High-Resolution Conformational Analysis of RGDechi-Derived Peptides Based on a Combination of NMR Spectroscopy and MD Simulations.

The crucial role of integrin in pathological processes such as tumor progression and metastasis formation has inspired intense efforts to design novel pharmaceutical agents modulating integrin functions in order to provide new tools for potential therapies. In the past decade, we have investigated the biological proprieties of the chimeric peptide RGDechi, containing a cyclic RGD motif linked to an echistatin C-terminal fragment, able to specifically recognize αvß3 without cross reacting with αvß5 and αIIbß3 integrin. Additionally, we have demonstrated using two RGDechi-derived peptides, called RGDechi1-14 and ψRGDechi, that chemical modifications introduced in the C-terminal part of the peptide alter or abolish the binding to the αvß3 integrin. Here, to shed light on the structural and dynamical determinants involved in the integrin recognition mechanism, we investigate the effects of the chemical modifications by exploring the conformational space sampled by RGDechi1-14 and ψRGDechi using an integrated natural-abundance NMR/MD approach. Our data demonstrate that the flexibility of the RGD-containing cycle is driven by the echistatin C-terminal region of the RGDechi peptide through a coupling mechanism between the N- and C-terminal regions.

Tissue-specific and mosaic imprinting defects underlie opposite congenital growth disorders in mice.

Differential DNA methylation defects of H19/IGF2 are associated with congenital growth disorders characterized by opposite clinical pictures. Due to structural differences between human and mouse, the mechanisms by which mutations of the H19/IGF2 Imprinting Control region (IC1) result in these diseases are undefined. To address this issue, we previously generated a mouse line carrying a humanized IC1 (hIC1) and now replaced the wildtype with a mutant IC1 identified in the overgrowth-associated Beckwith-Wiedemann syndrome. The new humanized mouse line shows pre/post-natal overgrowth on maternal transmission and pre/post-natal undergrowth on paternal transmission of the mutation. The mutant hIC1 acquires abnormal methylation during development causing opposite H19/Igf2 imprinting defects on maternal and paternal chromosomes. Differential and possibly mosaic Igf2 expression and imprinting is associated with asymmetric growth of bilateral organs. Furthermore, tissue-specific imprinting defects result in deficient liver- and placenta-derived Igf2 on paternal transmission and excessive Igf2 in peripheral tissues on maternal transmission, providing a possible molecular explanation for imprinting-associated and phenotypically contrasting growth disorders.

Interactome mapping defines BRG1, a component of the SWI/SNF chromatin remodeling complex, as a new partner of the transcriptional regulator CTCF.

The highly conserved zinc finger CCCTC-binding factor (CTCF) regulates genomic imprinting and gene expression by acting as a transcriptional activator or repressor of promoters and insulator of enhancers. The multiple functions of CTCF are accomplished by co-association with other protein partners and are dependent on genomic context and tissue specificity. Despite the critical role of CTCF in the organization of genome structure, to date, only a subset of CTCF interaction partners have been identified. Here we present a large-scale identification of CTCF-binding partners using affinity purification and high-resolution LC-MS/MS analysis. In addition to functional enrichment of specific protein families such as the ribosomal proteins and the DEAD box helicases, we identified novel high-confidence CTCF interactors that provide a still unexplored biochemical context for CTCF's multiple functions. One of the newly validated CTCF interactors is BRG1, the major ATPase subunit of the chromatin remodeling complex SWI/SNF, establishing a relationship between two master regulators of genome organization. This work significantly expands the current knowledge of the human CTCF interactome and represents an important resource to direct future studies aimed at uncovering molecular mechanisms modulating CTCF pleiotropic functions throughout the genome.

Ruta graveolens water extract inhibits cell-cell network formation in human umbilical endothelial cells via MEK-ERK1/2 pathway.

