Extracellular matrix deposition by primary human lung fibroblasts in response to TGF-beta1 and TGF-beta3.

Increased collagen and extracellular matrix (ECM) deposition within the lung is a characteristic feature of lung fibrosis. Transforming growth factor (TGF)-beta isoforms play a pivotal role in the production of collagen and ECM. In this study, we investigated the effects of TGF-beta1 and TGF-beta3 on the main processes controlling ECM deposition using primary human lung fibroblasts. We analyzed 1) collagen metabolism by [3H]proline incorporation, 2) matrix metalloproteinase (MMP) expression by substrate gel zymography, and 3) tissue inhibitor of metalloproteinases (TIMP) expression by Western blot analysis. TGF-beta1 and TGF-beta3 increased the percentage of secreted collagens in supernatants of primary fibroblasts from 8.0 +/- 1.2 (control) to 23.6 +/- 4.6 and 22.3 +/- 1.3%, respectively. The collagen percentage in deposited ECM was increased from 5.8 +/- 0.3 (control) to 9.0 +/- 0.5 and 8.8 +/- 0.5% by TGF-beta1 and TGF-beta3, respectively. Secretion of MMP-1 (interstitial collagenase) by fibroblasts was reduced by both TGF-beta isoforms, whereas secretion of MMP-2 (gelatinase A) was unaffected by either of the two isoforms. Both TGF-beta isoforms increased TIMP-1 protein expression, whereas TIMP-2 protein was decreased. We thus conclude that TGF-beta1 and TGF-beta3 are equally potent in increasing ECM deposition. Their fibrotic effect in lung fibroblasts results from 1) an increase in the secretion and deposition of total ECM and collagens, 2) a decrease in MMP-1 secretion, and 3) an increase of TIMP-1 expression.

Does aldosterone-induced cardiac fibrosis involve direct effects on cardiac fibroblasts?

To investigate the hypothesis that aldosterone plays a role in the development of fibrosis, cultured fibroblasts from adult rat heart have been examined for their expression of aldosterone receptors and the effects of aldosterone on collagen synthesis. Binding assays with both 3H-aldosterone and 3H-RU26752 in intact cardiac fibroblasts and cytosolic extracts from cardiac fibroblasts failed to reveal expression of aldosterone receptors. However, using the method of reverse transcription-polymerase chain reaction, we could demonstrate the expression of mRNA for the mineralocorticoid receptor in both cardiac fibroblasts and neonatal rat cardiomyocytes. Functional studies investigating the effect of aldosterone on collagen synthesis (3H-proline incorporation into collagenous protein) revealed that aldosterone does not stimulate collagen synthesis in cardiac fibroblasts at concentrations (10(-8) to 10(-9) M) observed in primary or secondary hyperaldosteronism. At higher concentrations (10(-6) to 10(-7) M) aldosterone inhibited collagen synthesis. Expression of collagen genes I alpha 1, III alpha 1, IV alpha 1 and of the collagenase gene was not affected by aldosterone. The collagen gene VI alpha 2 was also found to be expressed in cultured cardiac fibroblasts, and its expression was also independent of aldosterone. The data indicate that fibrosis is not due to a direct effect of aldosterone on fibroblast collagen synthesis.

Different translocation of three distinct PKC isoforms with tumor-promoting phorbol ester in human platelets.

Protein kinase C (PKC), a family of related but distinct enzymes whose cellular functions are poorly understood, acts in synergy with Ca2+ mobilization for the activation of platelets. Using specific antibodies for the different isoforms, immunoblot analysis revealed the presence in human platelets of three different PKC subtypes which specifically react with alpha, beta and zeta-PKC antibodies. Whereas the subcellular distribution of the alpha PKC remained unaffected, incubation of platelets with 1 microM PMA for 2 min resulted in a significant subcellular distribution from cytosol to membrane of beta-PKC (25%) and zeta (15%). The beta-PKC isoform is more sensitive than alpha and zeta-PKC to PMA, since 100 nM PMA resulted in a translocation of 85%, 64% and 66% respectively of a maximum translocation observed with 1 microM PMA.

