Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) is a fundamental determinant of cardiovascular homesotasis: it regulates systemic blood pressure, vascular remodelling and angiogenesis. Physiologically, the most important stimulus for the continuous formation of NO is the viscous drag (shear stress) generated by the streaming blood on the endothelial layer. Although shear-stress-mediated phosphorylation of eNOS is thought to regulate enzyme activity, the mechanism of activation of eNOS is not yet known. Here we demonstrate that the serine/threonine protein kinase Akt/PKB mediates the activation of eNOS, leading to increased NO production. Inhibition of the phosphatidylinositol-3-OH kinase/Akt pathway or mutation of the Akt site on eNOS protein (at serine 1177) attenuates the serine phosphorylation and prevents the activation of eNOS. Mimicking the phosphorylation of Ser 1177 directly enhances enzyme activity and alters the sensitivity of the enzyme to Ca2+, rendering its activity maximal at sub-physiological concentrations of Ca2+. Thus, phosphorylation of eNOS by Akt represents a novel Ca2+-independent regulatory mechanism for activation of eNOS.

Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation

Understanding wound healing today involves much more than simply stating that there are three phases: "inflammation, proliferation, and maturation." Wound healing is a complex series of reactions and interactions among cells and "mediators." Each year, new mediators are discovered and our understanding of inflammatory mediators and cellular interactions grows. This article will attempt to provide a concise report of the current literature on wound healing by first reviewing the phases of wound healing followed by "the players" of wound healing: inflammatory mediators (cytokines, growth factors, proteases, eicosanoids, kinins, and more), nitric oxide, and the cellular elements. The discussion will end with a pictorial essay summarizing the wound-healing process.

The basic science of wound healing.

We tested the hypothesis that endothelial nitric oxide synthase (eNOS) modulates angiogenesis in two animal models in which therapeutic angiogenesis has been characterized as a compensatory response to tissue ischemia. We first administered L-arginine, previously shown to augment endogenous production of NO, to normal rabbits with operatively induced hindlimb ischemia. Angiogenesis in the ischemic hindlimb was significantly improved by dietary supplementation with L-arginine, compared to placebo-treated controls; angiographically evident vascularity in the ischemic limb, hemodynamic indices of limb perfusion, capillary density, and vasomotor reactivity in the collateral vessel-dependent ischemic limb were all improved by oral L-arginine supplementation. A murine model of operatively induced hindlimb ischemia was used to investigate the impact of targeted disruption of the gene encoding for ENOS on angiogenesis. Angiogenesis in the ischemic hindlimb was significantly impaired in eNOS-/- mice versus wild-type controls evaluated by either laser Doppler flow analysis or capillary density measurement. Impaired angiogenesis in eNOS-/- mice was not improved by administration of vascular endothelial growth factor (VEGF), suggesting that eNOS acts downstream from VEGF. Thus, (a) eNOS is a downstream mediator for in vivo angiogenesis, and (b) promoting eNOS activity by L-arginine supplementation accelerates in vivo angiogenesis. These findings suggest that defective endothelial NO synthesis may limit angiogenesis in patients with endothelial dysfunction related to atherosclerosis, and that oral L-arginine supplementation constitutes a potential therapeutic strategy for accelerating angiogenesis in patients with advanced vascular obstruction.

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Nitric oxide synthase modulates angiogenesis in response to tissue ischemia.

Vascular endothelial growth factor (VEGF) is a regulator of vasculogenesis and angiogenesis. To investigate the role of nitric oxide (NO) in VEGF-induced proliferation and in vitro angiogenesis, human umbilical vein endothelial cells (HUVEC) were used. VEGF stimulated the growth of HUVEC in an NO-dependent manner. In addition, VEGF promoted the NO-dependent formation of network-like structures in HUVEC cultured in three dimensional (3D) collagen gels. Exposure of cells to VEGF led to a concentration-dependent increase in cGMP levels, an indicator of NO production, that was inhibited by nitro-L-arginine methyl ester. VEGF-stimulated NO production required activation of tyrosine kinases and increases in intracellular calcium, since tyrosine kinase inhibitors and calcium chelators attenuated VEGF-induced NO release. Moreover, two chemically distinct phosphoinositide 3 kinase (PI-3K) inhibitors attenuated NO release after VEGF stimulation. In addition, HUVEC incubated with VEGF for 24 h showed an increase in the amount of endothelial NO synthase (eNOS) protein and the release of NO. In summary, both short- and long-term exposure of human EC to VEGF stimulates the release of biologically active NO. While long-term exposure increases eNOS protein levels, short-term stimulation with VEGF promotes NO release through mechanisms involving tyrosine and PI-3K kinases, suggesting that NO mediates aspects of VEGF signaling required for EC proliferation and organization in vitro.

