Light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. In the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (SCN)-sympathetic nervous system. Moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamo-adenohypophysial axis. The abolishment after SCN lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. The magnitude of corticostereone response is dose dependently correlated with the light intensity. The light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment.

It has been proposed that hematopoietic and endothelial cells are derived from a common cell, the hemangioblast. In this study, we demonstrate that a subset of CD34+ cells have the capacity to differentiate into endothelial cells in vitro in the presence of basic fibroblast growth factor, insulin-like growth factor-1, and vascular endothelial growth factor. These differentiated endothelial cells are CD34+, stain for von Willebrand factor (vWF), and incorporate acetylated low-density lipoprotein (LDL). This suggests the possible existence of a bone marrow-derived precursor endothelial cell. To demonstrate this phenomenon in vivo, we used a canine bone marrow transplantation model, in which the marrow cells from the donor and recipient are genetically distinct. Between 6 to 8 months after transplantation, a Dacron graft, made impervious to prevent capillary ingrowth from the surrounding perigraft tissue, was implanted in the descending thoracic aorta. After 12 weeks, the graft was retrieved, and cells with endothelial morphology were identified by silver nitrate staining. Using the di(CA)n and tetranucleotide (GAAA)n repeat polymorphisms to distinguish between the donor and recipient DNA, we observed that only donor alleles were detected in DNA from positively stained cells on the impervious Dacron graft. These results strongly suggest that a subset of CD34+ cells localized in the bone marrow can be mobilized to the peripheral circulation and can colonize endothelial flow surfaces of vascular prostheses.

It has been proposed that hematopoietic and endothelial cells are derived from a common cell, the hemangioblast. In this study, we demonstrate that a subset of CD34+ cells have the capacity to differentiate into endothelial cells in vitro in the presence of basic fibroblast growth factor, insulin-like growth factor-1, and vascular endothelial growth factor. These differentiated endothelial cells are CD34+, stain for von Willebrand factor (vWF), and incorporate acetylated low-density lipoprotein (LDL). This suggests the possible existence of a bone marrow-derived precursor endothelial cell. To demonstrate this phenomenon in vivo, we used a canine bone marrow transplantation model, in which the marrow cells from the donor and recipient are genetically distinct. Between 6 to 8 months after transplantation, a Dacron graft, made impervious to prevent capillary ingrowth from the surrounding perigraft tissue, was implanted in the descending thoracic aorta. After 12 weeks, the graft was retrieved, and cells with endothelial morphology were identified by silver nitrate staining. Using the di(CA)n and tetranucleotide (GAAA)n repeat polymorphisms to distinguish between the donor and recipient DNA, we observed that only donor alleles were detected in DNA from positively stained cells on the impervious Dacron graft. These results strongly suggest that a subset of CD34+ cells localized in the bone marrow can be mobilized to the peripheral circulation and can colonize endothelial flow surfaces of vascular prostheses.

Abstract-Interactions between integrins and growth factor receptors play a critical role in the development and healing of the vasculature. This study mapped two binding domains on fibronectin (FN) that modulate the activity of the angiogenic factor, vascular endothelial growth factor (VEGF T he growth, repair, and regeneration of blood vessels are complex processes that involve coordinated regulation of endothelial cell proliferation, migration, and differentiation. 1 One of the most important vascular morphogens is vascular endothelial growth factor (VEGF). VEGF has been shown to play a major role in vasculogenesis and angiogenesis by gene deletion studies. 2,3 Targeted disruption of the VEGF receptor Flk-1 (VEGFR-2) in mice resulted in failure of blood-island formation and endothelial differentiation. 4 Flk-1 is also the first endothelial receptor tyrosine kinase to be expressed in the hemangioblast. 5 We and others recently demonstrated that the hematopoietic progenitor cell CD34 ϩ can differentiate into endothelial cells, and that VEGF was one of the critical factors promoting this differentiation. 6,7 Interactions between cells and their extracellular matrix (ECM) play an integral role in blood vessel development. The earliest ECM protein expressed in the embryo during vasculogenesis is fibronectin (FN). 8 Gene deletion studies have demonstrated that both FN and its major integrin receptor, ␣ 5 ␤ 1 , are critical for vasculogenesis and angiogenesis in the developing embryo. 9 -11 Collectively, these observations suggest important roles for FN and its integrin receptor, ␣ 5 ␤ 1 , in vasculogenesis and angiogenesis.In this study, we show that novel VEGF binding domains of FN are required for promoting the specific association of the FN receptor integrin ␣ 5 ␤ 1 with the VEGF receptor, Flk-1. This association between VEGF and FN is required for the full effects of VEGF-induced endothelial cell migration and proliferation. This study demonstrates that FN can profoundly affect VEGF biological activity and consequently the behavior of endothelial cells through their coordinated effects on Flk-1 and ␣ 5 ␤ 1 . Materials and Methods Solid-Phase VEGF Binding AssayECM proteins and FN peptides were purchased from Sigma and Gibco and were purified further by gel filtration and ion exchange chromatography. Microtiter plates were coated with the appropriate ECM proteins (50 L; 10 g/mL) in 100 mmol/L bicarbonate buffer (pH 9) overnight at 4°C.

