Erythropoietin (Epo) is produced by renal Epo-producing cells (REPs) in a hypoxia-inducible manner. The conversion of REPs into myofibroblasts and coincident loss of Epo-producing ability are the major cause of renal fibrosis and anemia. However, the hypoxic response of these transformed myofibroblasts remains unclear. Here, we used complementary in vivo transgenic and live imaging approaches to better understand the importance of hypoxia signaling in Epo production. Live imaging of REPs in transgenic mice expressing green fluorescent protein from a modified Epo-gene locus revealed that healthy REPs tightly associated with endothelium by wrapping processes around capillaries. However, this association was hampered in states of renal injury-induced inflammation previously shown to correlate with the transition to myofibroblast-transformed renal Epo-producing cells (MF-REPs). Furthermore, activation of hypoxia-inducible factors (HIFs) by genetic inactivation of HIF-prolyl hydroxylases (PHD1, PHD2, and PHD3) selectively in Epo-producing cells reactivated Epo production in MF-REPs. Loss of PHD2 in REPs restored Epo-gene expression in injured kidneys but caused polycythemia. Notably, combined deletions of PHD1 and PHD3 prevented loss of Epo expression without provoking polycythemia. Mice with PHD-deficient REPs also showed resistance to LPS-induced Epo repression in kidneys, suggesting that augmented HIF signaling counterbalances inflammatory stimuli in regulation of Epo production. Thus, augmentation of HIF signaling may be an attractive therapeutic strategy for treating renal anemia by reactivating Epo synthesis in MF-REPs.

Inhibition of plasminogen activator inhibitor (PAI)-1 is useful to treat several disorders including thrombosis. An inhibitor of PAI-1 (TM5275) was newly identified by an extensive study of structure-activity relationship based on a lead compound (TM5007) which was obtained through virtual screening by docking simulations. Its antithrombotic efficacy and adverse effects were tested in vivo in rats and nonhuman primates (cynomolgus monkey). TM5275, administered orally in rats (1 to 10 mg/kg), has an antithrombotic effect equivalent to that of ticlopidine (500 mg/kg) in an arterial venous shunt thrombosis model and to that of clopidogrel (3 mg/kg) in a ferric chloride-treated carotid artery thrombosis model. TM5275 does not modify activated partial thromboplastin time and prothrombin time or platelet activity and does not prolong bleeding time. Combined with tissue plasminogen activator, TM5275 improves the latter's therapeutic efficacy and reduces its adverse effect. Administered to a monkey model of photochemical induced arterial thrombosis, TM5275 (10 mg/kg) has the same antithrombotic effect as clopidogrel (10 mg/kg), without enhanced bleeding. This study documents the antithrombotic benefits of a novel, more powerful, PAI-1 inhibitor in rats and, for the first time, in nonhuman primates. These effects are obtained without adverse effect on bleeding time.

f Erythropoietin (Epo) is produced in the kidney and liver in a hypoxia-inducible manner via the activation of hypoxia-inducible transcription factors (HIFs) to maintain oxygen homeostasis. Accelerating Epo production in hepatocytes is one plausible therapeutic strategy for treating anemia caused by kidney diseases. To elucidate the regulatory mechanisms of hepatic Epo production, we analyzed mouse lines harboring liver-specific deletions of genes encoding HIF-prolyl-hydroxylase isoforms (PHD1, PHD2, and PHD3) that mediate the inactivation of HIF1␣ and HIF2␣ under normal oxygen conditions. The loss of all PHD isoforms results in both polycythemia, which is caused by Epo overproduction, and fatty livers. We found that deleting any combination of two PHD isoforms induces polycythemia without steatosis complications, whereas the deletion of a single isoform induces no apparent phenotype. Polycythemia is prevented by the loss of either HIF2␣ or the hepatocyte-specific Epo gene enhancer (EpoHE). Chromatin analyses show that the histones around EpoHE dissociate from the nucleosome structure after HIF2␣ activation. HIF2␣ also induces the expression of HIF3␣, which is involved in the attenuation of Epo production. These results demonstrate that the total amount of PHD activity is more important than the specific function of each isoform for hepatic Epo expression regulated by a PHD-HIF2␣-EpoHE cascade in vivo.