/pdf/nitric-oxide-production-contributes-to-the-angiogenic-12sl1d4g6y.pdf

Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells.

IS053 Endothelial nitric oxide synthase modulates angiogenesis in response to tissue ischemia

Background Vascular endothelial growth factor (VEGF)/vascular permeability factor (VPF) is an endothelial cell (EC) mitogen. This feature is considered central to the documented role of VEGF/VPF in promoting angiogenesis. More recent evidence suggests that VEGF/VPF may also serve a “maintenance” function, modulating various aspects of EC biology. In the present study, we sought to determine the extent to which VEGF/VPF may stimulate the release of NO from normal ECs. Methods and Results VEGF/VPF produced a dose-dependent rise in NO concentration ([NO]) from vascular segments of rabbit thoracic aorta, pulmonary artery, and inferior vena cava. In comparison to stimulation with acetylcholine, the onset of increased [NO] after administration of VEGF/VPF was slower, reaching a maximum value after 8 minutes. Preincubation of the aortic segments with l-arginine raised by twofold both baseline [NO] and [NO] stimulated by addition of 2.5 μg/mL VEGF/VPF. Removal of CaCl 2 from the Krebs solution, disruption of the endothelium, and administration of N G -monomethyl-l-arginine abrogated the stimulatory effect of 10 μg/mL VEGF/VPF. Similar findings were documented with an NO-specific polarographic electrode to measure NO released from cultured human umbilical vein ECs. Conclusions VEGF/VPF stimulates production of NO from rabbit and human ECs. This finding (1) constitutes inferential evidence for the presence of functional VEGF/VPF receptors on quiescent endothelium of the adult rabbit as well as human ECs and (2) supports the notion that putative maintenance functions of VEGF/VPF may include regulation of baseline synthesis and/or release of EC NO.

Vascular Endothelial Growth Factor/Vascular Permeability Factor Augments Nitric Oxide Release From Quiescent Rabbit and Human Vascular Endothelium

OBJECTIVE
Aldo-keto reductase 1C3 (AKR1C3) has been postulated to be involved in androgen, progesterone, and estrogen metabolism. AKR1C3 inhibition has been proposed for treatment of endometriosis and polycystic ovary syndrome. Clinical biomarkers of target engagement, which can greatly facilitate drug development, have not yet been described for AKR1C3 inhibitors. Here, we analyzed pharmacodynamic data from a phase 1 study with a new selective AKR1C3 inhibitor, BAY1128688, to identify response biomarkers and assess effects on ovarian function.


DESIGN
In a multiple-ascending-dose placebo-controlled study, 33 postmenopausal women received BAY1128688 (3 mg, 30 mg, or 90 mg once daily; or 60 mg twice daily) or placebo for 14 days. Eighteen premenopausal women received 60 mg BAY1128688 once or twice daily for 28 days.


METHODS
We measured 17 serum steroids by liquid chromatography-tandem mass spectrometry, alongside analysis of pharmacokinetics, menstrual cyclicity and safety parameters.


RESULTS
In both study populations, we observed substantial, dose-dependent increases in circulating concentrations of the inactive androgen metabolite androsterone and minor increases in circulating etiocholanolone and dihydrotestosterone concentrations.In premenopausal women, androsterone concentrations increased 2.95-fold on average (95% confidence interval: 0.35-3.55) during once- or twice-daily treatment. Note, no concomitant changes in serum 17β-estradiol and progesterone were observed, and menstrual cyclicity and ovarian function were not altered by the treatment.


CONCLUSIONS
Serum androsterone was identified as a robust response biomarker for AKR1C3 inhibitor treatment in women. AKR1C3 inhibitor administration for 4 weeks did not affect ovarian function.

Novel AKR1C3 inhibitor affects androgen metabolism but not ovarian function in healthy women: A phase 1 study.

http://www.jidonline.org/article/S0022202X2301299X/pdf

1112 An innovative phase 1b trial demonstrating proof-of-pharmacology for two novel IRAK4-inhibitors using a local (Imiquimod) and systemic (LPS) driven immune response

Frank Zollmann

Papers

Vascular Endothelial Growth Factor/Vascular Permeability Factor Augments Nitric Oxide Release From Quiescent Rabbit and Human Vascular Endothelium

Novel AKR1C3 inhibitor affects androgen metabolism but not ovarian function in healthy women: A phase 1 study.

1112 An innovative phase 1b trial demonstrating proof-of-pharmacology for two novel IRAK4-inhibitors using a local (Imiquimod) and systemic (LPS) driven immune response