ABSTRACT
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.
Subject(s)
Extracellular Matrix/metabolism , Fibroblasts/metabolism , Fibroblasts/ultrastructure , Transforming Growth Factor beta/pharmacology , Blotting, Western , Cells, Cultured , Collagen/metabolism , Collagenases/metabolism , Gelatinases/metabolism , Humans , Matrix Metalloproteinase 1 , Matrix Metalloproteinase 2 , Metalloendopeptidases/metabolism , Proline/metabolism , Tissue Inhibitor of Metalloproteinase-1/analysis , Tissue Inhibitor of Metalloproteinase-2/analysis , TritiumABSTRACT
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.
Subject(s)
Aldosterone/pharmacology , Collagen/biosynthesis , Endomyocardial Fibrosis/etiology , Fibroblasts/drug effects , Angiotensin II/pharmacology , Animals , Cell Division/drug effects , Cells, Cultured , Collagen/genetics , Endomyocardial Fibrosis/pathology , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Expression , Male , Myocardium/metabolism , Myocardium/pathology , Polymerase Chain Reaction , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Receptors, Mineralocorticoid/genetics , Receptors, Mineralocorticoid/metabolism , Spironolactone/analogs & derivatives , Spironolactone/metabolism , TritiumSubject(s)
Angiotensin II/blood , Blood Platelets/metabolism , Hypertension/blood , Receptors, Angiotensin/metabolism , Adult , Angiotensin Receptor Antagonists , Binding, Competitive , Biphenyl Compounds/metabolism , Humans , Imidazoles/metabolism , In Vitro Techniques , Kinetics , Losartan , Middle Aged , Oligopeptides/metabolism , Receptors, Angiotensin/classification , Tetrazoles/metabolismABSTRACT
The influence of the renin-angiotensin system on renal hemodynamics, tubular pressure and tubulo-glomerular feedback was investigated with the angiotensin converting enzyme inhibitor MK 421 (enalapril), in uninephrectomized rats with and without ischemia-induced acute renal failure. In animals with normal renal function proximal tubular pressure and tubulo-glomerular feedback response were lowered by enalapril long-term treatment, whereas glomerular filtration rate and renal blood flow were not influenced by the drug. After 45 and 70 minutes ischemia there was no difference between treated and untreated animals in the severely impaired glomerular filtration rate. Renal blood flow remained unaffected by the treatment. The histological damage due to ischemia (tubular casts, tubular necrosis and medullary capillary congestion) was not influenced by enalapril. As tubulo-glomerular feedback had been significantly inhibited during renin-angiotensin inhibition, its importance in mediating acute renal failure remains doubtful; other factors such as tubular obstruction and medullary congestion may be crucial.
