Anti-proliferative properties of the phosphodiesterase-4 inhibitor rolipram can supplement immunosuppressive effects of cyclosporine for treatment of obliterative bronchiolitis in heterotopic rat allografts.

BACKGROUND: Potent prevention and therapy of obliterative bronchiolitis may enhance long-term survival after lung transplantation. Phosphodiesterase-4 inhibitors have been established for anti-inflammatory treatment, particularly of pulmonary diseases. Using a heterotopic rat model, the effect of rolipram was investigated and compared with cyclosporine for epithelium disturbance and leukocyte infiltration and proliferation, which are key events in the development of obliterative bronchiolitis. METHODS: Tracheae were transplanted into the omentum of allo- and syngeneic animals. Four allogeneic groups were investigated: treatment with rolipram; treatment with cyclosporine; treatment with a combination of rolipram and cyclosporine; and untreated (60-day time course). Using histo- and immunohistochemical stainings, epithelium disturbance, leukocyte subsets, proliferating cells and luminal occlusion were quantified by digital morphometry. RESULTS: In rolipram-treated animals, the epithelium was completely disturbed until Day 14. It was temporarily preserved in rats that received cyclosporine until Day 60. In the acute phase (Day 5), infiltration of monocytes/macrophages was significantly inhibited by rolipram, but less effective than in cyclosporine-treated rats. At later timepoints (Days 28 and 60), rolipram significantly inhibited proliferation, in contrast to enhanced proliferation of fibroblast-like cells after cyclosporine treatment. The combination of rolipram and cyclosporine led to temporary epithelial preservation and effective inhibition of leukocyte infiltration (Day 5) and proliferation (Days 28 and 60). Luminal occlusion was significantly reduced in the combination group compared with the cyclosporine-only group. CONCLUSIONS: Although cyclosporine temporary protects epithelial integrity by the inhibition of acute rejection, rolipram showed greater potency for long-term inhibition of mesenchymal-cell proliferation. The combination of both drugs may be useful for limiting chronic obliterative changes after lung transplantation.

Evidence for the decreased expression of the latent TGF-beta binding protein and its splice form in human liver tumours.

BACKGROUND: Recently, a splice form of the latent TGF-beta binding protein (LTBP-1) was identified in the liver lacking potential important sequences for matrix association and proteinase cleavage (LTBP-1D, -1delta53). For a better understanding of the unknown (patho)physiological role, the expression levels of LTBP-1D and LTBP-1 (full length) were investigated in normal and malignant human liver on the mRNA and protein level. METHODS: Normal liver (5 specimens), hepatocellular carcinoma (4 specimens) and fibrolamellar carcinoma (2 specimens) were examined by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry, for which specific antibodies were generated. RESULTS: The mRNA levels of LTBP-1/-1D in malignant liver tissues are decreased in comparison to normal liver--more so in HCC than in FLC. This finding was confirmed by a strong decrease of immunostaining of LTBP-1/-1D in neoplastic parenchymal cells of HCC and FLC. However, the intensity of LTBP-1 (full length) protein staining was increased in the extracellular matrix of the carcinomas, while LTBP-1D was not detectable in the matrix. CONCLUSION: Since TGF-beta is known to be over-expressed in liver tumours, the results suggest its enhanced synthesis without binding to LTBP-1. This probably influences the availability of bioactive TGF-beta in the tumour tissue. The missing matrix localization of LTBP-1D indicates that the hinge region containing a heparin-binding site is essential for the binding of LTBP-1 in the extracellular matrix. LTBP-1D may fulfil specific functions for the latency of matrix-unbound TGF-beta.

Beta(2)-adrenoceptor stimulation enhances latent transforming growth factor-beta-binding protein-1 and transforming growth factor-beta1 expression in rat hippocampus after transient forebrain ischemia.

A protective capacity of transforming growth factor-beta1 (TGF-beta1) against various insults inducing neurone cell death in vitro and in vivo has been well established. We have recently shown the rapid up-regulation and persistent expression of TGF-beta1 in surviving CA1 pyramidal cells after cerebral ischemia suggesting an endogenous mechanism of neuroprotection by this multifunctional cytokine. In the present study, we demonstrated that intraperitoneal administration of clenbuterol, a lipophilic beta(2)-adrenoceptor agonist, caused an increase in TGF-beta1 expression in non-ischemic rats and further enhanced TGF-beta1 protein levels in rat CA1 pyramidal neurones after transient forebrain ischemia. In the hippocampus neuroprotection by clenbuterol (0.5 mg/kg) was accompanied by increased TGF-beta1 immunoreactivity as early as 3 h, and remained elevated up to 2 days after ischemia. The corresponding increased TGF-beta1 mRNA levels after ischemia were not further enhanced by clenbuterol, suggesting post-transcriptional regulation of TGF-beta1 protein after beta(2)-adrenoceptor stimulation. In saline-treated rats latent TGF-beta-binding protein-1 (LTBP-1) immunoreactivity was moderately elevated 3 and 6 h after ischemia, and returned to control levels after 1 day of reperfusion. In parallel with the up-regulation of TGF-beta1 immunoreactivity, LTBP-1 levels in the hippocampus were considerably increased by clenbuterol from 3 h to 2 days after ischemia. Our data demonstrate a concomitant increase in LTBP-1 and TGF-beta1 expression in the ischemic hippocampus after stimulation of beta(2)-adrenoceptors.