Angiogenesis is a process encompassing several steps such as endothelial cells proliferation, differentiation and migration to form a vascular network, involving different signal transduction pathways. Among these, ERK1/2 signaling mediates VEGF-dependent signaling pathway. Here we report that the water extract of Ruta graveolens (RGWE), widely known as a medicinal plant, is able to impair in a dose-dependent manner, cell network formation without affecting cell viability. Biochemical analysis showed that the major component of RGWE is rutin, unable to reproduce RGWE effect. We found that RGWE inhibits ERK1/2 phosphorylation and that this event is crucial in cell network formation since the transfection of HUVEC with a constitutively active MEK (caMEK), the ERK1/2 activator, induces a robust cell network formation as compared to untransfected and/or mock transfected cells and, more importantly, caMEK transfected cells became unresponsive to RGWE. Moreover, RGWE inhibits VEGF and nestin gene expression, necessary for vessel formation, and the caMEK transfection induces their higher expression. In conclusion, we report that RGWE is able to significantly impair vessels network formation without affecting cell viability, preventing ERK1/2 activation and, in turn, down-regulating VEGF and nestin expression. These findings point to RGWE as a potential therapeutic tool capable to interfere with pathologic angiogenesis.

Functional characterization of the neuron-restrictive silencer element in the human tryptophan hydroxylase 2 gene expression.

Tryptophan hydroxylase 2 (TPH2) is the key enzyme in the synthesis of neuronal serotonin. Although previous studies suggest that TPH2 neuron-restrictive silencer element (NRSE) functions as a negative regulator dependent on neuron-restrictive silencer factor (NRSF) activity, the underlying mechanisms are yet to be fully elucidated. Here, we show a detailed analysis of the NRSE-mediated repression of the human TPH2 (hTPH2) promoter activity in RN46A cells, a cell line derived from rat raphe neurons. Quantitative real-time RT-PCR analysis revealed the expression of serotonergic marker genes (Mash1, Nkx2.2, Gata2, Gata3, Lmx1b, Pet-1, 5-Htt, and Vmat2) and Nrsf gene in RN46A cells. Tph1 mRNA is the prevalent form expressed in RN46A cells; Tph2 mRNA is also expressed but at a lower level. Electrophoretic mobility shift assays and reporter assays showed that hTPH2 NRSE is necessary for the efficient DNA binding of NRSF and for the NRSF-dependent repression of the hTPH2 promoter activity. The hTPH2 promoter activity was increased by knockdown of NRSF, or over-expression of the engineered NRSF (a dominant-negative mutant or a DNA-binding domain and activation domain fusion protein). MS-275, a class I histone deacetylase (HDAC) inhibitor, was found to be more potent than MC-1568, a class II HDAC inhibitor, in enhancing the hTPH2 promoter activity. Furthermore, treatment with the ubiquitin-specific protease 7 deubiquitinase inhibitors, P-22077 or HBX 41108, increased the hTPH2 promoter activity. Collectively, our data demonstrate that the hTPH2 NRSE-mediated promoter repression via NRSF involves class I HDACs and is modulated by the ubiquitin-specific protease 7-mediated deubiquitination and stabilization of NRSF.

Neuro-Nutraceutical Polyphenols: How Far Are We?

The brain, composed of billions of neurons, is a complex network of interacting dynamical systems controlling all body functions. Neurons are the building blocks of the nervous system and their impairment of their functions could result in neurodegenerative disorders. Accumulating evidence shows an increase of brain-affecting disorders, still today characterized by poor therapeutic options. There is a strong urgency to find new alternative strategies to prevent progressive neuronal loss. Polyphenols, a wide family of plant compounds with an equally wide range of biological activities, are suitable candidates to counteract chronic degenerative disease in the central nervous system. Herein, we will review their role in human healthcare and highlight their: antioxidant activities in reactive oxygen species-producing neurodegenerative pathologies; putative role as anti-acetylcholinesterase inhibitors; and protective activity in Alzheimer's disease by preventing Aß aggregation and tau hyperphosphorylation. Moreover, the pathology of these multifactorial diseases is also characterized by metal dyshomeostasis, specifically copper (Cu), zinc (Zn), and iron (Fe), most important for cellular function. In this scenario, polyphenols' action as natural chelators is also discussed. Furthermore, the critical importance of the role exerted by polyphenols on microbiota is assumed, since there is a growing body of evidence for the role of the intestinal microbiota in the gut-brain axis, giving new opportunities to study molecular mechanisms and to find novel strategies in neurological diseases.

Tryptophan hydroxylase 2 (TPH2) in a neuronal cell line: modulation by cell differentiation and NRSF/rest activity.

