Casein kinase I (CKI) is an essential component of the biological clock, phosphorylating PER proteins, and in doing so regulating their turnover and nuclear entry in oscillator cells of the suprachiasmatic nucleus (SCN). Although hereditary decreases in PER phosphorylation have been well characterized, little is known about the consequences of acute enzyme inhibition by pharmacological means. A novel reagent, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine (PF-670462), proved to be both a potent (IC 50 ϭ 7.7 Ϯ 2.2 nM) and selective (Ͼ30-fold with respect to 42 additional kinases) inhibitor of CKI in isolated enzyme preparations; in transfected whole cell assays, it caused a concentrationrelated redistribution of nuclear versus cytosolic PER. When tested in free-running animals, 50 mg/kg s.c. PF-670462 produced robust phase delays when dosed at circadian time (CT)9 (Ϫ1.97 Ϯ 0.17 h). Entrained rats dosed in normal light-dark (LD) and then released to constant darkness also experienced phase delays that were dose-and time of dosing-dependent. PF-670462 yielded only phase delays across the circadian cycle with the most sensitive time at CT12 when PER levels are near their peak in the SCN. Most importantly, these druginduced phase delays persisted in animals entrained and maintained in LD throughout the entire experiment; re-entrainment to the prevailing LD required days in contrast to the rapid elimination of the drug (t 1/2 ϭ 0.46 Ϯ 0.04 h). Together, these results suggest that inhibition of CKI yields a perturbation of oscillator function that forestalls light as a zeitgeber, and they demonstrate that pharmacological tools such as PF-670462 may yield valuable insight into clock function.Circadian behavior is mediated by a timed sequence of intracellular events, genomic in nature, occurring in mammals within so-called pacemaker cells of the suprachiasmatic nucleus (SCN) (Antle and Silver, 2005). Here, rhythmicity relies on a common theme of precisely regulated gene transcription and translation as a means to perpetuate cycling, and in doing so, to dictate the timing of downstream events (Reppert and Weaver, 2001; for review, see Schibler, 2006, among others). Clock-related proteins rise and fall in concentration as a consequence of nuclear and cytosolic feedback loops, adjusted daily to maintain reproducible cycling timed at roughly 24-h intervals. Light is the primary zeitgeber, or "time-giver", in this process, and its actions on loop dynamics are thought to comprise the most important external influence on pacemaker function.In its simplest conception, the oscillations of the clock can run using four genes, Period (Per1, Per2) and Cryptochrome (Cry1, Cry2), that are activated in early circadian day by heterodimeric complexes of CLOCK and BMAL1 (Griffin et al., 1999;Kume et al., 1999;Cermakian and Boivin, 2003). Translated PER partners in the cytoplasm with CRY, the resultant heterodimer, readily gaining nuclear entry and opposing activation by Clock and BMAL1. A feedback loop is...

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common proangiogenic factors [PAF; VEGF-A and basic fibroblast growth factor (bFGF)]. We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (EC). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed Matrigel angiogenesis stimulated by PAFs and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Antiangiogenesis and antitumor efficacy were associated with disrupted co-localization of ECs with desmin þ perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current antiangiogenic modalities in the clinic.

The results from this study indicate that the combination of sunitinib and PF-562,271 TKI has the potential to target different aspects of angiogenesis and tumor aggressiveness and may have significantly greater effect than relevant single agent, blocking not only tumor growth, but also impacting the ability of the tumor to recover upon withdrawal of the therapy.

The lymphatic vasculature is involved in the pathogenesis of acute cardiac injuries, but little is known about its role in chronic cardiac dysfunction. Here, we demonstrate that angiotensin II infusion induced cardiac inflammation and fibrosis at 1 week and caused cardiac dysfunction and impaired lymphatic transport at 6 weeks in mice, while co-administration of VEGFCc156s improved these parameters. To identify novel mechanisms underlying this protection, RNA sequencing analysis in distinct cell populations revealed that VEGFCc156s specifically modulated angiotensin II-induced inflammatory responses in cardiac and peripheral lymphatic endothelial cells. Furthermore, telemetry studies showed that while angiotensin II increased blood pressure acutely in all animals, VEGFCc156s-treated animals displayed a delayed systemic reduction in blood pressure independent of alterations in angiotensin II-mediated aortic stiffness. Overall, these results demonstrate that VEGFCc156s had a multifaceted therapeutic effect to prevent angiotensin II-induced cardiac dysfunction by improving cardiac lymphatic function, alleviating fibrosis and inflammation, and ameliorating hypertension.

These findings suggest that the time of dosing, when held constant in both the monkey and rat studies, produced nearly identical effects despite the subjects' diurnal or nocturnal preference. Importantly, these changes in rhythm occurred in the presence of light, revealing the drug as a powerful zeitgeber in a non-human primate and, by extension, in man.

The magnitude of the shifts in activity onsets achieved with chronic dosing of PF-670462 appears to be a function of the dose and the previously established phase relationship. Its cumulative effect further suggests that the pharmacodynamic t (1/2) of the drug greatly exceeds its pharmacokinetic one. Most importantly, these changes in circadian behavior occurred in the presence of a fixed L:D cycle, confirming the drug to be a robust modulator of circadian phase in the presence of the natural zeitgeber.

No abstract

The ultrasound biomicroscope is an effective tool for the real-time characterization of the structure and function of embryo/fetal rat hearts. DMO causes significant deficits to in utero heart function for up to ten days (GD 21) following its final administration, suggesting long-term or possible permanent changes cardiac function.