Homocysteine decreases platelet NO level via protein kinase C activation

doi:10.1016/j.niox.2008.11.005

Nitric Oxide

Volume 20, Issue 2, 1 March 2009, Pages 104-113

https://doi.org/10.1016/j.niox.2008.11.005 Get rights and content

Abstract

Hyperhomocysteinaemia has been associated with increased risk of thrombosis and atherosclerosis. Homocysteine produces endothelial injury and stimulates platelet aggregation. Several molecular mechanisms related to these effects have been elucidated. The study aimed to deeply investigate the homocysteine effect on nitric oxide formation in human platelets. The homocysteine-induced changes on nitric oxide, cGMP, superoxide anion levels and nitrotyrosine formation were evaluated. The enzymatic activity and the phosphorylation status of endothelial nitric oxide synthase (eNOS) at thr495 and ser1177 residues were measured. The protein kinase C (PKC), assayed by immunofluorescence confocal microscopy technique and by phosphorylation of p47pleckstrin, and NADPH oxidase activation, tested by the translocation to membrane of the two cytosolic subunits p47^phox and p67^phox, were assayed. Results show that homocysteine reduces platelet nitric oxide and cGMP levels. The inhibition of eNOS activity and the stimulation of NADPH oxidase primed by PKC appear to be involved. PKC stimulates the eNOS phosphorylation of the negative regulatory residue thr495 and the dephosphorylation of the positive regulatory site ser1177. GF109203X and U73122, PKC and phospholipase Cγ2 pathway inhibitors, respectively, reverse this effect. Moreover, homocysteine stimulates superoxide anion elevation and NADPH oxidase activation. These effects are significantly decreased by GF109203X and U73122, suggesting the involvement of PKC in NADPH oxidase activation. Homocysteine induces formation of the peroxynitrite biomarker nitrotyrosine. Taken together these results suggest that the homocysteine-mediated responses leading to nitric oxide impairment are mainly coupled to PKC activation. Thus homocysteine stimulates platelet aggregation and decreases nitric oxide bioavailability.

Section snippets

Reagents

l-arginine, cysteine, dithiothreitol, Dowex AG 50W-X8, FAD, FMN, homocysteine, indomethacin, leupeptin, methionine, NADPH, PMSF (phenylmethanesulfonyl fluoride), PMA (phorbol-12-myristate-13-acetate), potent and direct activator of PKC, tetrahydrobiopterin, thrombin, U73122 (1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-ylamino)hexyl]-1H-pyrrole-2,5-dione) inhibitor of PLCγ2 and all chemical reagents were from Sigma–Aldrich, USA. Apocynin (4-hydroxy-3-methoxyacetophenone), inhibitor of NADPH

Effect of homocysteine on NO and cGMP basal level

Homocysteine reduces platelet NO basal level, being the effect significant (P < 0.001) at all tested concentrations (Fig. 1A). No effect was evaluated when platelets were incubated with 100 μM cysteine or 100 μM methionine (data not shown). As additional evidence for the homocysteine effect cGMP intracellular level was evaluated. Results (Fig. 1B) show that homocysteine also decreases cGMP formation. Values indicating cGMP and NO levels, measured in the presence of varying homocysteine

Discussion

Hyperhomocysteinaemia is considered an independent risk factor for various arterial thrombosis [34], [35], even if several intervention trials have failed to demonstrate any clinical benefit from homocysteine-lowering therapy [36], [37]. Hyperhomocysteinaemia has been associated with increased platelet activation [38], [39], [40] and increased sensitivity to agonists in peripheral vascular disease [41]. Patients with elevated plasma homocysteine concentration have increased circulating plasma

Acknowledgments

This study was supported by the Grant No. 020306006029 from the Ministero della Salute, Rome, Italy. We thank the “Centro Trasfusionale, Ospedale San Martino” of Genoa for blood drawing assistance.