Effect of cytokines on regulation of the production of transforming growth factor beta-1 in cultured human Tenon's capsule fibroblasts.

PURPOSE: Transforming growth factor-beta1 (TGF-beta1) is thought to play a pivotal role in the regulation of the wound healing process after glaucoma filtering surgery. The aim of the present study was to investigate whether platelet-derived growth factor isoforms (PDGF-AA, PDGF-BB), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin-1beta (IL-1beta) and transforming growth factor-beta1 (TGF-beta1) modulate the production of latent and/or active TGF-beta1 by cultured human Tenon's capsule fibroblasts (HTF). METHODS: Human Tenon's capsule fibroblasts were seeded at two different densities (30 cells/mm2 and 150 cells/mm2) and stimulated for five days with PDGF-AA, PDGF-BB, bFGF, EGF, IL-1beta and TGF-beta1. Control cells were treated with serum-free medium (WM/F12). The concentrations of latent and active TGF-beta1 in the medium were determined using an immunoassay before and after activation of TGF-beta1 by transient acidification. RESULTS: The concentration of latent TGF-beta1 in conditioned media from HTF seeded at high density (150 cells/mm2) significantly increased after stimulation with 5 ng/ml TGF-beta1 (151.5 +/-41.7 pg/ml) or 10 ng/ml IL-1beta (45.7+/-8.1 pg/ml). The concentration of active TGF-beta1 in conditioned media also significantly increased after stimulation of HTF with 5 ng/ml TGF-beta1 (48.4+/-27.5 pg/ml). CONCLUSIONS: The present results indicate that TGF-beta1 is the most potent inducer of its own synthesis in HTF. Activation of an autocrine TGF-beta1 loop may play a role in the wound healing response after glaucoma filtering surgery.

The expression of transforming growth factor-beta1 (TGF-beta1) in hippocampal neurons: a temporary upregulated protein level after transient forebrain ischemia in the rat.

Exogenous TGF-beta1 has been shown to protect neurons from damage induced in vitro and in vivo. In this study we attempted to examine the expression of endogenous TGF-beta1 mRNA and protein in the hippocampus of non-ischemic and ischemic rats, and to localize TGF-beta1 protein and DNA fragmentation by double-staining. Transient ischemia was induced for 10 min in Wistar rats by clamping both common carotid arteries and lowering blood pressure to 40 mmHg. Bioactive TGF-beta1 was selectively determined in CA1 pyramidal neurons of non-ischemic rats. It was upregulated after 3 h and 6 h of reperfusion corresponding to the increase in TGF-beta1 mRNA level detected by RT-PCR. Lectin and GFAP staining showed no detectable activated microglial cells and astrocytes in the hippocampus 3 h and 6 h after ischemia. When neuronal damage proceeded through day 2 to day 4 after ischemia as demonstrated by TUNEL-staining, TGF-beta1 immunoreactivity (ir) disappeared in damaged neurons but persisted in viable neurons although TGF-beta1 mRNA levels continuously increased. Double-staining revealed that TUNEL-positive neurons did not express TGF-beta1, while TUNEL-negative neurons in the CA1 subfield exhibited a distinct TGF-beta1 ir. These data indicate that hippocampal CA1 neurons can express TGF-beta1 under physiological conditions and upregulate its expression during the first hours after ischemia, that is independent of the activation of glial cells. The endogenous TGF-beta1 expressed in neurons may play a role in the pathological process of DNA degradation and delayed neuronal death after transient forebrain ischemia.

Cytokine regulation of hyaluronate production by human Tenon's capsule fibroblasts.

PURPOSE: The high-molecular weight glycosaminoglycan hyaluronate (HA), a component of the extracellular matrix, has been shown to play important roles in many biological processes including cell proliferation, migration and differentiation. In the present study, the effect of cytokines on production of hyaluronate (HA) by human tenon's capsule fibroblasts (HTF) was determined. METHODS: HTF (2(nd) passage) were seeded at a cell density of 30 cells/mm(2) and stimulated by six different cytokines (platelet-derived growth factor (PDGF)-AA, PDGF-BB, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin (IL)-1beta and transforming growth factor (TGF)-beta1). Controls were treated with aliquots of serum-free medium only. Concentrations of HA were determined using a radiometric assay based on the specific binding of HA to HA binding proteins. RESULTS: The concentration of HA in conditioned medium of HTF was significantly increased only after stimulation with PDGF-AA [10 and 100ng/ml], IL-1beta [1 and 10ng/ml] and TGF-beta1 [5ng/ml]. CONCLUSIONS: Production of HA by HTF is regulated by PDGF-AA, IL-1beta and TGF-beta1 and is speculated to be involved in the wound healing reaction after glaucoma filtration surgery.

