Influence of hepatocyte growth factor, epidermal growth factor, and mycophenolic acid on endothelin-1 synthesis in human endothelial cells.

BACKGROUND: Endothelin-1 (ET-1) is a potent vasoconstrictive peptide which plays an important pathophysiological role in ischaemic renal failure and drug-induced renal injury such as cyclosporin A (CsA)- and tacrolimus-associated nephrotoxicity. In contrast, hepatocyte growth factor (HGF) and epidermal growth factor (EGF) seem to accelerate renal regeneration after ischaemic and drug-induced renal injury. This study aimed to investigate the influence of HGF and EGF on ET-1 synthesis in cultured human umbilical vein endothelial cells (HUVEC) and renal artery endothelial cells (RAEC). In addition, we have investigated whether mycophenolic acid (MPA), a new immunosuppressive drug, which in contrast to CsA and tacrolimus lacks nephrotoxic side effects, modulates ET-1 synthesis in endothelial cells. METHODS: ET-1 release was measured with a specific enzyme-linked immunosorbent assay. ET-1 mRNA expression was investigated by reverse transcription polymerase chain reaction. RESULTS: HGF and EGF (0.001-10 nM) exerted a significant concentration-dependent inhibitory effect on ET-1 release by HUVEC and RAEC (minimum 56.1+/-4.3% of control, n=6, mean+/-SE). The suppressive effect of HGF and EGF on ET-1 synthesis was dose-dependently antagonized by the tyrosine kinase inhibitors tyrphostin AG1478, lavendustin A and methyl 2,5-dihydroxycinnamate. Incubation of HUVEC and RAEC with MPA (2.5, 10, 25, and 50 microg/ml) for 3-5 h induced a significant reduction of ET-1 mRNA expression. After 48 h incubation with MPA (1-50 microg/ml) a significant decrease of ET-1 release and DNA content per culture well was observed, whereas ET-1 release referred to the DNA content in the corresponding culture well did not differ significantly from controls. CONCLUSIONS: The present findings demonstrate that HGF and EGF reduce ET-1 synthesis in endothelial cells via their receptor tyrosine kinase activity and suggest that the renoprotective effects of HGF and EGF might be linked to their inhibitory action on ET-1 synthesis. This study also provides evidence that, in contrast to CsA and tacrolimus, MPA does not stimulate ET-1 synthesis. This might explain the clinical observation that renal function often improves when CsA or tacrolimus is replaced by mycophenolate mofetil.

Oxidized low-density lipoproteins bind to the scavenger receptor, CD36, of hepatic stellate cells and stimulate extracellular matrix synthesis.

Cumulating evidence suggests that oxidative stress resulting in lipid peroxidation and protein modification is involved in the pathogenesis of chronic liver injury and fibrogenesis. We investigated the effects of oxidized low-density lipoproteins (oxLDL) on collagen and fibronectin synthesis of cultured human and rat hepatic stellate cells (HSC). As shown on protein and mRNA levels, oxLDL dose-dependently stimulated the synthesis of collagen types I and III and fibronectin of cultured HSC. The effect was biphasic, with a maximum between 5 and 25 microg/mL oxLDL (c-fibronectin concentration in HSC supernatants increased 3.9-fold; collagen type I increased 4-fold). Higher oxLDL concentrations were cytotoxic. LDL modified with malondialdehyde (MDA) was not toxic, but stimulated extracellular matrix synthesis as well. As demonstrated by immunofluorescence microscopy (double staining of CD36 and iso-alpha-smooth muscle actin [iso-alpha-sm actin]), immunoblot, and reverse-transcription polymerase chain reaction (RT-PCR), respectively, cultured human HSC express the oxLDL receptor, CD36 (glycoprotein IIIb). Colocalization of CD36 and iso-alpha-sm actin on sinusoidal lining cells was further demonstrated using sections of human fibrotic liver. Preincubation of cultured human HSC with the monoclonal antibody, OKM5, known to block CD36-mediated oxLDL uptake, resulted in a reduction of the oxLDL-stimulated collagen type I synthesis by 56%. In summary, our results demonstrate that low concentrations of modified lipoproteins (oxLDL and MDA-LDL) represent fibrogenic mediators that bind to CD36 and stimulate matrix synthesis of HSC.

