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Thus, inhibition of proteo(fibrino)lysis and augmented tissue factor expression in the heart precede and may contribute to the coronary perivascular fibrosis seen with obesity and insulin resistance. Furthermore, inhibition of ACE activity can attenuate all 3 phenomena.

Plasminogen activator inhibitor-1 (PAI-1) inhibits fibrinolysis and proteolysis. Basic fibroblast growth factor (bFGF) stimulates angiogenesis, which requires regional proteolysis. Because modulation of vasculopathy requires tight control of proteolysis, effects of bFGF on PAI-1 expression in endothelial cells (ECs) were characterized. bFGF increased PAI-1 mRNA and accumulation of PAI-1 protein in conditioned media in human umbilical vein ECs. The bFGF-mediated increase in PAI-1 mRNA was attenuated by inhibition of extracellular signal-regulated kinase kinase in human ECV304 cells. The rate of decrease in PAI-1 mRNA after actinomycin D treatment was not affected by bFGF. Transient transfection assays of the human PAI-1 promoter-luciferase construct demonstrated that bFGF-induced PAI-1 transcription was dependent on the elements within the -313 to -260 bp relative to the transcription start site. This region contains an E26 transformation specific 1 (Ets-1)-like site. Electrophoretic mobility shift assay showed that bFGF increased nuclear translocation or DNA binding of the Ets-1-like transcription factor to the PAI-1 promoter. Nucleotide substitution to disrupt the Ets-1-like site reduced bFGF-stimulated promoter activity. Fenofibric acid, an agonist ligand for the peroxisome proliferator-activated receptor-alpha, inhibited basal and bFGF-stimulated PAI-1 expression. By inducing PAI-1 expression from ECs, bFGF may control proteolysis and fibrinolysis in vessel walls.

The growth of bacteria is often enhanced by addition of carbon materials such as graphite or activated charcoal to the growth medium. In this work, bacterial strains that strictly require such carbon materials under the ordinarily lethal stress caused by high concentrations of salt were isolated. The organisms were gram-positive, spore-forming, sugar-nonfermenting aerobic bacilli and were provisionally designated ''Bacillus carbophilus'' Kasumi after examination of their phenotypic traits. The growth-and germination-promoting effects of graphite and activated charcoal were demonstrated either quantitatively on agar plates containing fine crystals of the carbon materials mixed with a nonpermissive growth medium or qualitatively on agar plates on nonpermissive growth media half-covered with fine carbon particles. Further experiments demonstrated a novel feature of the phenomenon; i.e., the ability to induce colony formation on the nonpermissive plate was transmissible through the air, as well as through plastic or glass barriers. The mechanism probably involves transmission of physical signals regulating cell growth.The unique biological effects of carbon materials, such as graphite and activated charcoal, have frequently been recognized: e.g., charcoal powders are often applied to the soil to enhance plant growth or used to facilitate the proliferation of bacteria such as Neisseria gonorrhoeae (4), Haemophilus pertussis (7), and Legionella pneumophila (3); artificial cardiac valves made of carbon displayed antithrombogenecity (1, 5); and carbon tooth implants displayed a favorable affinity to gingival tissue (10). It seems to be generally accepted that these carbon materials act indirectly on living organisms, e.g., by adsorbing inhibitory substances from the medium. We report here that carbon exerts a novel effect on the proliferation of at least some bacteria, involving an unexpected mechanism. MATERIALS AND METHODSCarbon and other materials. Graphite preparations were provided by the Toyo Carbon Co., Osaka, Japan (graphites A to C), and the Mitsubishi Pencil Co., Tokyo, Japan (graphites 1 to 4). Their sources and properties are listed in Table 1. Two activated-charcoal preparations, one washed with HCl and the other washed with HCl and then neutralized, were Sigma products. Aluminum oxide (Wako Pure Chemicals, Kyoto, Japan) and levigated alumina (Norton Co., Worcester, Mich.) were commercial products. Glass powders were prepared by grinding microscope slide coverslips in a ceramic mortar, washed repeatedly with water, and sterilized by autoclaving. Carbon materials scattered on agar plates were used after exhaustive sterilization by heating at about 700ЊC for 1 min on a gas burner. In other cases, they were mixed with nutrient agar and autoclaved at 120ЊC for 15 min.Bacteria and growth conditions. Isolation and preliminary characterization of bacteria with high graphite requirements are described below. These are Bacillus strains and are maintained in our collections (Tokai University and Fujisawa Pharmaceutic...