Plasminogen activator inhibitor-1 (PAI-1), an endogenous inhibitor of a major fibrinolytic factor, tissue-type plasminogen activator, can both promote and inhibit angiogenesis. However, the physiologic role and the precise mechanisms underlying the angiogenic effects of PAI-1 remain unclear. In the present study, we report that pharmacologic inhibition of PAI-1 promoted angiogenesis and prevented tissue necrosis in a mouse model of hind-limb ischemia. Improved tissue regeneration was due to an expansion of circulating and tissue-resident granulocyte-1 marker (Gr-1 ؉ ) neutrophils and to increased release of the angiogenic factor VEGF-A, the hematopoietic growth factor kit ligand, and G-CSF. Immunohistochemical analysis indicated increased amounts of fibroblast growth factor-2 (FGF-2) in ischemic gastrocnemius muscle tissues of PAI-1 inhibitor-treated animals. Ab neutralization and genetic knockout studies indicated that both the improved tissue regeneration and the increase in circulating and ischemic tissue-resident Gr-1 ؉ neutrophils depended on the activation of tissuetype plasminogen activator and matrix metalloproteinase-9 and on VEGF-A and FGF-2. These results suggest that pharmacologic PAI-1 inhibition activates the proangiogenic FGF-2 and VEGF-A pathways, which orchestrates neutrophil-driven angiogenesis and induces cell-driven revascularization and is therefore a potential therapy for ischemic diseases. IntroductionApproximately 500 to 1000 people per million per year are diagnosed with critical ischemia of the limb, which in most cases results in serious morbidity and mortality. Therapeutic restoration of blood flow by, for example, the induction of the formation of new capillaries (angiogenesis) is the ultimate goal for critical limb ischemia patients. Growth of new blood vessels in the adult occurs through angiogenesis or arteriogenesis (vessel maturation via recruitment of smooth muscle cells) and vasculogenesis (mobilization of BM-derived cells). 1,2 In contrast to promising results from animal studies, administration of proangiogenic factors such as fibroblast growth factor 2 (FGF-2, also known as basic FGF) or VEGF-A failed to induce significant improvement in ischemia in several phase 1 clinical trials. 3 The plasminogen activation system and matrix metalloproteinases (MMPs), which can cleave growth factors, growth factor receptors, and adhesion molecules and mediate the extracellular matrix degradation that is necessary for cell migration, are widely recognized as being involved in the process of angiogenesis. 2,4 Although plasminogen activator inhibitor-1 (PAI-1) is one of the primary regulators of the fibrinolytic system, it also has dramatic effects on cell adhesion, detachment, and migration 5 and can inhibit cellular migration by affecting cell adhesion. 6,7 PAI-1-deficient (PAI-1 Ϫ/Ϫ ) mice showed improved vascular wound healing in models of perivascular electric or transluminal mechanical injury 8 due to improved migration of PAI-1 Ϫ/Ϫ smooth muscle cells. The 52-kDa serine protease inh...

SUMMARY 3To find an optimal range of urate concentrations wherein the risk of attacks during the initial 6 months of treatment is minimized, data from 350 gouty patients treated with antihyperuricemic drugs were retrospectively analyzed. We determined the optimal range of urate concentrations to be 4.fr--{).6 mg/dl. If urate concentrations were within this range, the risk ratio of an attack as opposed to outside of the range was 0.705 (95% confidence interval, 0.629---D. 791). The increase (or decrease) in urate concentration in one month associated with minimal risk of gouty attacks was also determined. The lowest risk ratio of attack (0.451) occurred at a range of -0.1 to 0.6 mg/dllmonth increase in urate concentrations (95% confidence interval, 0.31 O-D.655). In conclusion, we propose that urate concentrations during the initial 6 months of antihyperuricemic therapy should be maintained within a range of 4.fr--{).6 mg/dl, and reduction in the urate concentrations during treatment should be as slow as possible.