Endothelin-1 synthesis and endothelin B receptor expression in human coronary artery smooth muscle cells and monocyte-derived macrophages is up-regulated by low density lipoproteins.

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide exerting its effects predominantly by paracrine and autocrine mechanisms. ET-1 acts as a mitogen and co-mitogen on vascular smooth muscle cells, and accumulating evidence suggests that ET-1 is involved in the pathogenesis of atherosclerosis. Deposition of low density lipoproteins (LDL) in the vessel wall is known to play a crucial role in the development of atherosclerotic lesions. In the present study, we have investigated the effect of native LDL (nLDL) and oxidatively modified LDL (oxLDL) on ET-1 synthesis and endothelin receptor expression in cultured human coronary artery smooth muscle cells and human monocyte-derived macrophages. Native LDL and oxLDL induced a significant stimulation of ET-1 release and ET-1 mRNA expression in human coronary artery smooth muscle cells and monocyte-derived macrophages. Antibodies against the scavenger receptor CD36 significantly reduced the oxLDL-induced stimulation of ET-1 synthesis. The antioxidants trolox and probucol did not significantly inhibit the LDL-induced rise of ET-1 release. Endothelin B receptor expression was up-regulated in both cell types after incubation with nLDL and oxLDL. In coronary smooth muscle cells, endothelin A receptor expression was slightly increased by LDL, whereas endothelin A receptor was not detectable in monocyte-derived macrophages. Coronary artery smooth muscle cells secreted a more than 150-fold higher amount of immunoreactive ET-1 into the cell culture medium than monocyte-derived macrophages. In summary, the present data, demonstrating a LDL-induced up-regulation of the endothelin system in coronary smooth muscle cells and in monocyte-derived macrophages, provide further support for a pathophysiological role of endothelin in coronary atherosclerosis and suggest that ET-1 might be involved in mediating the atherogenic effects of LDL.

Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells.

The aim of this study was to identify fibrogenic mediators stimulating activation, proliferation, and/or matrix synthesis of rat pancreatic stellate cells (PSC). PSC were isolated from the pancreas of normal Wistar rats and from rats with cerulein pancreatitis. Cell activation was demonstrated by immunofluorescence microscopy of smooth muscle alpha-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin, and transforming growth factor (TGF)-beta(1). Proliferation was measured by bromodeoxyuridine incorporation. Matrix synthesis was demonstrated on the protein and mRNA level. Within a few days in primary culture, PSC changed their phenotype from fat-storing to SMA-positive myofibroblast-like cells expressing platelet-derived growth factor (PDGF) alpha- and PDGF beta-receptors. TGF-beta(1) and tumor necrosis factor (TNF)-alpha accelerated the change in the cells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basic fibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 +/- 0.49- and 2.96 +/- 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 +/- 1.13-fold), 5 ng/ml TGF-beta(1) (2.46 +/- 0.89-fold), 20 ng/ml PDGF (2.27 +/- 0.68-fold), and 50 ng/ml TGF-alpha (1.87 +/- 0.19-fold). As shown by RT-PCR, PSC express predominantly the splice variant EIII-A of fibronectin. Immunofluorescence microscopy and Northern blot confirmed that in particular bFGF and TGF-beta(1) stimulated the synthesis of fibronectin and collagens type I and III. In conclusion, our data demonstrate that 1) TGF-beta(1) and TNF-alpha accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF, TGF-beta(1), PDGF, and, to a lesser extent, TGF-alpha stimulate extracellular matrix synthesis of cultured rat PSC.

Characterization of modified low density lipoprotein subfractions by capillary isotachophoresis.