Hypertension is an important risk factor for coronary atherosclerosis, which is accelerated by inflammation and diminished fibrinolysis. We have previously shown that levels of plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of fibrinolysis, are increased with atherogenic metabolic derangement.Because the liver is one of the major sources of circulating PAI-1, we here examined the effects of two proin- major physiologic inhibitor of fibrinolysis, may play a pivotal role in the deposition of fibrin in the affected vessels. We have previously reported that circulating PAI-1 levels were increased in an animal model of atherogenic metabolic derangement (3). Liver is one of the major sources of circulating PAI-1 (4) and insulin stimulates PAI-1 synthesis in liver cells (5), suggesting that the insulin resistance and subsequent hyperinsulinemia typically seen in hypertensives may contribute to a prothrombotic risk state.

Objective-We sought to determine the etiologic mechanism of pleiotropic growth factor, hepatocyte growth factor (HGF), as a regulator of hepatic synthesis of plasminogen activator inhibitor (PAI)-1, the physiological inhibitor of fibrinolysis and a potential inducer of atherothrombosis. Methods and Results-HGF increased PAI-1 mRNA expression and PAI-1 protein accumulation in the conditioned media of human liver-derived HepG2 cells, and increased hepatic PAI-1 mRNA expression in vivo in mice. HGF-inducible PAI-1 mRNA was attenuated by U0126, a specific inhibitor of mitogen-activated protein kinase (MAPK) kinase, and genistein, an inhibitor of tyrosine kinase. HGF increased the human PAI-1 promoter (Ϫ829 to ϩ36 bp) activity, and deletion and mutation analysis uncovered a functional E box (5Ј-CACATG-3Ј) at positions Ϫ158 to Ϫ153 bp. Electrophoretic mobility shift assays demonstrated that this E box binds upstream stimulatory factors (USFs). HGF phosphorylated USFs through MAPK and tyrosine kinase pathways. Co-transfection of USF1 expression vector increased PAI-1 promoter activity. Sterol regulatory element-binding protein-1 attenuated HGF-inducible PAI-1 promoter activity. Conclusions-Because

Obesity is associated with an increased risk of atherosclerotic coronary artery disease. Cytokines and oxygen-centered free radicals implicated in insulin resistance stimulate adipocyte and endothelial production of plasminogen activator inhibitor type-1 (PAI-1), the primary physiologic inhibitor of fibrinolysis, in vitro. In obese hyperinsulinemic animal models simulating insulin resistance, plasma PAI-1 activity is increased. As the cardiovascular risk profile in specific populations may differ, endogenous fibrinolysis in lean and obese subjects was characterized and the mechanisms underlying differences were identified. Obese subjects (body mass index > 26) exhibited increased blood levels of PAI-1 antigen compared with corresponding values in lean controls. Blood t-PA antigen differed as well, yet basal endogenous fibrinolytic activity was decreased because of the high PAI-1 activity. The increased PAI-1 level was associated with increased levels of immunoreactive insulin (IRI). In diabetic subjects, coronary atherectomy specimens exhibited strong positive PAI-1 immunostaining, suggesting that in the diabetic vascular wall, intramural fibrinolytic activity is diminished. Using the oral glucose tolerance test, patients with significant stenosis confirmed by coronary angiography exhibited increased sigmaIRI, sigmaBS, sigmaIRI/sigmaBS, and IRI at 120 min compared to subjects without significant stenosis. IRI at 120 min was closely correlated with the severity of coronary artery disease. These results indicate that adipocyte overproduction of PAI-1 by insulin induces decreased endogenous fibrinolytic activity and contributes to the accelerated coronary macroangiopathy in hyperinsulinemic obese subjects with insulin resistance.

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