Mario B. Marrero

Vascular Endothelial Growth Factor Signals Endothelial Cell Production of Nitric Oxide and Prostacyclin through Flk-1/KDR Activation of c-Src

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mitogen that promotes angiogenesis, vascular hyperpermeability, and vasodilation by autocrine mechanisms involving nitric oxide (NO) and prostacyclin (PGI2) production. These experiments used immunoprecipitation and immunoassay procedures to characterize the signaling pathways by which VEGF induces NO and PGI2 formation in cultured endothelial cells. The data showed that VEGF stimulates complex formation of the flk-1/kinase-insert domain-containing receptor (KDR) VEGF receptor with c-Src and that Src activation is required for VEGF induction of phospholipase C γ1 activation and inositol 1,4,5-trisphosphate formation. Reporter cell assays showed that VEGF promotes a ∼50-fold increase in NO formation, which peaks at 5–20 min. This effect is mediated by a signaling cascade initiated by flk-1/KDR activation of c-Src, leading to phospholipase C γ1 activation, inositol 1,4,5-trisphosphate formation, release of [Ca2+] i and nitric oxide synthase activation. Immunoassays of VEGF-induced 6-keto prostaglandin F1α formation as an indicator of PGI2 production revealed a 3–4-fold increase that peaked at 45–60 min. The PGI2 signaling pathway follows the NO pathway through release of [Ca2+] i , but diverges prior to NOS activation and also requires activation of mitogen-activated protein kinase. These results suggest that NO and PGI2 function in parallel in mediating the effects of VEGF.

Novel Mechanism of Activation of NADPH Oxidase 5 CALCIUM SENSITIZATION VIA PHOSPHORYLATION

In contrast to other Nox isoforms, the activity of Nox5 does not require the presence of accessory proteins and is entirely dependent on the elevation of intracellular calcium. Previous studies have shown that the EC50 of Nox5 for calcium is relatively high and raises the question of whether Nox5 can be sufficiently activated in cells that do not experience extreme elevations of intracellular calcium. In the current study, we have identified a novel mechanism governing the activity of Nox5. Exposure of cells expressing Nox5 to phorbol 12-myristate 13-acetate (PMA) resulted in a slow and sustained increase in ROS, which was markedly different from the rapid response to ionomycin. PMA greatly potentiated the activity of Nox5 in response to low concentrations of ionomycin. The ability of PMA to increase Nox5 activity was abolished by calcium chelation and was a direct effect on enzyme activity, since PMA increased the calcium sensitivity of Nox5 in a cell-free assay. PMA stimulated the time-dependent phosphorylation of Nox5 on Thr494 and Ser498. Mutation of these residues to alanine abolished both PMA-dependent phosphorylation and calcium sensitization. Conversely, mutation of Thr494 and Ser498 to glutamic acid produced a gain of function mutant that had increased activity at low concentrations of ionomycin. Within the cell, Nox5 was detected in detergent-resistant microdomains of the endoplasmic reticulum. In summary, the phosphorylation of Nox5 at key residues facilitates enzyme activation at lower levels of intracellular calcium and may provide an avenue for enzyme activation in response to a greater variety of extracellular stimuli.

Inhibitory Interactions of the Bradykinin B2 Receptor with Endothelial Nitric-oxide Synthase

It has been shown previously that the endothelial nitric-oxide synthase (eNOS) interacts reversibly with the plasmalemmal caveolae structural protein, caveolin-1. The eNOS-caveolin-1 interaction inhibits eNOS catalytic activity. In the present study, we show that eNOS also participates in reversible inhibitory interactions with the G protein-coupled bradykinin B2 receptor. eNOS and the B2 receptor are coimmunoprecipitated from endothelial cell lysates by antibodies directed against either of the two proteins. A glutathioneS-transferase fusion protein containing intracellular domain 4 of the receptor is bound by purified recombinant eNOS inin vitro binding assays. The fusion protein selectively inhibits the activity of purified eNOS. A synthetic peptide corresponding to membrane-proximal residues 310–334 in intracellular domain 4 also potently inhibits eNOS activity (IC50 < 1 μm). Treatment of cultured endothelial cells with bradykinin or Ca2+ ionophore promotes a rapid dissociation of the eNOS·B2 receptor complex. These data demonstrate that the bradykinin B2 receptor physically associates with eNOS in a ligand- and Ca2+-dependent manner. Reversible and inhibitory membrane-docking interactions of eNOS, therefore, are not restricted to those with caveolin-1 but also occur with the bradykinin B2 receptor.