Serotonin (5-HT) is a neurotransmitter involved in many aspects of the neuronal function. The synthesis of 5-HT is initiated by the hydroxylation of tryptophan, catalyzed by tryptophan hydroxylase (TPH). Two isoforms of TPH (TPH1 and TPH2) have been identified, with TPH2 almost exclusively expressed in the brain. Following TPH2 discovery, it was reported that polymorphisms of both gene and non-coding regions are associated with a spectrum of psychiatric disorders. Thus, insights into the mechanisms that specifically regulate TPH2 expression and its modulation by exogenous stimuli may represent a new therapeutic approach to modify serotonergic neurotransmission. To this aim, a CNS-originated cell line expressing TPH2 endogenously represents a valid model system. In this study, we report that TPH2 transcript and protein are modulated by neuronal differentiation in the cell line A1 mes-c-myc (A1). Moreover, we show luciferase activity driven by the human TPH2 promoter region and demonstrate that upon mutation of the NRSF/REST responsive element, the promoter activity strongly increases with cell differentiation. Our data suggest that A1 cells could represent a model system, allowing an insight into the mechanisms of regulation of TPH2 and to identify novel therapeutic targets in the development of drugs for the management of psychiatric disorders.

Angiogenesis and Multiple Sclerosis Pathogenesis: A Glance at New Pharmaceutical Approaches.

Multiple sclerosis is a chronic disease of the central nervous system characterized by demyelination and destruction of axons. The most common form of the disease is the relapsing-remitting multiple sclerosis in which episodic attacks with typical neurological symptoms are followed by episodes of partial or complete recovery. One of the underestimated factors that contribute to the pathogenesis of multiple sclerosis is excessive angiogenesis. Here, we review the role of angiogenesis in the onset and in the development of the disease, the molecular mechanisms underlying angiogenesis, the current therapeutic approaches, and the potential therapeutic strategies with a look at natural compounds as multi-target drugs with both neuroprotective and anti-angiogenic properties.

Genotype-Phenotype Correlations in Neurofibromatosis Type 1: Identification of Novel and Recurrent NF1 Gene Variants and Correlations with Neurocognitive Phenotype.

Neurofibromatosis type 1 (NF1) is one of the most common genetic tumor predisposition syndrome, caused by mutations in the NF1. To date, few genotype-phenotype correlations have been discerned in NF1, due to a highly variable clinical presentation. We aimed to study the molecular spectrum of NF1 and genotype-phenotype correlations in a monocentric study cohort of 85 NF1 patients (20 relatives, 65 sporadic cases). Clinical data were collected at the time of the mutation analysis and reviewed for accuracy in this investigation. An internal phenotypic categorization was applied. The 94% of the patients enrolled showed a severe phenotype with at least one systemic complication and a wide range of associated malignancies. Spine deformities were the most common complications in this cohort. We also reported 66 different NF1 mutations, of which 7 are novel mutations. Correlation analysis identified a slight significant inverse correlation between age at diagnosis and delayed acquisition of psychomotor skills with residual multi-domain cognitive impairment. Odds ratio with 95% confidence interval showed a higher prevalence of learning disabilities in patients carrying frameshift mutations. Overall, our results aim to offer an interesting contribution to studies on the genotype-phenotype of NF1 and in genetic management and counselling.

Ruta graveolens water extract (RGWE) ameliorates ischemic damage and improves neurological deficits in a rat model of transient focal brain ischemia.

INTRODUCTION AND AIMS: The limited therapeutic options for ischemic stroke treatment render necessary the identification of new strategies. In recent years, it has been shown that natural compounds may represent a valid therapeutic opportunity. Therefore, the present study aimed to evaluate the protective effect of Ruta graveolens water extract (RGWE) in an in vivo experimental model of brain ischemia. METHODS: RGWE effects on ischemic damage and neurological function were evaluated in adult rats subjected to transient occlusion of the Middle Cerebral Artery (tMCAO), receiving two intraperitoneal injections of RGWE, 100 and 300 min after the induction of ischemia. In addition, astroglial and microglial activation was measured as GFAP and IBA-1 expression by immunofluorescence and confocal microscopy analysis. RESULTS: Treatment with RGWE containing 10 mg/kg of Rutin, the major component, ameliorates the ischemic damage and improves neurological performances. Interestingly, the pro-inflammatory states of astrocytes and microglia, respectively detected by using C3 and iNOS markers, were significantly reduced in ipsilateral cortical and striatal areas in ischemic RGWE-treated rats. CONCLUSIONS: RGWE shows a neuroprotective effect on brain infarct volume extent in a transient focal cerebral ischemia model and this effect was paralleled by the prevention of pro-inflammatory astroglial and microglial activation. Collectively, our findings support the idea that natural compounds may represent potential therapeutic opportunities against ischemic stroke.