Oxidative modification of low density lipoproteins (LDLs) is an important pathogenetic factor in atherosclerosis. The various steps in oxidative modifications of LDL can be monitored using different methodologies with varying degrees of complexity. In this study, we propose capillary isotachophoresis (CITP) as a suitable tool to detect and measure the degree of oxidation of LDL. LDL was isolated from pooled plasma of healthy volunteers by sequential ultracentrifugation, and oxidation was performed in vitro as well as in cell culture experiments. Native LDL and oxidatively modified LDL were characterized by apo B-100 fluorescence and conjugated diene formation. Samples were separated by CITP combined with sudan black B staining. To underline the inherent advantages of this approach, CITP was compared with classical lipoprotein electrophoresis using agarose gel. We demonstrate the CITP method to be highly sensitive, as changes in peak area of the separated LDL subfractions were detected after only 2 h of oxidation. The leading LDL peaks increased, while the terminating LDL peaks decreased in parallel throughout the duration of oxidation. The LDL samples, oxidized for 4-24 h, also exhibited an increased migration velocity of the fractions. In summary, we present the first study investigating LDL-subfractions separated by CITP and the alterations of these LDL-subfractions after gradual in vitro oxidation and after oxidative modification by monocyte-derived macrophages and vascular smooth muscle cells.

Inhibitory effect of epidermal growth factor and hepatocyte growth factor on endothelin-1 release by rabbit proximal tubule cells.

Several studies have demonstrated an upregulation of endothelin-1 (ET-1) synthesis in acute and chronic renal failure. Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) have been shown to stimulate renal tubular cell proliferation and to accelerate renal regeneration after drug-induced and ischemia-induced renal injury. This study aimed to investigate the effect of EGF and HGF on ET-1 release, and whether the effect of EGF and HGF is antagonized by the tyrosine kinase inhibitor lavendustin A. Rabbit proximal tubule cells were incubated for 48 h with EGF or HGF (0.1-10.0 nM), lavendustin A (0.1-10.0 microM) or co-incubated with EGF or HGF (1 nM) and lavendustin A. ET-1 concentrations in the culture medium were measured with a specific enzyme-linked immunosorbent assay (ELISA). EGF and HGF exerted a significant (p < 0.001) dose-dependent inhibitory effect on ET-1 release. Lavendustin A induced a dose-dependent stimulation of ET-1 release and antagonized the inhibitory effect of EGF and HGF on ET-1 release. The inhibition of EGF and HGF receptor tyrosine kinase activity by lavendustin A was confirmed by Western blotting. These data suggest that EGF and HGF reduce ET-1 release via EGF and HGF receptor tyrosine kinase activity. The inhibitory action of EGF and HGF on ET-1 release might be involved in mediating the protective effects of EGF and HGF in renal injury.

Hepatocyte growth factor is upregulated by low-density lipoproteins and inhibits endothelin-1 release.

Low-density lipoproteins (LDL) are known to cause endothelial injury and to promote the development of atherosclerotic lesions. This study demonstrates a significant concentration-dependent stimulatory effect of LDL on hepatocyte growth factor (HGF) synthesis (maximum release: 423 +/- 16% of control) and HGF receptor mRNA expression in cultured human coronary artery endothelial cells (HCAEC). HGF is a potent mitogen for endothelial cells but does not affect smooth muscle cell proliferation. In contrast, endothelin-1 (ET-1) acts as a mitogen on vascular smooth muscle cells and seems to be upregulated in coronary atherosclerosis. In this study, the basal ET-1 synthesis in HCAEC was concentration-dependently reduced by HGF (minimum: 54 +/- 3% of control). This inhibitory effect seems to be mediated via the tyrosine kinase activity of the HGF receptor c-met, since it was antagonized by the tyrosine kinase inhibitor lavendustin A. In addition, HGF also significantly reduced the LDL-stimulated ET-1 release. The LDL-induced upregulation of HGF synthesis in HCAEC and the inhibitory effect of HGF on ET-1 synthesis suggest a protective role of HGF in coronary atherosclerosis.

