Oxide Synthase In Endothelial Cells Research Articles

Harnessing the pleiotropic effects of atorvastatin-fenofibrate combination for cardiovascular stents

Atorvastatin and fenofibrate have been conventionally employed as lipid-lowering agents. They also exhibit beneficial effects in the treatment of endothelial dysfunction, oxidative stress and vascular inflammation due to their pleiotropic effects that include vasodilatory and anti-inflammatory effects. These pleiotropic effects may serve to overcome the drawbacks of late stent thrombosis and delayed endothelialization that plague conventional drug eluting stents. However, the combination has not been explored yet as therapeutic coatings in drug eluting stents. The present study aims to investigate the potential of atorvastatin-fenofibrate combination loaded in a biodegradable poly(l-lactide-co-caprolactone) polymer film to inhibit thrombus formation and macrophage activation apart from exploring their effect on the proliferation of smooth muscle cells and endothelial cells. The dual drug-loaded polymer films were characterized by spectroscopy and calorimetry. In vitro studies revealed that the combination effectively retarded the proliferation of only smooth muscle cells but not the endothelial cells which augers well for stent applications where rapid re-endothelialization is preferred. Further, the dual drug-loaded films exhibited a marked decrease in the adhesion and activation of platelets and macrophages revealing the potent anti-thrombogenic and anti-inflammatory effects of the combination. The pleiotropic effects of the combination may be attributed to their ability to activate nitric oxide synthase in endothelial cells while mTOR levels remained unaltered by the combination.

Aerobic exercise training-induced changes in serum C1q/TNF-related protein levels are associated with reduced arterial stiffness in middle-aged and older adults

Adiponectin regulates endothelial nitric oxide synthase in endothelial cells, and body fat loss by aerobic exercise training promotes adiponectin secretion. Recently, C1q/tumor necrosis factor-related proteins (CTRPs) have been identified as novel adipokines and are paralogs of adiponectin, but the association between exercise training-induced reduction of arterial stiffness and circulating CTRPs levels remains unclear. This study aimed to clarify whether the reduction of arterial stiffness in middle-aged and older adults is associated with the change in serum levels of CTRPs induced by exercise training. A total of 52 middle-aged and older participants were randomly divided into two groups: a training group ( n = 26) and a sedentary control group ( n = 26). Participants in the training group completed 8 wk of aerobic exercise training (60-70% peak oxygen uptake for 45 min, 3 days/wk). The reduction of percent whole body fat, abdominal visceral fat area, and carotid-femoral pulse-wave velocity (cfPWV) was significantly greater in the training group than in the control group ( P < 0.05). Moreover, the increase in serum adiponectin, CTRP3, and CTRP5 from baseline to 8 wk was significantly higher in the training group compared with the control group ( P < 0.05). Additionally, the training-induced change in cfPWV was negatively correlated with the training-induced change in serum adiponectin, CTRP3, and CTRP5 levels ( r = -0.51, r = -0.48, r = -0.42, respectively, P < 0.05), and increased plasma nitrite/nitrate level by exercise training was correlated only with adiponectin levels ( r = 0.41, P < 0.05). These results suggest that the exercise training-induced increase in serum CTRPs levels may be associated with the reduction of arterial stiffness in middle-aged and older adults.

Comparison of morphofunctional features of the ventral hippocampus in adult and old rats after combined stress

Animals were exposed to 7 days of combined stress in a special device consisting of six isolated compartments of similar volumes. During the 30-min exposure, the stressors of various modalities, including pulse lighting, heavy sound, and vibration, were presented and stochastically changed every 5 min. This treatment was coincided with restraint and elevated temperature in the compartments. We observed more severe dystrophic lesion and highly expressed inducible nitric oxide synthase in neurons of the pyramidal layer of the CA3 field, signs of impaired blood flow, perivascular edema, and decreased expression of endothelial nitric oxide synthase in endothelial cells of the microvasculature, and lower expression of serine racemase in neuropil of the radial layer of the CA1 field in the ventral hippocampi after the exposure of 24-month-old rats to combined stress as compared to adult 12-month-old rats.