Protection from angiotensin II-mediated vasculotoxic and hypertensive response in mice lacking PI3Kgamma.

Hypertension affects nearly 20% of the population in Western countries and strongly increases the risk for cardiovascular diseases. In the pathogenesis of hypertension, the vasoactive peptide of the renin-angiotensin system, angiotensin II and its G protein-coupled receptors (GPCRs), play a crucial role by eliciting reactive oxygen species (ROS) and mediating vessel contractility. Here we show that mice lacking the GPCR-activated phosphoinositide 3-kinase (PI3K)gamma are protected from hypertension that is induced by administration of angiotensin II in vivo. PI3Kgamma was found to play a role in angiotensin II-evoked smooth muscle contraction in two crucial, distinct signaling pathways. In response to angiotensin II, PI3Kgamma was required for the activation of Rac and the subsequent triggering of ROS production. Conversely, PI3Kgamma was necessary to activate protein kinase B/Akt, which, in turn, enhanced L-type Ca(2+) channel-mediated extracellular Ca(2+) entry. These data indicate that PI3Kgamma is a key transducer of the intracellular signals that are evoked by angiotensin II and suggest that blocking PI3Kgamma function might be exploited to improve therapeutic intervention on hypertension.

A novel approach for studying receptor-ligand interactions on living cells surface by using NUS/T1ρ-NMR methodologies combined with computational techniques: The RGDechi15D-αvß5 integrin complex.

Structural investigations of receptor-ligand interactions on living cells surface by high-resolution Nuclear Magnetic Resonance (NMR) are problematic due to their short lifetime, which often prevents the acquisition of experiments longer than few hours. To overcome these limitations, we developed an on-cell NMR-based approach for exploring the molecular determinants driving the receptor-ligand recognition mechanism under native conditions. Our method relies on the combination of high-resolution structural and dynamics NMR data with Molecular Dynamics simulations and Molecular Docking studies. The key point of our strategy is the use of Non Uniform Sampling (NUS) and T1ρ-NMR techniques to collect atomic-resolution structural and dynamics information on the receptor-ligand interactions with living cells, that can be used as conformational constraints in computational studies. In fact, the application of these two NMR methodologies allows to record spectra with high S/N ratio and resolution within the lifetime of cells. In particular, 2D NUS [1H-1H] trNOESY spectra are used to explore the ligand conformational changes induced by receptor binding; whereas T1ρ-based experiments are applied to characterize the ligand binding epitope by defining two parameters: T1ρ Attenuation factor and T1ρ Binding Effect. This approach has been tested to characterize the molecular determinants regulating the recognition mechanism of αvß5-integrin by a selective cyclic binder peptide named RGDechi15D. Our data demonstrate that the developed strategy represents an alternative in-cell NMR tool for studying, at atomic resolution, receptor-ligand recognition mechanism on living cells surface. Additionally, our application may be extremely useful for screening of the interaction profiling of drugs with their therapeutic targets in their native cellular environment.

ZBTB2 protein is a new partner of the Nucleosome Remodeling and Deacetylase (NuRD) complex.

