A Small Molecule Inhibitor of Plasminogen Activator Inhibitor-1 Reduces Brain Amyloid-ß Load and Improves Memory in an Animal Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a major cause of dementia in the elderly with no effective treatment. Accumulation of amyloid-ß peptide (Aß) in the brain is a pathological hallmark of AD and is believed to be a central disease-causing and disease-promoting event. In a previous study, we showed that deletion of plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of tissue type and urokinase type plasminogen activators (tPA and uPA), significantly reduced brain Aß load in APP/PS1 mice, an animal model of familial AD. In this study, we further show that oral administration of TM5275, a small molecule inhibitor of PAI-1, for a period of 6 weeks, inhibits the activity of PAI-1 and increases the activities of tPA and uPA as well as plasmin, which is associated with a reduction of Aß load in the hippocampus and cortex and improvement of learning/memory function in APP/PS1 mice. Protein abundance of low density lipoprotein related protein-1 (LRP-1), a multi ligand endocytotic receptor involved in transporting Aß out of the brain, as well as plasma Aß42 are increased, whereas the expression and processing of full-length amyloid-ß protein precursor is not affected by TM5275 treatment in APP/PS1 mice. In vitro studies further show that PAI-1 increases, whereas TM5275 reduces, Aß40 level in the culture medium of SHSY5Y-APP neuroblastoma cells. Collectively, our data suggest that TM5275 improves memory function of APP/PS1 mice, probably by reducing brain Aß accumulation through increasing plasmin-mediated degradation and LRP-1-mediated efflux of Aß in the brain.

Plasminogen activator inhibitor 1, fibroblast apoptosis resistance, and aging-related susceptibility to lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disorder with unknown cause and no effective treatment. The incidence of and mortality from IPF increase with age, suggesting that advanced age is a major risk factor for IPF. The mechanism underlying the increased susceptibility of the elderly to IPF, however, is unknown. In this study, we show for the first time that the protein level of plasminogen activator inhibitor 1 (PAI-1), a protease inhibitor which plays an essential role in the control of fibrinolysis, was significantly increased with age in mouse lung homogenate and lung fibroblasts. Upon bleomycin challenge, old mice experienced augmented PAI-1 induction and lung fibrosis as compared to young mice. Most interestingly, we show that fewer (myo)fibroblasts underwent apoptosis and more (myo)fibroblasts with increased level of PAI-1 accumulated in the lung of old than in young mice after bleomycin challenge. In vitro studies further demonstrate that fibroblasts isolated from lungs of old mice were resistant to H2O2 and tumor necrosis factor alpha-induced apoptosis and had augmented fibrotic responses to TGF-ß1, compared to fibroblasts isolated from young mice. Inhibition of PAI-1 activity with a PAI-1 inhibitor, on the other hand, eliminated the aging-related apoptosis resistance and TGF-ß1 sensitivity in isolated fibroblasts. Moreover, we show that knocking down PAI-1 in human lung fibroblasts with PAI-1 siRNA significantly increased their sensitivity to apoptosis and inhibited their responses to TGF-ß1. Together, the results suggest that increased PAI-1 expression may underlie the aging-related sensitivity to lung fibrosis in part by protecting fibroblasts from apoptosis.

Transforming growth factor ß suppresses glutamate-cysteine ligase gene expression and induces oxidative stress in a lung fibrosis model.

The concentration of glutathione (GSH), the most abundant intracellular free thiol and an important antioxidant, is decreased in the lung in both fibrotic diseases and experimental fibrosis models. The underlying mechanisms and biological significance of GSH depletion, however, remain unclear. Transforming growth factor ß (TGF-ß) is the most potent and ubiquitous profibrogenic cytokine and its expression is increased in almost all fibrotic diseases. In this study, we show that increasing TGF-ß1 expression in mouse lung to a level comparable to those found in lung fibrotic diseases by intranasal instillation of AdTGF-ß1(223/225), an adenovirus expressing constitutively active TGF-ß1, suppressed the expression of both catalytic and modifier subunits of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in de novo GSH synthesis, decreased GSH concentration, and increased protein and lipid peroxidation in mouse lung. Furthermore, we show that increasing TGF-ß1 expression activated JNK and induced activating transcription factor 3, a transcriptional repressor involved in the regulation of the catalytic subunit of GCL, in mouse lung. Control virus (AdDL70-3) had no significant effect on any of these parameters, compared to saline-treated control. Concurrent with GSH depletion, TGF-ß1 induced lung epithelial apoptosis and robust pulmonary fibrosis. Importantly, lung GSH levels returned to normal, whereas fibrosis persisted at least 21 days after TGF-ß1 instillation. Together, the data suggest that increased TGF-ß1 expression may contribute to the GSH depletion observed in pulmonary fibrosis diseases and that GSH depletion may be an early event in, rather than a consequence of, fibrosis development.

Therapeutic value of small molecule inhibitor to plasminogen activator inhibitor-1 for lung fibrosis.

Fibrosis is a final stage of many lung diseases, with no effective treatment. Plasminogen activator inhibitor-1 (PAI-1), a primary inhibitor of tissue-type and urokinase-type plasminogen activators (tPA and uPA, respectively), plays a critical role in the development of fibrosis. In this study, we explored the therapeutic potential of an orally effective small molecule PAI-1 inhibitor, TM5275, in a model of lung fibrosis induced by transforming growth factor-ß1 (TGF-ß1), the most potent and ubiquitous profibrogenic cytokine, and in human lung fibroblasts (CCL-210 cells). The results show that an intranasal instillation of AdTGF-ß1(223/225), an adenovirus expressing constitutively active TGF-ß1, increased the expression of PAI-1 and induced fibrosis in murine lung tissue. On the other hand, treating mice with 40 mg/kg of TM5275 for 10 days, starting 4 days after the instillation of AdTGF-ß1(223/225), restored the activities of uPA and tPA and almost completely blocked TGF-ß1-induced lung fibrosis, as shown by collagen staining, Western blotting, and the measurement of hydroxyproline. No loss of body weight was evident under these treatment conditions with TM5275. Furthermore, we show that TM5275 induced apoptosis in both myofibroblasts (TGF-ß1-treated) and naive (TGF-ß1-untreated) human lung fibroblasts, and this apoptosis was associated with the activation of caspase-3/7, the induction of p53, and the inhibition of α-smooth muscle actin, fibronectin, and PAI-1 expression. Such an inhibition of fibrotic responses by TM5275 occurred even in cells pretreated with TGF-ß1 for 6 hours. Together, the results suggest that TM5275 is a relatively safe and potent antifibrotic agent, with therapeutic potential in fibrotic lung disease.