Targeted overexpression of endothelial nitric oxide synthase in endothelial cells improves cerebrovascular reactivity in Ins2Akita-type-1 diabetic mice

Reduced bioavailability of nitric oxide due to impaired endothelial nitric oxide synthase (eNOS) activity is a leading cause of endothelial dysfunction in diabetes. Enhancing eNOS activity in diabetes is a potential therapeutic target. This study investigated basal cerebral blood flow and cerebrovascular reactivity in wild-type mice, diabetic mice (Ins2Akita+/−), nondiabetic eNOS-overexpressing mice (TgeNOS), and the cross of two transgenic mice (TgeNOS-Ins2Akita+/−) at six months of age. The cross was aimed at improving eNOS expression in diabetic mice. The major findings were: (i) Body weights of Ins2Akita+/− and TgeNOS-Ins2Akita+/− were significantly different from wild-type and TgeNOS mice. Blood pressure of TgeNOS mice was lower than wild-type. (ii) Basal cerebral blood flow of the TgeNOS group was significantly higher than cerebral blood flow of the other three groups. (iii) The cerebrovascular reactivity in the Ins2Akita+/− mice was significantly lower compared with wild-type, whereas that in the TgeNOS-Ins2Akita+/− was significantly higher compared with the Ins2Akita+/− and TgeNOS groups. Overexpression of eNOS rescued cerebrovascular dysfunction in diabetic animals, resulting in improved cerebrovascular reactivity. These results underscore the possible role of eNOS in vascular dysfunction in the brain of diabetic mice and support the notion that enhancing eNOS activity in diabetes is a potential therapeutic target.

Polygonum viviparum L. induces vasorelaxation in the rat thoracic aorta via activation of nitric oxide synthase in endothelial cells.

BackgroundIn the past several decades, Polygonum viviparum L. (PV) was reported to have antibacterial, antiulcer, antioxidant, antitumor, anti-inflammatory, and antiarthritic properties. The anti-inflammatory pathway was recently elucidated through cytosolic nuclear factor E2-related factor 2 (Nrf2) activation and heme oxygenase (HO)-1 protein expression. PV is a perennial herb and widely distributed in high-elevation mountain regions, such as the Tibetan Plateau. In Tibetan traditional medicine, PV is usually used to boost the blood circulation to dissipate blood stasis. Therefore, this study focused on how PV improves the vascular circulation and acts on vascular tissues.MethodsIn this study, we isolated aortas from Sprague-Dawley rats (male, weight about 250 ~ 350 g), and detected the effects of PV on phenylephrine (PE)-induced contraction and cyclic guanosine 3′,5′-monophosphate (cGMP) formation using aortic rings. In addition, human umbilical vein endothelial cells (HUVECs) were used to exam nitric oxygen (NO) synthase (NOS) activity by directly measuring NO production in the culture medium. Endothelial (e) NOS phosphorylation, and cytosolic Nrf2 and HO-1 expressions were measured using a Western blot analysis.ResultsPV dose-dependently relaxed PE-induced contractions in endothelial-intact but not -denuded aorta. The concentration to produce 50% relaxation was 22.04 ± 1.77 μg/ml. PV-induced vasorelaxation was markedly blocked by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), an NOS inhibitor, methylene blue (MB), a guanylyl cyclase inhibitor, and hemoglobin, an NO scavenger. PV increased cGMP formation; however, this effect was also suppressed by co-pretreatment with l-NAME, MB, hemoglobin, and Ca2+-free medium. In HUVECs, PV increased NO formation, which was greatly attenuated by NOS inhibitors (L-NAME and L-NMMA) and by removing extracellular Ca2+ and chelating intracellular Ca2+ with BAPTA-AM. In addition, PV promoted eNOS phosphorylation, Nrf2 degradation, and HO-1 protein expression according to a Western blot analysis.ConclusionsThe results suggest that PV possesses vasorelaxing action in an endothelium-dependent manner and works through activating Ca2+/calmodulin- dependent NO synthesis; when NO is released and then transferred to smooth muscle cells, NO activates guanylyl cyclase and increases cGMP formation, ultimately resulting in vasorelaxation. Thus, PV can be considered for application as a potential therapeutic approach for vascular-associated disorders.

