Nitric oxide (NO), the smallest signalling molecule known, is produced by three isoforms of NO synthase (NOS; EC 1.14.13.39). They all utilize l-arginine and molecular oxygen as substrates and require the cofactors reduced nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and (6R-)5,6,7,8-tetrahydrobiopterin (BH(4)). All NOS bind calmodulin and contain haem. Neuronal NOS (nNOS, NOS I) is constitutively expressed in central and peripheral neurons and some other cell types. Its functions include synaptic plasticity in the central nervous system (CNS), central regulation of blood pressure, smooth muscle relaxation, and vasodilatation via peripheral nitrergic nerves. Nitrergic nerves are of particular importance in the relaxation of corpus cavernosum and penile erection. Phosphodiesterase 5 inhibitors (sildenafil, vardenafil, and tadalafil) require at least a residual nNOS activity for their action. Inducible NOS (NOS II) can be expressed in many cell types in response to lipopolysaccharide, cytokines, or other agents. Inducible NOS generates large amounts of NO that have cytostatic effects on parasitic target cells. Inducible NOS contributes to the pathophysiology of inflammatory diseases and septic shock. Endothelial NOS (eNOS, NOS III) is mostly expressed in endothelial cells. It keeps blood vessels dilated, controls blood pressure, and has numerous other vasoprotective and anti-atherosclerotic effects. Many cardiovascular risk factors lead to oxidative stress, eNOS uncoupling, and endothelial dysfunction in the vasculature. Pharmacologically, vascular oxidative stress can be reduced and eNOS functionality restored with renin- and angiotensin-converting enzyme-inhibitors, with angiotensin receptor blockers, and with statins.

Inflammation is usually analysed from the perspective of tissue-infiltrating leukocytes. Microvascular endothelial cells at a site of inflammation are both active participants in and regulators of inflammatory processes. The properties of endothelial cells change during the transition from acute to chronic inflammation and during the transition from innate to adaptive immunity. Mediators that act on endothelial cells also act on leukocytes and vice versa. Consequently, many anti-inflammatory therapies influence the behaviour of endothelial cells and vascular therapeutics influence inflammation. This Review describes the functions performed by endothelial cells at each stage of the inflammatory process, emphasizing the principal mediators and signalling pathways involved and the therapeutic implications.

Abstract-Dicer is a key enzyme involved in the maturation of microRNAS (miRNAs). miRNAs have been shown to be regulators of gene expression participating in the control of a wide range of physiological pathways. To assess the role of Dicer and consequently the importance of miRNAs in the biology and functions of human endothelial cells (EC) during angiogenesis, we globally reduced miRNAs in ECs by specific silencing Dicer using siRNA and examined the effects on EC phenotypes in vitro. The knockdown of Dicer in ECs altered the expression (mRNA and/or protein) of several key regulators of endothelial biology and angiogenesis, such as TEK/Tie-2, KDR/VEGFR2, Tie-1, endothelial nitric oxide synthase and IL-8. Although, Dicer knockdown increased activation of the endothelial nitric oxide synthase pathway it reduced proliferation and cord formation of EC in vitro. The miRNA expression profile of EC revealed 25 highly expressed miRNAs in human EC and using miRNA mimicry, miR-222/221 regulates endothelial nitric oxide synthase protein levels after Dicer silencing. Collectively, these results indicate that maintenance and regulation of endogenous miRNA levels via Dicer mediated processing is critical for EC gene expression and functions in vitro. (Circ Res. 2007;100:1164-1173.)Key Words: endothelium Ⅲ Dicer Ⅲ miRNA Ⅲ angiogenesis M icroRNAs (miRNAs) are short noncoding RNAs that have been identified in a variety of organisms and have been shown to regulate gene expression. 1,2 In mammalian cells, these small RNAs (Ϸ22 nt) are transcribed as parts of longer molecules that are processed in the nucleus into hairpins RNAs by the protein Drosha. 3,4 These premiRNAs are then transported to the cytoplasm, via an exportin 5-dependent mechanism, where they are digested by a second, doubled-stranded specific ribonuclease called Dicer. 5,6 The mature miRNAs are incorporated into a ribonucleoprotein complex 7,8 or RISC complex,9 that mediates the downregulation of target gene activity by translational inhibition or target mRNA degradation, resulting in reduced levels of the corresponding protein or transcript, respectively. 7,10,11 miRNAs have been implicated in the control of a wide range of physiological pathways 12,13 such as development, differentiation, growth and metabolism. 14 -17 Moreover, tissue-specific patterns of miRNAs are providing insights into their possible functions. Many miRNAs exhibit striking organ specific expression patterns, or even expression restricted to single tissue layer within an organ 18 and different miRNAs have been specifically cloned from heart, brain, embryonic stem cells and pancreatic islet cells. 19 -23 To dissect the significance of miRNAs in mammalian biology, several groups have disrupted the Dicer gene in mice 24,25 and the loss of Dicer resulted in embryonic lethality, demonstrating that Dicer is necessary for normal mouse development. Other reports using conditional knockout approaches have demonstrated that Dicer plays essential roles in the maintenance of hair follicles, 26 lung epi...