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Homocysteine (Hcy) is a sulphur amino-acid, metabolite of methionine catabolism and folate cycle. Increased plasma levels of Hcy have been related to a negative impact on the pathophysiology of vascular disease [8–11], including endothelial dysfunction, impairment of methylation, and enhanced oxidative stress [12] and platelet activation [13,14], by raising the production of thromboxane A2 (TxA2) [15] and reducing nitric oxide (NO) [16]. In fact, previous reports documented an association between suboptimal inhibition with antiplatelet agents (high-on treatment residual platelet reactivity, HRPR) and elevated Hcy [17].
Dual antiplatelet therapy (DAPT) and Renin-angiotensin system inhibitors (RASi) represent the cornerstone in the treatment of patients undergoing percutaneous coronary interventions (PCI), mainly after an acute ischemic event. However, high-on treatment residual platelet reactivity (HRPR), is not infrequent despite optimal medical treatment. Homocysteine (Hcy) is a metabolite of methionine catabolism linked to atherothrombosis. Recently, a potential crosstalk between RAS and Hcy has been suggested, potentially favouring platelet aggregation and cardiovascular disease.Therefore, we aimed to investigate the impact of RASi on Hcy levels and platelet aggregation in patients on DAPT after PCI.
Patients undergoing PCI on DAPT with ASA plus an ADP-antagonist (clopidogrel, ticagrelor or prasugrel), were included. RASi comprised angiotensin converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARB). Aggregation tests were performed by Multiple Electrode Aggregometry. We included 1210 patients, of whom 862 (71.2%) were on treatment with RASi. Overall, DAPT composition was ASA+clopidogrel in 566 (46.8%) patients, ASA+ticagrelor in 428 (35.4%) and ASA+prasugrel in 216 (17.9%). Median values of Hcy were higher in RASi patients (p = 0.006), who displayed a higher percentage of Hcy above the median value (52.4% vs. 44.8%, p = 0.019, adjustedOR [95%CI] = 1.40 [1.04–1.88], p = 0.027). No differences in HRPR rate were found according to RASi use for ASPI test (3.6% vs. 3.3%, p = 0.88) and ADP test (25.6% vs. 24.3%,p = 0.62; adjustedOR [95%CI] = 1.23 [0.89–1.70], p = 0.220) and according to ADP-antagonist type. A direct linear relationship was observed between platelet reactivity and Hcy in both patients receiving RASi and untreated ones, with higher values of platelet aggregation being observed in patients with Hcy above the median, independently from RASi administration and DAPT strategy.
In patients on DAPT after PCI, RASi treatment did not emerge as an independent predictor of HRPR. However, the levels of Hcy were significantly elevated in patients on RASi and related to higher values of platelet reactivity, independently from the DAPT strategy.
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Enhanced levels of homocysteine during pregnancy induce oxidative stress and contribute to many age-related diseases. In this study, we analyzed age-dependent synaptic modifications in developing neuromuscular synapses of rats with prenatal hyperhomocysteinemia (hHCY). One of the main findings indicate that the intensity and the timing of transmitter release in synapses of neonatal (P6 and P10) hHCY rats acquired features of matured synaptic transmission of adult rats. The amplitude and frequency of miniature end-plate currents (MEPCs) and evoked transmitter release were higher in neonatal hHCY animals compared to the control group. Analysis of the kinetics of neurotransmitter release demonstrated more synchronized release in neonatal rats with hHCY. At the same time lower release probability was observed in adults with hHCY. Spontaneous transmitter release in neonates with hHCY was inhibited by hydrogen peroxide (H₂O₂) whereas in controls this oxidant was effective only in adult animals indicating a higher susceptibility of motor nerve terminals to oxidative stress. The morphology and the intensity of endocytosis of synaptic vesicles in motor nerve endings was assessed using the fluorescence dye FM 1-43. Adult-like synapses were found in neonates with hHCY which were characterized by a larger area of presynaptic terminals compared to controls. No difference in the intensity of FM 1-43 fluorescence was observed between two groups of animals. Prenatal hHCY resulted in reduced muscle strength assessed by the Paw Grip Endurance test. Using biochemical assays we found an increased level of H₂O₂ and lipid peroxidation products in the diaphragm muscles of hHCY rats. This was associated with a lowered activity of superoxide dismutase and glutathione peroxidase. Our data indicate that prenatal hHCY induces oxidative stress and apparent faster functional and morphological "maturation" of motor synapses. Our results uncover synaptic mechanisms of disrupted muscle function observed in hHCY conditions which may contribute to the pathogenesis of motor neuronal diseases associated with enhanced level of homocysteine.