Mechanism of hypertension and proteinuria during angiogenesis inhibition

Angiogenesis inhibition by blocking vascular endothelial growth factor (VEGF)-mediated signalling with monoclonal antibodies or tyrosine kinase inhibitors has become an established treatment of various forms of cancer. This treatment is frequently associated with the development of hypertension and proteinuria. As VEGF increases the expression and the activity of nitric oxide synthase in endothelial cells, a decrease in the bioavailability of nitric oxide has been proposed as a key mechanism leading to hypertension during angiogenesis inhibition. However, results of clinical and experimental studies exploring this possibility are conflicting. Rarefaction, that is a structural decrease of microcirculatory vessels, has been reported during antiangiogenic treatment, but evidence that it plays a role in development of hypertension is lacking. Elevated circulating and urinary levels of endothelin-1 have been observed in clinical and experimental studies with angiogenesis inhibitors. Furthermore, the observation that endothelin receptor blockers can prevent or revert the rise in blood pressure during angiogenesis inhibition and attenuate proteinuria provides strong evidence that an activated endothelin-signalling pathway is a final common mediator of angiogenesis inhibition-induced rise in blood pressure and renal toxicity.

Hydrogen sulfide increases nitric oxide production with calcium-dependent activation of endothelial nitric oxide synthase in endothelial cells

Hydrogen sulfide (H2S) was recently discovered to be synthesized in mammalian tissues by several different enzymes. Numerous studies have shown that H2S has vasodilator and antihypertensive effects in the cardiovascular system. However, intracellular mechanisms of the H2S-induced vasodilation and its interactions with other endothelium-derived relaxing factors, such as nitric oxide (NO), remain unclear. We investigated whether H2S directly regulates endothelial NO synthase (eNOS) activity and NO production in endothelial cells. NaHS, a H2S donor, dose-dependently increased NO production in cultured endothelial cells. This effect was abolished by a calcium chelator (BAPTA-AM), but not by the absence of extracellular calcium. The NaHS-induced NO production was partially blocked by inhibitors of ryanodine receptor (dantrolene) or inositol 1,4,5-triphosphate receptor (xestospongin C). NaHS significantly increased intracellular calcium concentrations, and this effect was attenuated by dantrolene or xestospongin C. NaHS induced phosphorylation of eNOS at the activating phosphoserine residue 1179. The NaHS-induced eNOS phosphorylation and NO production were not affected by a PI3K/Akt inhibitor (wortmannin). The data of this study suggest that H2S directly acts on endothelial cells to induce eNOS activation and NO production by releasing calcium from the intracellular store in endoplasmic reticulum, which may explain one of mechanisms of its vasodilator function.

Abstract 114: Pomegranate Regressed Atheroma in Carotid and Coronary Arteries: Fiction or Fact?

