and enlarged our study group in order to perform a multivariate statistical analysis. We analyzed 26 laryngeal squamous cell carcinomas immunohistochemically for the distribution of the BM-components collagen IV, collagen VII, laminin-1, laminin-5, perlecan and fibronectin. The resulting data were correlated with clinical prognostic factors and statistical correlation coefficients were determined for independent uni- and multivariate analysis. All carcinomas analyzed revealed defects of the peritumoral BM with more extensive loss of collagen VII than collagen IV, laminin-1, perlecan and fibronectin. Laminin-5 in contrast was present even in poorly differentiated tumors showing an enhanced intracytoplasmatic staining in the tumor cells. Furthermore, our statistical analysis did not show independent prognostic significance of any of the BM-components. Our observations indicate a divergence between the loss of several major BM-components (collagens IV, VII, laminin-1, perlecan) and an enhanced deposition of laminin-5. This suggests a severely altered cell-matrix interaction, since laminin-5 links the collagen VII-containing anchoring fibrils to cell receptors of the integrin type.

Defective basement membrane in laryngeal carcinomas with heterogeneous loss of distinct components.

In this study, we evaluated immunohistochemically the composition of the tumor-associated epithelial basement membrane (BM) in a series of 66 laryngeal squamous cell carcinomas (SCC) and compared these results with those from 10 cases with laryngeal dysplasia and five cases with normal mucosa (controls). The major BM components collagen IV and VII, laminin-1, perlecan (heparan sulfate proteoglycan), and fibronectin were evaluated. The extent of the retained BM material was quantified by semiautomated morphometry. A subsequent statistical analysis correlated the immunohistochemical findings with the histopathologically evaluated degree of tumor cell differentiation. In our series, we observed a distinct correlation between the degree of tumor cell differentiation and the amount of retained BM material. The loss of BM affected the various components differently, with a more extensive loss of collagen VII even in highly differentiated tumors and a progressive loss of collagen IV immunostaining with decreasing tumor cell differentiation. With respect to the other BM components, a stepwise loss of BM material also was seen with decreasing degree of the tumor cell differentiation. This loss, however was not at a statistically significant level, so these parameters did not show further statistically relevant data. In dysplastic lesions (regardless of the degree of dysplasia), focal BM disruptions were seen that affected the various BM components to a very similar extent. Our observations provide evidence that laryngeal carcinomas show a progressive loss of BM material along with decreasing tumor cell differentiation. This loss of BM, however, affects the various BM components differently. This indicates a dysregulation of the BM, either induced by uncoordinated neosynthesis or selectively enhanced degradation by proteases or both. Finally, the BM analysis may provide information on the biological course of the tumors. The loss of collagen VII may serve as a marker for "early" invasive tumor growth, whereas the amount of collagen IV provides significant information on the loss of tumor cell differentiation.

Role of the hexosamine biosynthetic pathway in diabetic nephropathy.

The hexosamine biosynthetic pathway has been hypothesized to be involved in the development of insulin resistance and diabetic vascular complications. In particular, it was demonstrated that hyperglycemia-induced production of transforming growth factor-beta (TGF-beta1), a prosclerotic cytokine causally involved in the development of diabetic nephropathy. Several lines of evidence indicate that TGF-beta1 induction is mediated by the hexosamine pathway. In cultured mesangial cells, high glucose levels induce TGF-beta1 production. This effect is eliminated by inhibition of glutamine: fructose-6-phosphate-amidotransferase (GFAT), the rate-limiting enzyme of this pathway. Furthermore, stable overexpression of GFAT increased levels of TGF-beta1 protein, mRNA, and promoter activity. Inasmuch as stimulation or inhibition of GFAT increased or decreased high glucose-stimulated activity of protein kinase C (PKC), respectively, the observed effects appear to be transduced by PKC. In similar experiments, involvement of the hexosamine pathway in hyperglycemia-induced production of cytokines (TGF-alpha and basic fibroblast growth factor [bFGF]) was demonstrated in vascular smooth muscle cells. These studies also revealed a rapid increase in GFAT activity by treatment with agents that elevated levels of cyclic adenosine 3',5' monophosphate (cAMP), thus indicating that GFAT activity is tightly regulated by cAMP-dependent phosphorylation. Using immunohistochemistry and in situ hybridization, high expression of GFAT was found in human adipocytes, skeletal muscle, vascular smooth muscle cells, and renal tubular epithelial cells. whereas glomerular cells remained essentially unstained. However, significant staining occurred in glomerular cells of patients with diabetic nephropathy. Current data indicate that the flux through the hexosamine pathway, regulated by GFAT, may be causally involved in the development of diabetic vascular disease, particularly diabetic nephropathy.