Platelet-derived growth factors stimulate proliferation and extracellular matrix synthesis of pancreatic stellate cells: implications in pathogenesis of pancreas fibrosis.

At present, the cell-cell interactions and molecular mechanisms of pancreas fibrogenesis are largely unknown. The purpose of this study was to investigate paracrine stimulatory loops between platelets and pancreatic stellate cells (PSC). Human PSC were obtained by outgrowth from fibrotic human pancreas. Native platelet lysate (nPL) and transiently acidified platelet lysate (aPL) were added to cultured PSC (passage 4 to 7) in the absence of serum. The synthesis of collagen types I and III and c-fibronectin (cFN) was demonstrated on protein (immunofluorescence and quantitative immunoassay) and mRNA (Northern blot) level. Using sections of human pancreas with acute pancreatitis, platelet aggregates in capillaries were demonstrated by transmission electron microscopy. nPL, and to an even greater extent aPL, significantly increased the synthesis of collagen types I and III and of c-FN (120 microl/ml aPL increased collagen type I concentration in PSC supernatants by 1.99 +/- 0.17 times and c-FN of 2.49 +/- 0.28 times, mean +/- SD, n = 3). nPL and aPL also significantly stimulated cell proliferation (increased bromodeoxyuridine (BrdU) incorporation by 6.4 +/- 0.78 times and 10 +/- 0.29 times, respectively). By preincubating aPL with transforming growth factor beta (TGFbeta)- and platelet-derived growth factor (PDGF)-neutralizing antibodies and the TGFbeta-latency associated peptide, respectively, TGFbeta1 was identified as the main mediator stimulating matrix synthesis and PDGF as the responsible mitogen. Our data demonstrate that platelets contain fibrogenic mediators that stimulate proliferation (PDGF) and matrix synthesis (TGFbeta1) of cultured PSC. We suggest that platelets and PSC cooperate in the development of pancreas fibrosis.

Lipopolysaccharide-activated macrophages stimulate the synthesis of collagen type I and C-fibronectin in cultured pancreatic stellate cells.

We have recently identified and characterized pancreatic stellate cells (PSC) in rats and humans (Gastroenterology 1998, 15:421-435). PSC are suggested to represent the main cellular source of extracellular matrix in chronic pancreatitis. Now we describe a paracrine stimulatory loop between human macrophages and PSC (rat and human) that results in an increased extracellular matrix synthesis. Native and transiently acidified supernatants of cultured macrophages were added to cultured PSC in the presence of 0.1% fetal calf serum. Native supernatants of lipopolysaccharide-activated macrophages stimulated the synthesis of collagen type I 1.38 +/- 0.09-fold of control and c-fibronectin 1.89 +/- 0.18-fold of control. Transiently acidified supernatants stimulated collagen type I and c-fibronectin 2.10 +/- 0.2-fold and 2.80 +/- 0.05-fold of control, respectively. Northern blot demonstrated an increased expression of the collagen-I-(alpha-1)-mRNA and fibronectin-mRNA in PSC 10 hours after addition of the acidified macrophage supernatants. Cell proliferation measured by bromodeoxyuridine incorporation was not influenced by the macrophage supernatants. Unstimulated macrophages released 1.97 pg TGFbeta1/microgram of DNA over 24 hours and lipopolysaccharide-activated macrophages released 6.61pg TGFbeta1/microgram of DNA over 24 hours. These data together with the results that, in particular, transiently acidified macrophage supernatants increased matrix synthesis, identify TGFbeta as the responsible mediator. In conclusion, our data demonstrate a paracrine stimulation of matrix synthesis of pancreatic stellate cells via TGFbeta1 released by activated macrophages. We suggest that macrophages might play a pivotal role in the development of pancreas fibrosis.

Depending on their concentration oxidized low density lipoproteins stimulate extracellular matrix synthesis or induce apoptosis in human coronary artery smooth muscle cells.