Background: Atherosclerosis is a disease of large and medium-sized muscular arteries and is characterized by endothelial dysfunction, vascular inflammation, and the buildup of lipids, cholesterol, calcium, and cellular debris within the intima of the vessel wall, oxidized low-density lipoprotein (LDL) can reduce the expression of nitric oxide synthase, the enzyme that produces nitric oxide. Nitric oxide reduces injury to the vessel walls, which also helps prevent the development of atherosclerosis Recently, they discovered that pomegranate juice enhances the bioactivity of nitric oxide synthase in endothelial cells. Furthermore, pomegranate’s antioxidant properties protect nitric oxide from oxidative destruction, thus augmenting its biological actions. Therefore the objective of present study was to assess the effect of pomegranateon atherosclerosis regression via interfering with inflammatory and oxidative pathways. Methods: 60 local domestic rabbits were assigned to three groups: Group I (n = 30) fed normal diet,Group II ( n = 30) Rabbit fed high cholesterol-diet (induced untreated group), Group III ( n = 30) high cholesterol-diet for 12 weeks but adding oral pomegranate juice at 6 weeks .Blood samples were collected at (0 time ), after 6 and 12 weeks for measurement of serum triglycerides (TG), total cholesterol (TC), HDL-C and serum high sensitive C-Reactive Protein (hsCRP), serum IL-6 and serum TNF-a. At the end of 12 weeks,both carotid and right coronary arteries was removed, part of it for histopathology to assess the atherosclerotic change according to AmericanHeart Association classification of atherosclerosis phases and for aortic intema-media thickness also part of it homogenized for malondialdehyde (MDA) and reduced glutathione (GSH). Results: Compared with the control, levels of TC, TG, LDL-C, VLDL-C, atherogenic index, hsCRP, IL-6, TNF-a and arterial MDA were increased and arterial GSH and serum HDL-C were decreased in the animals with a high-choleterole diet ( P &lt; 0.01). Histologically all induced-untreated rabbit showed significant atherosclerosis lesions in both arteries ( P &lt; 0.01) all occurred at 6 and 12 weeks. While in pomegranate treated group at 12 weeks, levels of TC, TG, LDL-C, VLDL-C, atherogenic index, hsCRP, IL-6, TNF-a and arterial tissue of MDA were decreased and coronary and carotid GSH and serum HDL-C were increased relatively ( P &lt; 0.01).Low power microscopic morphologic analysis revealed that pomegranate treated group markedly regressed ( P &lt; 0.01) the atherosclerotic lesion in form of decrease intima-media thickness in histomorphometric measurements in the studied arteries at 12 weeks compared with rabbits on a high-fat diet at same period. In conclusion pomegranate juiceregressed atherosclerosis in carotid and coronary arteries in hypercholesterolemic rabbit via inhibition of inflammatory, oxidative pathways, reduced level of lipid parameters and decreased aortic intima-media thickness.

The Use of l-Arginine in the Management of Pre-Eclampsia and Intrauterine Growth Restriction

The Use of l-Arginine in the Management of Pre-Eclampsia and Intrauterine Growth Restriction

Identification of Magnetic Nanoparticles for Combined Positioning and Lentiviral Transduction of Endothelial Cells

The combination of magnetic nanoparticles (MNPs) with a magnetic field is a powerful approach to enable cell positioning and/or local gene therapy. Because physical requirements for MNPs differ between these two applications we have explored whether the use of different MNPs can provide site-specific positioning combined with efficient viral transduction of endothelial cells (ECs). A variety of MNPs was screened for magnetic cell labeling and lentivirus binding. Then two different MNPs were chosen and their combined application was evaluated regarding EC magnetization and transduction efficiency. The combined use of PEI-Mag2 and NDT-Mag1 particles provided both efficient lentiviral transduction and high magnetic responsiveness of ECs that could be even retained within the vascular wall under flow conditions. The use of these MNPs did not affect biological characteristics of ECs like surface marker expression and vascular network formation. Importantly, with this method we could achieve an efficient functional overexpression of endothelial nitric oxide synthase in ECs. The application of two different MNPs provides optimal results for magnetic labeling of ECs in combination with viral transduction. This novel approach could be very useful for targeted gene therapy ex vivo and site-specific cell replacement in the vascular system.

Tat-Mediated p66shc Transduction Decreased Phosphorylation of Endothelial Nitric Oxide Synthase in Endothelial Cells

We evaluated the role of Tat-mediated p66shc transduction on the activation of endothelial nitric oxide synthase in cultured mouse endothelial cells. To construct the Tat-p66shc fusion protein, human full length p66shc cDNA was fused with the Tat-protein transduction domain. Transduction of TAT-p66shc showed a concentration- and time-dependent manner in endothelial cells. Tat-mediated p66shc transduction showed increased hydrogen peroxide and superoxide production, compared with Tat-p66shc (S/A), serine 36 residue mutant of p66shc. Tat-mediated p66shc transduction decreased endothelial nitric oxide synthase phosphorylation in endothelial cells. Furthermore, Tat-mediated p66shc transduction augmented TNF-α-induced p38 MAPK phosphorylation in endothelial cells. These results suggest that Tat-mediated p66shc transduction efficiently inhibited endothelial nitric oxide synthase phosphorylation in endothelial cells.