Serine/threonine kinase Akt is thought to mediate many biological actions toward anti-apoptotic responses. Screening of drugs that could interfere with the Akt signaling pathway revealed that Hsp90 inhibitors (e.g. geldanamycin, radicicol, and its analogues) induced Akt dephosphorylation, which resulted in Akt inactivation and apoptosis of the cells. Hsp90 inhibitors did not directly affect Akt kinase activity in vitro. Thus, we examined the effects of Hsp90 inhibitors on upstream Akt kinases, phosphatidylinositide-3-OH kinase (PI3K) and 3-phosphoinositide-dependent protein kinase-1 (PDK1). Hsp90 inhibitors had no effect on PI3K protein expression. In contrast, treatment of the cells with Hsp90 inhibitors decreased the amount of PDK1 without directly inhibiting PDK1 kinase activity. We found that the kinase domain of PDK1 was essential for complex formation with Hsp90 and that Hsp90 inhibitors suppressed PDK1 binding to Hsp90. PDK1 degradation mechanisms revealed that inhibition of PDK1 binding to Hsp90 caused proteasome-dependent degradation of PDK1. Treatment of proteasome inhibitors increased the amount of detergent-insoluble PDK1 in Hsp90 inhibitor-treated cells. Therefore, the association of PDK1 with Hsp90 regulates its stability, solubility, and signaling. Because Akt binding to Hsp90 is also involved in the maintenance of Akt kinase activity, Hsp90 plays an important role in PDK1-Akt survival signaling pathway.The characterization of the survival signal transduction pathways stimulated by growth factors and cytokines has revealed that phosphatidylinositide-3-OH kinase (PI3K) 1 is involved in the pathway (1-3). PI3K is a heterodimeric lipid kinase, which consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. After stimulation with growth factors and cytokines, PI3K is activated by the interaction of the 85-kDa subunit with phosphotyrosines of activated intracellular domain of growth factor receptors or with the receptorassociated adapter proteins. Subsequently, PI3K associates with the plasma membrane where the 110-kDa catalytic subunit phosphorylates phosphoinositides. The generated phospholipid second messenger molecule, phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P 3 ), raises a diverse set of cellular responses (4 -6). The major targets of PtdIns(3,4,5)P 3 are pleckstrin homology (PH) domain-containing proteins, including serine/threonine kinase Akt (also known as protein kinase B (PKB) or RAC-PK) (7, 8).The interaction of PtdIns(3,4,5)P 3 with the PH domain of Akt recruits Akt to the plasma membrane, where it is phosphorylated at two key regulatory residue sites, Thr 308 and Ser 473 . Phosphorylation at both residues is necessary for full activation of Akt and the subsequent regulation of many PI3K-regulated biological responses, including glucose uptake, protein synthesis, and apoptosis inhibition (7,8). Akt phosphorylation at Thr 308 is catalyzed by the ubiquitously expressed 3-phosphoinositide-dependent protein kinase-1 (PDK1) (9 -11). The kinase responsible for phosph...

Synchronous oscillations of thousands of cellular clocks in the suprachiasmatic nucleus (SCN), the circadian centre, are coordinated by precisely timed cell–cell communication, the principle of which is largely unknown. Here we show that the amount of RGS16 (regulator of G protein signalling 16), a protein known to inactivate Gαi, increases at a selective circadian time to allow time-dependent activation of intracellular cyclic AMP signalling in the SCN. Gene ablation of Rgs16 leads to the loss of circadian production of cAMP and as a result lengthens circadian period of behavioural rhythm. The temporally precise regulation of the cAMP signal by clock-controlled RGS16 is needed for the dorsomedial SCN to maintain a normal phase-relationship to the ventrolateral SCN. Thus, RGS16-dependent temporal regulation of intracellular G protein signalling coordinates the intercellular synchrony of SCN pacemaker neurons and thereby defines the 24 h rhythm in behaviour.

Vascular Endothelial Growth Factor (VEGF) has been typically considered to be an endothelial-specific growth factor. However, it was recently demonstrated that VEGF can interact with non endothelial cells. In this study, we tested whether vascular smooth muscles cells (VSMCs) can express VEGF receptors, such as flk-1, flt-1, and neuropilin (NP)-1, and respond to VEGF in vitro. In cultured VSMCs, flk-1 and flt-1 expression was inversely related to cell density. The expression of flk-1 was down-regulated with increasing passage numbers. However, NP-1 levels were not affected by cell density or passage numbers. Flk-1, Flt-1, and NP-1 protein levels were confirmed by Western Blotting. Although the functional mature form of Flk-1 protein is expressed at low levels in VSMCs, phosphorylation of Flk-1 following VEGF(165) stimulation was still observed. SMCs migrated significantly in response to VEGF(165) and VEGF-E, whereas Placenta Growth Factor (PlGF) induced migration only at higher concentrations. Since VEGF-E is a specific activator of flk-1 while PlGF specifically activates only flt-1, SMC migration induced by VEGF(165) is likely to be mediated primarily through the flk-1 receptor. VSMCs did not significantly proliferate in response to VEGF(165), PlGF, and VEGF-E. In conclusion, our studies demonstrate the presence of VEGF receptors on VSMCs that are functional. These studies also indicate that in vivo, VEGF may play a role in modulating the response of VSMCs.

Advanced age was a major risk factor for postoperative AF. Furthermore, inflammatory response induced by surgical trauma was also associated with the development of AF after off-pump CABG.