Transformation-dependent calcium influx by voltage-operated calcium channels in stellate cells of rat liver.

BACKGROUND/AIMS: The transformation of hepatic stellate cells into myofibroblasts is a key step in the pathogenesis of fibrotic liver diseases. The intracellular signaling associated with hepatic stellate cell transformation becomes a point of interest, especially the role of cytosolic free calcium concentration ([Ca2+]i). The aim of the study was to investigate possible differences between various transformation phenotypes of hepatic stellate cells with regard to the calcium influx mediated by L-type voltage-operated calcium channels (L-type VOC). METHODS: Hepatic stellate cells were isolated from rat liver by pronase-collagenase reperfusion and cultured under standard conditions. The transformation of hepatic stellate cells was stimulated by treatment with transforming growth factor-beta (TGF-beta) or inhibited with interferon-gamma (IFN-gamma) and characterized by immunocytochemistry for smooth muscle alpha-actin and determination of hyaluronan in the culture media with a ligand binding assay. [Ca2+]i was measured in individual cells with fluorescence microscopy using fura-2. VOCs were activated by the standard procedure of extracellular potassium elevation, to achieve depolarization, and identified by various controls. RESULTS: In transformed myofibroblasts the activation of VOCs by potassium elevation from 5.4 mmol/l to 50.4 mmol/l led to a 19% increase in [Ca2+]i in contrast to 0.2% in hepatic stellate cells cultured for 3 days. In 7-day old hepatic stellate cells, after stimulation of cell transformation with TGF-beta-1, an enhanced [Ca2+]i response to potassium elevation was detected, while inhibition of transformation with IFN-gamma for the same time caused a decreased calcium signal compared with untreated control cultures. Short-term treatment with the cytokines (1 day) did not influence depolarization-dependent calcium signals. CONCLUSION: The results show the [Ca2+]i increase via L-type VOCs to be dependent on the transformation level of hepatic stellate cells into myofibroblasts which can be influenced by the long-term treatment of hepatic stellate cells with TGF-beta or IFN-gamma. In contrast, there is no evidence for direct regulation of VOC activity by TGF-beta or IFN-gamma after short-term exposure.

Subcellular localization of (latent) transforming growth factor beta and the latent TGF-beta binding protein in rat hepatocytes and hepatic stellate cells.

Recently, the existence of the large latent transforming growth factor beta (TGF-beta) complex, consisting of TGF-beta, the N-terminal part of its precursor (latency-associated peptide [LAP]), and the latent TGF-beta binding protein (LTBP), was demonstrated in rat liver parenchymal cells (PC) and stellate cells (HSC). However, in contrast to HSC, in freshly isolated PC, no message of these proteins is detectable. This study was performed to investigate the subcellular distribution of the proteins forming the latent TGF-beta complex in PC and HSC from rat liver to obtain more information about their origin and potential intracellular functions. PC and HSC were isolated from rat liver by protease reperfusion and investigated for TGF-beta1,-2,-3, beta1-LAP, and LTBP-1 after cultivation using double-immunofluorescent staining, followed by high-resolution confocal microscopic analysis. Subcellular fractions obtained by standard differential centrifugation of rat liver homogenate were analyzed using a TGF-beta1 enzyme-linked immunosorbent assay (ELISA) and Western blotting for beta1-LAP and LTBP-1. By confocal microscopy, a diffuse distribution of TGF-beta and LAP in the cytoplasm of PC is noticed, whereas the LTBP immunostaining predominates at plasma membranes. In PC, distinct intracellular granules were superimposed with TGF-beta, LAP, and LTBP stainings identified as lysosomal compartments and mitochondria by ELISA and immunoblotting of subcellular fractions. In HSC, stainings of colocalized TGF-beta, LAP, and LTBP are strongest in the perinuclear area, indicating synthesis and secretion via endoplasmic reticulum and Golgi, respectively. Partially, the proteins were also found in HSC nuclei. During the transformation of HSC to myofibroblasts, LAP and LTBP become strongly colocalized with other components of the cytoskeletal network like smooth muscle--actin, desmin, and talin. The results confirm biochemical data about the existence and expression of the large latent TGF-beta complex in PC and HSC, respectively. Baseline information is provided from which new hypotheses regarding intracellular functions of TGF-beta, LAP, and LTBP in liver parenchymal and stellate cells can be concluded.