Gap Junction Protein Cx37 Interacts With Endothelial Nitric Oxide Synthase in Endothelial Cells

The gap junction protein connexin37 (Cx37) plays an important role in cell-cell communication in the vasculature. A C1019T Cx37 gene polymorphism, encoding a P319S substitution in the regulatory C terminus of Cx37 (Cx37CT), correlates with arterial stenosis and myocardial infarction in humans. This study was designed to identify potential binding partners for Cx37CT and to determine whether the polymorphism modified this interaction. Using a high-throughput phage display, we retrieved 2 binding motifs for Cx37CT: WHK ... [K,R]XP ... and FHK ... [K,R]XXP ... , the first being more common for Cx37CT-319P and the second more common for Cx37CT-319S. One of the peptides (WHRTPRLPPPVP) showed 77.7% homology with residues 843 to 854 of endothelial nitric oxide synthase (eNOS). In vitro binding of this peptide or of the homologous eNOS sequence to both Cx37CT isoforms was confirmed by cross-linking and surface plasmon resonance. Electrophysiological analysis of Cx37 single channel activity in transfected N2a cells showed that eNOS-like and eNOS(843-854) increased the frequency of events with conductances higher than 300 pS. We demonstrated that eNOS coimmunoprecipitated with Cx37 in a mouse endothelial cell (EC) line (bEnd.3), human primary ECs, and a human EC line transfected with Cx37-319P or Cx37-319S. Cx37 and eNOS colocalized at EC membranes. Moreover, a dose-dependent increase in nitric oxide production was observed in ECs treated with Cx37 antisense. Overall, our data show for the first time a functional and specific interaction between eNOS and Cx37. This interaction may be relevant for the control of vascular physiology both in health and in disease.

Gene Therapy With the Angiogenic Cytokine Secretoneurin Induces Therapeutic Angiogenesis by a Nitric Oxide–Dependent Mechanism

The neuropeptide secretoneurin induces angiogenesis and postnatal vasculogenesis and is upregulated by hypoxia in skeletal muscle cells. We sought to investigate the effects of secretoneurin on therapeutic angiogenesis. We generated a secretoneurin gene therapy vector. In the mouse hindlimb ischemia model secretoneurin gene therapy by intramuscular plasmid injection significantly increased secretoneurin content of injected muscles, improved functional parameters, reduced tissue necrosis, and restored blood perfusion. Increased muscular density of capillaries and arterioles/arteries demonstrates the capability of secretoneurin gene therapy to induce therapeutic angiogenesis and arteriogenesis. Furthermore, recruitment of endothelial progenitor cells was enhanced by secretoneurin gene therapy consistent with induction of postnatal vasculogenesis. Additionally, secretoneurin was able to activate nitric oxide synthase in endothelial cells and inhibition of nitric oxide inhibited secretoneurin-induced effects on chemotaxis and capillary tube formation in vitro. In vivo, secretoneurin induced nitric oxide production and inhibition of nitric oxide attenuated secretoneurin-induced effects on blood perfusion, angiogenesis, arteriogenesis, and vasculogenesis. Secretoneurin also induced upregulation of basic fibroblast growth factor and platelet-derived growth factor-B in endothelial cells. In summary, our data indicate that gene therapy with secretoneurin induces therapeutic angiogenesis, arteriogenesis, and vasculogenesis in the hindlimb ischemia model by a nitric oxide-dependent mechanism.

Mechanism of Purinergic Activation of Endothelial Nitric Oxide Synthase in Endothelial Cells