Alpha7 nicotinic receptors as novel therapeutic targets for inflammation-based diseases

In recent years the etiopathology of a number of debilitating diseases such as type 2 diabetes, arthritis, atherosclerosis, psoriasis, asthma, cystic fibrosis, sepsis, and ulcerative colitis has increasingly been linked to runaway cytokine-mediated inflammation. Cytokine-based therapeutic agents play a major role in the treatment of these diseases. However, the temporospatial changes in various cytokines are still poorly understood and attempts to date have focused on the inhibition of specific cytokines such as TNF-α. As an alternative approach, a number of preclinical studies have confirmed the therapeutic potential of targeting alpha7 nicotinic acetylcholine receptor-mediated anti-inflammatory effects through modulation of proinflammatory cytokines. This “cholinergic anti-inflammatory pathway” modulates the immune system through cholinergic mechanisms that act on alpha7 receptors expressed on macrophages and immune cells. If the preclinical findings translate into human efficacy this approach could potentially provide new therapies for treating a broad array of intractable diseases and conditions with inflammatory components.

Vascular Endothelial Growth Factor Activates STAT Proteins in Aortic Endothelial Cells

Vascular endothelial growth factor (VEGF) intracellular signaling in endothelial cells is initiated by the activation of distinct tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1/KDR). Because the tyrosine kinase-dependent transcription factors known as STAT (signal transducers and activators of transcription) proteins are important modulators of cell growth responses induced by other growth factor receptors, we have determined the effects VEGF of on STAT activation in BAEC (bovine aortic endothelial cells). Here, we show that VEGF induces tyrosine phosphorylation and nuclear translocation of STAT1 and STAT6. VEGF also stimulates STAT3 tyrosine phosphorylation, but nuclear translocation does not occur. We found that placenta growth factor, which selectively activates VEGFR1, has no effect on the STATs. However, upon VEGF stimulation, STAT1 associates with the VEGFR2 in a tyrosine kinase-dependent manner, indicating that VEGF-induced STAT1 activation is mediated primarily by VEGFR2. Thus, our study shows for the first time that VEGF activates the STAT pathway through VEGFR2. Because the growth-promoting activity of VEGF depends upon VEGFR2 activation, these findings suggest a role for the STATs in the regulation of gene expression associated with the angiogenic effects of VEGF.

Convergence of alpha 7 nicotinic acetylcholine receptor-activated pathways for anti-apoptosis and anti-inflammation: Central role for JAK2 activation of STAT3 and NF-κB

Our laboratories have previously identified the alpha7 nAChR-JAK2 pathway as playing a central role in nicotine-induced neuroprotection. We have also reported that the angiotensin II (Ang II) AT(2) receptor induced activation of SHP-1 induces the tyrosine dephosphorylation of JAK2 that results in a complete neutralization of the alpha7 nAChR-JAK2 pro-survival cascade. In this study, we investigated the effects of inhibiting the alpha7 nAChR-JAK2 pro-survival cascade on the nicotine-induced production of the survival factor Bcl-2 and the transcriptional activation of NF-kappaB, AP-1, STAT1, STAT3, and STAT5. We report that nicotine induced the production of Bcl-2 and increased the transcriptional activation of NF-kappaB, AP-1, STAT1, and STAT3, and with the exception of AP-1, the other transcription factors (NF-kappaB, STAT1, and STAT3) were significantly reduced by JAK2 inhibition. We also demonstrate that, via transfection of either Bcl-2 antisense or NF-kappaB, STAT1 and STAT3 transcription factor decoys oligodeoxyribonucleotides into PC12 cells, nicotine induces its neuroprotection in PC12 cells via activation of the alpha7 nAChR-JAK2-(NF-kappaB; STAT3)-Bcl-2 pro-survival pathway. Finally, the neuroprotective nicotine-induced production of Bcl-2 appears to fully counteract the Abeta (1-42)-induced apoptosis of PC12 cells by blocking Abeta (1-42)-induced mitochondrial release of cytosolic cytochrome C.