The interaction of aminogroups with pyrroloquinoline quinone as detected by the reduction of nitroblue tetrazolium.

The interaction of pyrroloquinoline quinone (PQQ) with amino groups was followed by measuring the capacity of adducts to reduce nitroblue tetrazolium (NBT). Of the natural amino acids only glycine, ornithine, and lysine interacted strongly with PQQ. The reducing activity of other less reactive amino acids, but not of lysine, was increased by ammonia, primary or secondary amines. Divalent cations, in contrast inhibited development of NBT-reducing activity. PQQ also developed NBT-reactivity in the presence of serotonin and albumin. A reaction scheme is proposed which explains these findings. It is suggested that the NBT-reducing activity of plasma which is not caused by glycation of plasma proteins, arises from PQQ adducts inherent to plasma. This NBT-reducing activity corresponds to approximately 10 micrograms PQQ/ml plasma.

Evaluation of the fructosamine test for the measurement of plasma protein glycation.

Repeated estimation of plasma protein glycation by the fructosamine assay gave more variable results than expected from analytical variability (coefficient of variation approximately 2%). Fructosamine results obtained on plasma samples drawn at different times of the day differed by up to 1 mmol/l, corresponding to a coefficient of variation of greater than 10%. As a consequence, the information concerning averaged glycaemia of a fructosamine determination is subject to an uncertainty of 7.8 mmol/l. Fructosamine concentrations were linearly related to the protein concentration. Correction for the protein concentration decreased this variability; however, factors other than protein concentration, such as lipid content, also influence results of fructosamine determinations.

[HbA1c determination in capillary blood]. / Die HbA1c-Bestimmung im Kapillarblut.

A practical procedure is described for the determination of HbA1c in capillary blood collected in micro-hematocrit-capillaries. These blood samples can be mailed to the analytical laboratory prior to consultation with the physician. The results of such determinations in 23 juvenile diabetic patients obtained during a two-year observation period are presented.

Effect of temperature on quantifying glycated (glycosylated) hemoglobin by cation-exchange chromatography.

As a consequence of nonideal chromatographic conditions, values for stable glycated hemoglobin (HbA1c) determined by cation-exchange chromatography in a commercial minicolumn system (y) or by "high-performance" liquid chromatography (x) differ markedly, yielding the regression line y = 0.82x + 0.6. With use of the protocol specified by the manufacturer, 20% of the HbA1c peak is not collected in the HbA1c fraction. Increasing the ionic strength of the eluting buffer by increasing the operating temperature to 28 degrees C increases the rate of elution from the minicolumn, making results of the two methods more closely comparable (y = 0.98x - 0.22). Because at a given pH the elution volume is determined primarily by the ionic strength, close limits on the composition of the eluting buffer are set by the temperature-dependence of its ionic strength. At a specified temperature and pH the position of a peak can be judged to within a volume of 1 mL if the conductivity of the eluent does not vary by more than +/- 0.05 mS.

Quantitation of glycosylated hemoglobin by boronate affinity chromatography.

Total hemoglobin glycosylation and the contribution of glycosylation at the N-terminus of the beta-chains and at "non"-beta-N-terminal positions were quantitated by use of boronate affinity and ion exchange chromatography. Glycohemoglobin (y) was found to correlate linearly (y = 1.92x + 0.53; r = 0.96) with HbA1c (x) and to contain approximately 50% beta-N-terminally glycosylated hemoglobin. This result is in agreement with the binding on boronate agarose of the various hemoglobin components resolved by cation exchange chromatography. An amount of glycohemoglobin similar to that of HbA1c was isolatable from HbA. A slope of less than 2 results because HbA1c is retained only to 93% and the intercept of the regression line reflects the partial adherence (65%) of HbA1a + b to the resin. These results confirm the occurrence of significant "non"-beta-N-terminal glycosylation and show that under optimal chromatographic conditions total glycohemoglobin can be determined with boronate affinity chromatography.