Decreased endothelial nitric oxide (NO) synthase (eNOS) activity and NO production are critical contributors to the endothelial dysfunction and vascular complications observed in many diseases, including diabetes mellitus. Extracellular nucleotides activate eNOS and increase NO generation; however, the mechanism of this observation is not fully clarified. To elucidate the signaling pathway(s) leading to nucleotide-mediated eNOS phosphorylation at Ser-1177, human umbilical vein endothelial cells were treated with several nucleotides, including ATP, UTP, and ADP, in the presence or absence of selective inhibitors. These experiments identified P2Y1, P2Y2, and possibly P2Y4 as the purinergic receptors involved in eNOS phosphorylation and demonstrated that this process was adenosine independent. Nucleotide-induced eNOS phosphorylation and activity were inhibited by BAPTA-AM (an intracellular free calcium chelator), rottlerin (a protein kinase Cdelta inhibitor), and protein kinase Cdelta siRNA. In contrast, blockade of AMP-activated protein kinase, calcium/calmodulin-dependent kinase II, calcium/calmodulin-dependent kinase kinase, serine/threonine protein kinase B, protein kinase A, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase did not affect nucleotide-mediated eNOS phosphorylation. The present study indicates that extracellular nucleotide-mediated eNOS phosphorylation is calcium and protein kinase Cdelta dependent. This newly identified signaling pathway opens new therapeutic avenues for the treatment of endothelial dysfunction.

A novel fluorescent probe for the detection of nitric oxide in vitro and in vivo

Fluorescence imaging of nitric oxide (NO) in vitro and in vivo is essential to developing our understanding of the role of nitric oxide in biology and medicine. Current probes such as diaminofluorescein depend on reactions with oxidized NO products, but not with nitric oxide directly, and this limits their applicability. Here we report the formation of an imaging probe for nitric oxide by coordinating the highly fluorescent chemical 4-methoxy-2-(1 H-naphtho[2,3- d]imidazol-2-yl)phenol (MNIP) with Cu(II). The coordination compound MNIP–Cu reacts rapidly and specifically with nitric oxide to generate a product with blue fluorescence that can be used in vitro and in vivo. In the present study MNIP–Cu was used to reveal nitric oxide produced by inducible nitric oxide synthase in lipopolysaccharide (LPS)-activated macrophages (Raw 264.7 cells) and by endothelial nitric oxide synthase in endothelial cells (HUVEC). MNIP–Cu was also used to evaluate the distribution of nitric oxide synthesis in a model of acute liver injury induced by LPS and d-galactosamine in mice. The results demonstrate that MNIP–Cu can act as a novel fluorescent probe for nitric oxide and has many potential applications in biomedical research.

Protein Never in Mitosis Gene A Interacting-1 (PIN1) regulates degradation of inducible nitric oxide synthase in endothelial cells

The peptidyl-proline isomerase Protein Never in Mitosis Gene A Interacting-1 (PIN1) increases the level or activity of several transcription factors that can induce the inducible nitric oxide (NO) synthase (iNOS). PIN1 can also regulate mRNA and protein turnover. Here, the effect of depletion of PIN1 on induction of iNOS by Escherichia coli endotoxin (LPS) and interferon-gamma (IFNgamma) in murine aortic endothelial cells (MAEC) was determined. Suppression of PIN1 by 85% with small hairpin RNA enhanced the induction of NO and iNOS protein by LPS-IFNgamma. There was no effect on induction of iNOS mRNA, suggesting a posttranscriptional effect. The enhanced levels of iNOS protein were functionally significant since LPS-IFNgamma was cytotoxic to MAEC lacking PIN1 but not MAEC harboring an inactive control construct, and because cytotoxicity was blocked by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester. Consistent with posttranscriptional action, knockdown of PIN1 increased the stability of iNOS protein in cycloheximide-treated cells. Furthermore, loss of iNOS was blocked by the calpain inhibitor carbobenzoxy-valinyl-phenylalaninal but not by the selective proteasome inhibitor epoxomicin. Immunoprecipitation indicated that PIN1 can interact with iNOS. Pull down of iNOS with a wild-type glutathione-S-transferase-PIN1 fusion protein, but not with a mutant of the amino terminal phospho-(serine/threonine)-proline binding WW domain of PIN1, indicated that this domain mediates interaction. The results suggest that PIN1 associates with iNOS and can limit its induction by facilitating calpain-mediated degradation in MAEC.