Hyperglycemia Enhances Angiotensin II-induced Janus-activated Kinase/STAT Signaling in Vascular Smooth Muscle Cells

We have shown previously that angiotensin II (Ang II) activates the janus-activated kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in vascular smooth muscle cells (VSMCs) and that activation of the JAK/STAT pathway is required for Ang II induction of VSMC proliferation. In the present study, we examined the effects of hyperglycemia (HG) on Ang II-induced JAK/STAT signaling events in cultured VSMCs. HG increases Ang II-induced JAK2 tyrosine phosphorylation and promotes a partial tyrosine phosphorylation of the enzyme under basal conditions. In addition, HG increases both basal and Ang II-induced complex formation of JAK2 with the Ang II AT1 receptor. The extent of STAT1 and STAT3 tyrosine and serine phosphorylation are also increased under HG conditions. Furthermore, the tyrosine phosphorylation and activities of the SHP-1 and SHP-2 tyrosine phosphatases, enzymes that regulate Ang II-induced JAK2 tyrosine phosphorylation, are altered by HG. SHP-1, which is responsible for JAK2 tyrosine dephosphorylation in VSMC, is completely deactivated in HG, resulting in a prolonged duration of JAK2 phosphorylation under HG conditions. HG also enhances Ang II induction of VSMC proliferation. Taken together, these data suggest that HG augments Ang II induction of VSMC proliferation by increasing signal transduction through the JAK/STAT pathway.

Paradoxical Activation of Endothelial Nitric Oxide Synthase by NADPH Oxidase

Objectives—Increased formation of reactive oxygen species (ROS) has been identified as a causative factor in endothelial dysfunction by reducing NO bioavailability and uncoupling endothelial nitric oxide synthase (eNOS). However, the specific contribution of ROS to endothelial function is not well understood. Methods and Results—A major source of intracellular ROS is the NADPH oxidase (Nox) family of enzymes. The goal of the current study was to directly assess the contribution of NADPH oxidase derived superoxide to eNOS function by expressing Nox5, a single gene product that constitutively produces superoxide within cells. Paradoxically, we found that instead of inhibiting eNOS, coexpression of Nox5 increased NO release from both bovine and human endothelial cells. To establish the functional significance of this observation in intact blood vessels, the endothelium of mouse aorta was transduced with Nox5 or control adenoviruses. Nox5 potently inhibited Ach-induced relaxation and potentiated contractile responses to phenylephrine. In precontracted aortae, acute exposure to superoxide dismutase induced significant vascular relaxation in vessels exposed to Nox5 versus control and unmasked the ability of Nox5 to activate eNOS in blood vessel endothelium. Conclusions—These findings suggest that ROS inhibit eNOS function via consumption of NO rather than direct inhibition of enzymatic activity.

Loss of Jak2 selectively suppresses DC-mediated innate immune response and protects mice from lethal dose of LPS-induced septic shock.

Given the importance of Jak2 in cell signaling, a critical role for Jak2 in immune cells especially dendritic cells (DCs) has long been proposed. The exact function for Jak2 in DCs, however, remained poorly understood as Jak2 deficiency leads to embryonic lethality. Here we established Jak2 deficiency in adult Cre+/+Jak2fl/fl mice by tamoxifen induction. Loss of Jak2 significantly impaired DC development as manifested by reduced BMDC yield, smaller spleen size and reduced percentage of DCs in total splenocytes. Jak2 was also crucial for the capacity of DCs to mediate innate immune response. Jak2−/− DCs were less potent in response to inflammatory stimuli and showed reduced capacity to secrete proinflammatory cytokines such as TNFα and IL-12. As a result, Jak2−/− mice were defective for the early clearance of Listeria after infection. However, their potency to mediate adaptive immune response was not affected. Unlike DCs, Jak2−/− macrophages showed similar capacity secretion of proinflammatory cytokines, suggesting that Jak2 selectively modulates innate immune response in a DC-dependent manner. Consistent with these results, Jak2−/− mice were remarkably resistant to lethal dose of LPS-induced septic shock, a deadly sepsis characterized by the excessive innate immune response, and adoptive transfer of normal DCs restored their susceptibility to LPS-induced septic shock. Mechanistic studies revealed that Jak2/SATA5 signaling is pivotal for DC development and maturation, while the capacity for DCs secretion of proinflammatory cytokines is regulated by both Jak2/STAT5 and Jak2/STAT6 signaling.

Application of Alpha7 Nicotinic Acetylcholine Receptor Agonists in Inflammatory Diseases: An Overview

ABSTRACTInflammatory disorders are characterized by the influx of immune cells into the vascular wall of veins and/or arteries in response to stimuli such as oxidized-LDL and various pathogens. These factors stimulate the local production of pro-inflammatory cytokines by macrophages and other cells that promote various inflammatory diseases such as atherosclerosis, Crohn’s, Alzheimer’s and diabetes. Numerous cytokines play a significant role in this process, though tumor necrosis factor (TNF) and various interleukins are thought to be among the most important regulators. These proinflammatory cytokines promote the above-described diseases by inducing endothelial cell dysfunction. In this brief commentary we will discuss some of the latest advances and discoveries in the treatment of these inflammatory diseases, making use of alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) agonists.