ZBTB2 is a protein belonging to the BTB/POZ zinc-finger family whose members typically contain a BTB/POZ domain at the N-terminus and several zinc-finger domains at the C-terminus. Studies have been carried out to disclose the role of ZBTB2 in cell proliferation, in human cancers and in regulating DNA methylation. Moreover, ZBTB2 has been also described as an ARF, p53 and p21 gene repressor as well as an activator of genes modulating pluripotency. In this scenario, ZBTB2 seems to play many functions likely associated with other proteins. Here we report a picture of the ZBTB2 protein partners in U87MG cell line, identified by high-resolution mass spectrometry (MS) that highlights the interplay between ZBTB2 and chromatin remodeling multiprotein complexes. In particular, our analysis reveals the presence, as ZBTB2 candidate interactors, of SMARCA5 and BAZ1B components of the chromatin remodeling complex WICH and PBRM1, a subunit of the SWI/SNF complex. Intriguingly, we identified all the subunits of the NuRD complex among the ZBTB2 interactors. By co-immunoprecipitation experiments and ChIP-seq analysis we definitely identify ZBTB2 as a new partner of the NuRD complex.

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis.

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Histone Deacetylase Inhibitors Impair Vasculogenic Mimicry from Glioblastoma Cells.

Glioblastoma (GBM), a high-grade glioma (WHO grade IV), is the most aggressive form of brain cancer. Available treatment options for GBM involve a combination of surgery, radiation and chemotherapy but result in a poor survival outcome. GBM is a high-vascularized tumor and antiangiogenic drugs are widely used in GBM therapy as adjuvants to control abnormal vasculature. Vasculogenic mimicry occurs in GBM as an alternative vascularization mechanism, providing a means whereby GBM can escape anti-angiogenic therapies. Here, using an in vitro tube formation assay on Matrigel®, we evaluated the ability of different histone deacetylase inhibitors (HDACis) to interfere with vasculogenic mimicry. We found that vorinostat (SAHA) and MC1568 inhibit tube formation by rat glioma C6 cells. Moreover, at sublethal doses for GBM cells, SAHA, trichostatin A (TSA), entinostat (MS275), and MC1568 significantly decrease tube formation by U87MG and by patient-derived human GBM cancer stem cells (CSCs). The reduced migration and invasion of HDACis-treated U87 cells, at least in part, may account for the inhibition of tube formation. In conclusion, our results indicate that HDACis are promising candidates for blocking vascular mimicry in GBM.

Selective Rac-1 inhibition protects from diabetes-induced vascular injury.

Diabetes mellitus is a main risk factor for vascular diseases. Vascular injury induced by diabetes mellitus is characterized by endothelial dysfunction attributable to an increased oxidative stress. So far, the molecular mechanisms involved in the vasculotoxic effects of diabetes are only partially known. We examined the effect of diabetes mellitus on oxidative stress and Rac-1 activation, a small G-protein involved in the activation of NADPH oxidase. Our results show that oxidative stress in vessels of different murine models of diabetes mellitus and in endothelial cells treated with high glucose is associated with an increased Rac-1/PAK binding and Rac-1 translocation from cytosol to plasma membrane, thus demonstrating an enhanced Rac-1 activity. More important, selective Rac-1 inhibition by an adenoviral vector carrying a dominant negative mutant of Rac-1 protected from oxidative stress and vascular dysfunction induced by diabetes mellitus. Our study demonstrates that Rac-1 plays a crucial role in diabetes-induced vascular injury, and it could be a target of novel therapeutic approaches to reduce vascular risk in diabetes mellitus.

Plgf-/-eNos-/- mice show defective angiogenesis associated with increased oxidative stress in response to tissue ischemia.

Neo-angiogenesis is a complex phenomenon modulated by the concerted action of several molecular factors. We have generated a congenic line of knockout mice carrying null mutations of both placental growth factor (PlGF) and endothelial nitric oxide synthase (eNOS), two genes that play a pivotal role in the regulation of pathological angiogenesis. In the present study, we describe the phenotype of this new experimental animal model after surgically induced hind-limb ischemia. Plgf-/-, eNos-/-, Plgf-/- eNos-/-, and wild-type C57BL/6J mice were studied. Plgf-/- eNos-/- mice showed the most severe phenotype: self-amputation, and death occurred in up to 47% of the animals studied; in ischemic legs, capillary density was severely reduced; macrophage infiltration and oxidative stress increased as compared to the other groups of animals. These changes were associated with an up-regulation of both inducible NOS (iNOS) expression and vascular endothelial growth factor (VEGF) protein levels in ischemic limbs, and to an increased extent of protein nitration. Our results demonstrate that the deletion of these two genes, Plgf, which acts in synergism with VEGF, and eNos, a downstream mediator of VEGF, determines a significant change in the vascular response to an ischemic stimulus and that oxidative stress within the ischemic tissue represents a crucial factor to maintain tissue homeostasis.