The target of rapamycin (TOR), as part of the rapamycinsensitive TOR complex 1 (TORC1), regulates various aspects of protein synthesis. Whether TOR functions in this process as part of TORC2 remains to be elucidated. Here, we demonstrate that mTOR, SIN1 and rictor, components of mammalian (m)TORC2, are required for phosphorylation of Akt and conventional protein kinase C (PKC) at the turn motif (TM) site. This TORC2 function is growth factor independent and conserved from yeast to mammals. TM site phosphorylation facilitates carboxyl-terminal folding and stabilizes newly synthesized Akt and PKC by interacting with conserved basic residues in the kinase domain. Without TM site phosphorylation, Akt becomes protected by the molecular chaperone Hsp90 from ubiquitination-mediated proteasome degradation. Finally, we demonstrate that mTORC2 independently controls the Akt TM and HM sites in vivo and can directly phosphorylate both sites in vitro. Our studies uncover a novel function of the TOR pathway in regulating protein folding and stability, processes that are most likely linked to the functions of TOR in protein synthesis.

Posttranscriptional gene regulation by microRNAs (miRNAs) is important for many aspects of development, homeostasis, and disease. Here, we show that reduction of endothelial miRNAs by cell-specific inactivation of Dicer, the terminal endonuclease responsible for the generation of miRNAs, reduces postnatal angiogenic response to a variety of stimuli, including exogenous VEGF, tumors, limb ischemia, and wound healing. Furthermore, VEGF regulated the expression of several miRNAs, including the upregulation of components of the c-Myc oncogenic cluster miR-17-92. Transfection of endothelial cells with components of the miR-17-92 cluster, induced by VEGF treatment, rescued the induced expression of thrombospondin-1 and the defect in endothelial cell proliferation and morphogenesis initiated by the loss of Dicer. Thus, endothelial miRNAs regulate postnatal angiogenesis and VEGF induces the expression of miRNAs implicated in the regulation of an integrated angiogenic response.endothelium ͉ VEGF M icroRNAs (miRNAs) are short (Ϸ22 nt) noncoding RNAs derived from long primary transcripts through sequential processing by the enzymes Drosha and Dicer. Dicer-generated miRNAs are incorporated into the RNA-induced silencing complex that mediates miRNA-dependent translational suppression or in some instances cleavage of respective mRNA targets or translational activation (1, 2). The significance of miRNAs in mammalian biology has been dissected by Dicer gene disruption in mice. Mutant and disrupted Dicer alleles caused embryonic lethality associated with a loss of pluripotent stem cells (3) and defective blood vessel formation (4). Tissue-specific inactivation of Dicer has led to the conclusion that Dicer is essential for several processes, for example, limb, lung, and skin morphogenesis, the maintenance of hair follicles, T cell development/ differentiation, and neuronal survival (5-11).The growth of blood vessels is essential for organ growth and tissue repair. During adulthood, most blood vessels remain quiescent to fulfill their main function of conducting nutritive blood flow to organs; however, during pathological events such as tissue ischemia, inflammation, and tumor progression, endothelial cells (ECs) become activated and angiogenesis ensues to provide conduits for blood flow (12). An imbalance in the growth of blood vessels contributes to the pathogenesis of numerous disorders (13), and the growth of vessels is a complex process, requiring a finely tuned balance between numerous stimulatory and inhibitory signals (14). VEGF has been identified as a central mediator of angiogenesis (15). We (16) and others (17) have recently shown that reduction of miRNA levels via Dicer silencing strongly impacts EC functions in vitro, suggesting a critical role for miRNAs in angiogenesis. The role of Dicer-regulated miRNAs in ovarian angiogenesis is suggested by data obtained in mice expressing a global hypomorphic Dicer1 allele, where female mice are infertile because of corpus luteum insufficiency and defective ovarian angiogenesis...