Novel platinum pyridinehydroxamic acid complexes: Synthesis, characterisation, X-ray crystallographic study and nitric oxide related properties

Herein, we describe the synthesis and characterisation of a novel class of Pt II and Pt IV pyridinehydroxamic acid (pyhaH) complexes of general formula cis-[Pt IICl 2( x-pyhaH) 2] and cis-[Pt IVCl 4( x-pyhaH) 2], respectively (where x = 3 or 4) in which the pyridinehydroxamic acid is coordinated to the platinum ion via the pyridine nitrogen only leaving the hydroxamic acid free to potentially release cytotoxic nitric oxide (NO). The crystal structure of the Pt IV derivative, cis-[PtCl 4(4-pyhaH) 2] · 2CH 3OH is reported. To establish the biological effect of the uncoordinated hydroxamic acid moiety in the Pt II compounds synthesised, the corresponding pyridinecarboxylic acid (pycaH) complexes of general formula cis-[Pt IICl 2( x-pycaH) 2] (where x = 3 or 4) and the Pt II pyridine (py) complex, cis-[Pt IICl 2(py) 2] were synthesised and served as reference standards. The NO-releasing properties of each of the Pt II compounds, the pyhaH and the pycaH ligands were studied. The Pt II pyridinehydroxamic acid derivatives were found to induce potent in vitro effects attributable to either NO-release from the hydroxamic acid moiety and/or stimulation of inducible nitric oxide synthase of endothelial cells.

Regulation of SR-BI-mediated selective lipid uptake in Chinese hamster ovary-derived cells by protein kinase signaling pathways

Scavenger receptor, class B, type I (SR-BI) mediates binding and internalization of a variety of lipoprotein and nonlipoprotein ligands, including HDL. Studies in genetically engineered mice revealed that SR-BI plays an important role in HDL reverse cholesterol transport and protection against atherosclerosis. Understanding how SR-BI's function is regulated may reveal new approaches to therapeutic intervention in atherosclerosis and heart disease. We utilized a model cell system to explore pathways involved in SR-BI-mediated lipid uptake from and signaling in response to distinct lipoprotein ligands: the physiological ligand, HDL, and a model ligand, acetyl LDL (AcLDL). In Chinese hamster ovary-derived cells, murine SR-BI (mSR-BI) mediates lipid uptake via distinct pathways that are dependent on the lipoprotein ligand. Furthermore, HDL and AcLDL activate distinct signaling pathways. Finally, mSR-BI-mediated selective lipid uptake versus endocytic uptake are differentially regulated by protein kinase signaling pathways. The protein kinase C (PKC) activator PMA and the phosphatidyl inositol 3-kinase inhibitor wortmannin increase the degree of mSR-BI-mediated selective lipid uptake, whereas a PKC inhibitor has the opposite effect. These data demonstrate that SR-BI's selective lipid uptake activity can be acutely regulated by intracellular signaling cascades, some of which can originate from HDL binding to murine SR-BI itself.

Retraction Note to: Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells

This article is retracted by the request of the corresponding author. The corresponding author is deeply sorry for any inconvenience this may have caused to the Experimental and Molecular Medicine and to the readers.

Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells

Fluid shear stress plays a critical role in vascular health and disease. While protein kinase A (PKA) has been implicated in shear-stimulated signaling events in endothelial cells, it remains unclear whether and how PKA is stimulated in response to shear stress. This issue was addressed in the present study by monitoring the phosphorylation of endogenous substrates of PKA. Shear stress stimulated the phosphorylation of cAMP responsive element binding protein (CREB) in a PKA-dependent manner. Western blot analysis using the antibody reactive against the consensus motif of PKA substrates detected two proteins, P135 and P50, whose phosphorylation was increased by shear stress. The phosphorylation of P135 was blocked by a PKA inhibitor, H89, but not by a phosphoinositide 3-kinase inhibitor, wortmannin. Expression of a constitutively active PKA subunit stimulated P135 phosphorylation, supporting the potential of P135 as a PKA substrate. P135 was identified as endothelial nitric oxide synthase (eNOS) by immunoprecipitation study. PKA appeared to mediate shear stress-stimulated eNOS activation. Shear stress stimulated intracellular translocation of PKA activity from 'soluble' to 'particulate' fractions without involving cellular cAMP increase. Taken together, this study suggests that shear stress stimulates PKA-dependent phosphorylation of target proteins including eNOS, probably by enhancing intracellular site-specific interactions between protein kinase and substrates.