GRN deletion in familial frontotemporal dementia showing association with clinical variability in 3 familial cases.

Progranulin (GRN) gene mutations have been genetically associated with frontotemporal dementia (FTD) and are present in about 23% of patients with familial FTD. However, the neurobiology of this secreted glycoprotein remains unclear. Here, we report the identification of 3 pedigrees of Southern Italian extraction in whom FTD segregates with autosomal dominant inheritance patterns. We present evidence that all the available patients in these 3 familial cases are carrying the rare GRN gene exon 6 deletion g10325_10331delCTGCTGT (relative to nt 1 inNG_007886.1), alias Cys157LysfsX97. This mutation was previously described in 2 sporadic cases but was never associated with familial cases. Our patients demonstrate heterogeneous clinical phenotypes, such as the behavioral variant (bvFTD) in the affected men and the nonfluent/agrammatic variant of primary progressive aphasia (nfvPPA) in the affected woman. Haploinsufficiency was revealed by both quantitative real-time PCR of the gene and protein analyses. These findings provide further support for a previously proposed role for the GRN gene in the genetic etiology of FTD and its phenotypic variability.

Characterization of nitric oxide release by nebivolol and its metabolites.

BACKGROUND: Nebivolol is a selective beta(1)-adrenergic receptor antagonist that causes a direct vasodilator effect attributed to the action on vascular nitric oxide (NO). This study aimed to investigate whether nebivolol or its metabolites induces NO production and to explore the mechanisms underlying this pharmacologic effect. METHODS: Conductance and resistance arteries from Wistar-Kyoto rats (WKY) (n = 33) incubated with the fluorescent probe diaminofluorescein-2 (DAF-2) were stimulated with increasing concentrations of nebivolol or its enantiomers and metabolites, and NO release was histologically evaluated. RESULTS: Nebivolol induced a dose-dependent increase in NO levels in the endothelium of both arteries. Levels of NO were significantly increased at 10(-6)mol/L and reached a plateau state at 10(-5)mol/L. Induction of NO is not a general action of beta-adrenoceptor antagonists, as atenolol had no effects. Nebivolol action on NO release was mainly caused by the D-isomer. Moreover NO production is also maintained after hepatic metabolism, as the three main metabolites of nebivolol were able to induce a significant increase in endothelial NO release. Finally, nebivolol-activated calcium mobilization is crucial to NO production. CONCLUSION: Our study shows the effects of D-nebivolol and its metabolites on endothelial NO production in both conductance and resistance arteries, and clarifies that this effect is realized through a calcium-dependent mechanism.

Leptin effect on endothelial nitric oxide is mediated through Akt-endothelial nitric oxide synthase phosphorylation pathway.

Recent evidence suggests that besides its action on the central nervous system, leptin can modulate vascular tone through local mechanisms involving nitric oxide (NO) release. In this study, using a fluorescent probe for direct determination of NO, we demonstrated both in endothelial cells and in vessels that leptin is able to stimulate NO release. The effect of leptin on NO is abolished by erbstatin A, a Ca(2+)-independent tyrosine kinase inhibitor, whereas it is not influenced by calcium removal or by other protein phosphorylation inhibitors, such as genistein (an ATP-dependent tyrosine-kinase inhibitor) or wortmannin and LY294002 (two different phosphatidylinositol [PI] 3-kinase inhibitors). Accordingly, leptin-induced vasorelaxation in aortic rings was abolished only by erbstatin A. Furthermore, immunoblotting studies revealed that leptin evokes Akt phosphorylation, with a comparable time course in both endothelial cells and vessels. Also in this experimental system, the effect of leptin was abolished by erbstatin A and not by other inhibitors. Finally, a considerable increase in endothelial NO synthase (eNOS) phosphorylation in Ser(1177) was found when vessels were treated with leptin. In conclusion, leptin induces NO production by activating a PI 3-kinase-independent Akt-eNOS phosphorylation pathway.