Various lines of evidence indicate that oxidative stress resulting in lipid peroxidation and protein modification is involved in the pathogenesis of atherosclerosis and coronary heart disease. We have investigated the effect of modified (oxidized) low-density lipoproteins (oxLDL) on collagen and fibronectin synthesis in cultured human coronary artery smooth muscle cells (HCA-SMC). As shown by immunofluorescence microscopy and time-resolved fluorescence immunoassay, oxLDL dose-dependently stimulated collagen type I and fibronectin synthesis in cultured HCA-SMC. The effect on matrix synthesis was biphasic, with a maximum effect at concentrations between 1 and 10 microg/ml oxLDL. Higher oxLDL concentrations (>25 microg/ml) were cytotoxic. Beside oxLDL, malondialdehyde-modified LDL also stimulated extracellular matrix synthesis. In the presence of 100 microg/ml ascorbic acid, 25, 50 and 100 microg/ml oxLDL induced apoptosis within 6-8 hours (demonstrated by TUNEL-reaction, annexin-V binding and APO-2.7-expression). Apoptosis was not induced by normal (unmodified) LDL and malondialdehyde-modified LDL. The radical scavengers and antioxidants TROLOX and probucol and the hydrogen peroxide eliminator catalase significantly reduced oxLDL-induced apoptosis. Our results demonstrate that low concentrations of oxLDL are profibrogenic by stimulating extracellular matrix synthesis, whereas higher oxLDL concentrations induce oxidative stress and apoptosis in coronary artery smooth muscle cells. The profibrogenic effect might be relevant in the formation of atherosclerotic plaques, and the proapoptotic effect might contribute to an increased plaque vulnerability.

Endothelin release by rabbit proximal tubule cells: modulatory effects of cyclosporine A, tacrolimus, HGF and EGF.

BACKGROUND: Previous studies have suggested that endothelins, a family of 21 amino acid peptides with potent vasoconstrictive and mitogenic properties, are involved in the pathogenesis of acute and chronic renal failure. In addition, endothelin seems to play an important role in mediating the nephrotoxic side effects of cyclosporine A (CsA) and tacrolimus. The present study investigated the production of endothelin-1 (ET-1) and endothelin-3 (ET-3) by bipolar differentiated rabbit proximal tubule cells (PT-1 cells), and the modulatory effect of CsA, tacrolimus, hepatocyte growth factor (HGF) and epidermal growth factor (EGF) on ET-1 and ET-3 release. METHODS: ET-1 and ET-3 mRNA was detected by RT-PCR, immunoreactive endothelin was localized to PT-1 cells by immunofluorescence staining with antibodies against ET-1 and ET-3. ET-1 and ET-3 release into the culture medium was determined by specific radioimmunoassays after solid phase extraction. RESULTS: PT-1 cells exhibited a time-dependent increase of ET-1 release up to an incubation period of 36 hours, whereas ET-3 release already reached a steady state level after four hours. PT-1 cells, cultured on filter membranes, released a significantly higher amount of immunoreactive ET-1 into the basolateral compartment than into the apical compartment. ET-3 release did not differ significantly between the basolateral and the apical compartment. Supplementation of the cell culture medium with 10% fetal calf serum induced a marked increase of the basolateral and apical ET-1 release, whereas ET-3 release was only slightly increased. CsA and tacrolimus (0.5 to 5000 microgram/liter) induced a significant, dose-dependent increase of ET-1 and ET-3 release by PT-1 cells with a maximum stimulation at a CsA concentration of 500 microgram/liter (P < 0.001) and a tacrolimus concentration of 50 microgram/liter (P < 0.001). HGF and EGF (10-10 to 10-8 mol/liter) exerted a significant (P < 0.001) dose-dependent inhibitory effect on ET-1 release, whereas ET-3 release was not significantly reduced. Coincubation of PT-1 cells with CsA or tacrolimus and HGF or EGF also resulted in a marked reduction of ET-1 release. CONCLUSIONS: The present data suggest that ET-1 and ET-3 release by cultured rabbit proximal tubule cells are regulated differently, and that the stimulatory effect of CsA and tacrolimus on ET-1 release is antagonized by HGF and EGF.