Molecular mechanism of diabetic nephropathy.

Diabetic nephropathy is one of the main causes of renal end-stage disease. Morphologically, the development of diabetic nephropathy is characterized by progressive thickening of the glomerular basement membrane and by expansion of the mesangial matrix which correlates to glomerular filtration function. In vitro studies with cultured mesangial cells revealed that elevated glucose concentrations increase collagen synthesis similar to the in vivo situation. These studies showed that hyperglycemia may be toxic either by non-enzymatic reaction of glucose with proteins and subsequent formation of advanced glucosylation end products or by increased metabolism leading to increased oxidative stress and activation of protein kinase C resulting in increased production of cytokines. Particularly, de novo synthesis of transforming growth factor beta1 (TGF-beta1) is induced and TGF-beta1 appears also involved since blockage of this prosclerotic factor inhibits high glucose-induced collagen synthesis. Interestingly, it could be demonstrated that angiotensin II also stimulates TGF-beta1 production possibly via the same signal transduction pathway. Besides the classical clinical chemical parameters for evaluation of renal function, the measurement of urinary albumin excretion is now widely used for detection of developing diabetic nephropathy. Since diabetes causes glomerular and tubular changes, tubular marker proteins may be used to detect early renal damage. An increased urinary excretion of matrix proteins (e.g. collagen) and cytokines (e.g. TGF-beta1) was found in early diabetic nephropathy. However, the diagnostic value of these new parameters remains to be established.

Analysis of total urinary catecholamines by liquid chromatography: methodology, routine experience and clinical interpretations of results.

A simple routine method is described for simultaneous assay of total urinary adrenaline, noradrenaline and dopamine. The catecholamines are pre-purified on a small ion-exchange column, separated by reversed phase ion-pair liquid chromatography, and are quantitated by electrochemical detection. The method was routinely applied to 422 urines. Elevated values were found in four urine specimens obtained from patients with histologically proven phaeochromocytomas. Virtually no interference by endogenous or exogenous compounds was found. Values for urinary catecholamines determined by fluorimetric analysis agreed with those obtained by high pressure liquid chromatography with electrochemical detection. Within-day CVs for the compounds ranged from 5.2-11.9%, between-day CVs from 3.3-6.6%. The normal range (95% confidence level) was 20-230 micrograms/24 h for noradrenaline and 1-35 micrograms/24 h for adrenaline.

Gene expression and protein deposition of major basement membrane components and TGF-beta 1 in human breast cancer.

In the present study we used immunohistochemistry and in-situ hybridization for the localization of major basement membrane (BM) components and their mRNA, respectively, in order to determine the extent of BM production and deposition in normal mammary tissue as well as in invasive mamma carcinomas. While normal mammary tissue showed an intact epithelial BM, as evidenced by a continuous linear staining for collagen i.v., laminin, heparan sulfate proteoglycan (perlecan) and fibronectin, this staining was widely lost in the invasive carcinomas. Non-invasive intraductal areas of the carcinomas (carcinoma-in-situ) revealed focal fragmentation and duplication of the epithelial BM. Using in-situ hybridization, we observed only focally positive mRNA-expression for collagen i.v.-, perlecan- and fibronectin-mRNA in normal glands, while mRNA-signals were significantly enhanced in one case of fibroadenoma and particularly in invasive and non-invasive carcinomas, regardless of the degree of tumor cell differentiation. In these instances both tumor and stroma cells were positively labelled. In addition, we could demonstrate a significant increase in the level of TGF-beta 1-mRNA--as the most active cytokine for the induction of matrix component production--by carcinoma cells and to lesser extent by stroma cells. The discrepancy between significantly enhanced mRNA-synthesis and loss in protein deposition points either to an upregulated activity of matrix degrading proteinases (matrix-metalloproteinases) or a posttranslational block of protein synthesis or both.

Prognostic aspects of the loss of epithelial basement membrane components in preinvasive and invasive laryngeal carcinomas.

The integrity of the epithelial basement membrane (BM) is an essential criterion or the biological behaviour of tumors. Previous studies on various types of carcinomas have demonstrated a good correlation between the amount of retained BM and the course of tumor growth. We therefore evaluated the prognostic significance of the tumor BM in laryngeal carcinomas. In this study, we analyzed 66 cases of laryngeal carcinomas using immunohistochemistry for the visualization of the major BM components collagen IV and VII, laminin-1, heparan sulfate proteoglycan (HSPG, perlecan) and fibronectin. The extent of retained BM-material was quantified morphometrically. A subsequent statistical analysis correlated the immunohistochemical findings with clinical and routine histological parameters, such as the mode of tumor infiltration. All carcinomas showed a defective epithelial BM. In addition, we observed a correlation between the degree of tumor cell-differentiation and the amount of BM material retained. The loss of BM, however, affected the various components differently with an "early" loss of collagen VII. In non-infiltrative dysplastic lesions focal BM disruptions were seen which affected the various BM components very similarly. When we statistically analyzed the correlation between the BM staining pattern and prognostically relevant parameters, collagen VII represented a marker for "early" stroma invasion. It also positively correlated with tumor size/stage, presence of lymph node metastasis and the recurrence of tumor growth. The collagen IV expression was positively correlated with the degree of tumor cell differentiation. The other parameters did not show further prognostically relevant data. Our observations provide significant information on the biological course of the disease. Thus, collagen VII may be a marker for "early" invasive tumor growth, as well as for lymphatic metastasis and local tumor recurrence, while the amount of collagen IV correlates with the tumor cell differentiation.

Morphological aspects of altered basement membrane metabolism in invasive carcinomas of the breast and the larynx.

In the present study we compared the localization of major basement membrane (BM) components and their mRNAs between invasive carcinomas of the breast (adenocarcinomas) and larynx carcinomas (squamous cell carcinomas, SCC), in order to determine the extent of BM production and deposition in malignant tumors of biologically different behaviour. Thus, breast carcinomas usually show a rapid locoregional/systemic spread, while the laryngeal SCCs normally show a more locally restricted growth pattern. While normal mammary glands and laryngeal mucosa revealed an intact epithelial BM as evidenced by a continuous linear staining for collagen IV, laminin-1, heparan sulfate proteoglycan (perlecan) and fibronectin-as well as collagen VII in the larynx mucosa-, this continuous staining was lost in the invasive carcinomas, however, affecting the two tumor types differently. In the breast carcinomas, a complete loss was seen even in well differentiated tumors affecting the various BM components similarly, while in the SCCs well differentiated carcinomas had retained significantly more BM material than poorly differentiated ones. In the SCCs, an "early" loss of collagen VII contrasted with a "later" loss of collagen IV, laminin, perlecan and fibronectin the extent of which was, however, associated with a decreasing degree of differentiation. In contrast to the protein findings, by use of the in-situ hybridization we observed a significant expression of mRNA for collagen IV, perlecan and fibronectin. The resulting pattern was comparable between both tumor types and not significantly related to the tumor cell differentiation. Both tumor cells and stroma cells were positively labelled with a more extensive labelling of the stroma cells. Our observations indicate a similar upregulation of the mRNAs for BM-components in breast and larynx carcinomas, but significant differences in the BM-protein deposition so that either major differences in presumed BM-proteolysis or further translational defects are suggested. Furthermore, it can be speculated that the far lesser amount of BM-material in the breast carcinomas may be linked to the more aggressive metastatic spread of those tumors, particularly when compared to the SCCs.