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Although the limitation of the study was the use of relatively high compared to physiological concentrations of Hcy, the short-term exposure (6 h) 10 mM Hcy may reflect a life-long exposure to moderately elevated levels of Hcy. Hcy suppresses activity of endothelial nitric oxide synthase (eNOS) and cellular arginine transport resulting in decreases bioavailability of nitric oxide (Jin et al., 2007; Signorello et al., 2009). The inhibition of eNOS induces endothelial-to-mesenchymal transition characterized by the loss of cell polarity and endothelial specific morphology and acquisition of myofibroblast-like features (O’Riordan et al., 2007).
There are numerous theories of aging, a process which still seems inevitable. Aging leads to cancer and multi-systemic disorders as well as chronic diseases. Decline in age- associated cellular functions leads to neurodegeneration and cognitive decline that affect the quality of life. Accumulation of damage, mutations, metabolic changes, failure in cellular energy production and clearance of altered proteins over the lifetime, and hyperhomocysteinemia, ultimately result in tissue degeneration. The decline in renal functions, nutritional deficiencies, deregulation of methionine cycle and deficiencies of homocysteine remethylation and transsulfuration cofactors cause elevation of homocysteine with advancing age. Abnormal accumulation of homocysteine is a risk factor of cardiovascular, neurodegenerative and chronic kidney disease. Moreover, approximately 50% of people, aged 65 years and older develop hypertension and are at a high risk of developing cardiovascular insufficiency and incurable neurodegenerative disorders. Increasing evidence suggests inverse relation between cognitive impairment, cerebrovascular and cardiovascular events and renal function. Oxidative stress, inactivation of nitric oxide synthase pathway and mitochondria dysfunction associated with impaired homocysteine metabolism lead to aging tissue degeneration. In this review, we examine impact of high homocysteine levels on changes observed with aging that contribute to development and progression of age associated diseases.
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In addition, phosphorylation on serine (Ser1177) and theonine (Thr495) residues [8] have been discussed extensively to regulate eNOS. The phosphorylation of Ser1177 is involved in multiple signaling pathways in the cardiovascular conditions [9]. Overall, we hypothesize that under oxidative stress, eNOS expression and activity are regulated through a VPO1 mediated signaling pathway.
Vascular peroxidase 1 (VPO1) is a member of the peroxidase family which aggravates oxidative stress by producing hypochlorous acid (HOCl). Our previous study demonstrated that VPO1 plays a critical role in endothelial dysfunction through dimethylarginine dimethylaminohydrolase2 (DDAH2)/asymmetric Dimethylarginine (ADMA) pathway. Hereby we describe the regulatory role of VPO1 on endothelial nitric oxide synthase (eNOS) expression and activity in human umbilical vein endothelial cells (HUVECs). In HUVECs AngiotensinII (100 nM) treatment reduced Nitric Oxide (NO) production, decreased eNOS expression and activity, which were reversed by VPO1 siRNA. Knockdown of VPO1 also attenuated ADMA production and eNOS uncoupling while enhancing phosphorylated ser1177 eNOS expression level. Furthermore, HOCl stimulation was shown to directly induce ADMA production and eNOS uncoupling, decrease phosphorylated ser1177 eNOS expression. It also significantly suppressed eNOS expression and activity together with NO production. Therefore, VPO1 plays a vital role in regulating eNOS expression and activity via hydrogen peroxide (H2O2)-VPO1-HOCl pathway.
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In vivo, pENW inhibited thrombus formation induced by endothelial injury in a dose-dependent manner, with a significantly prolonged time to the occurrence of arterial occlusion. It was shown that pENW offered protection for blood vessels from oxidative injury. pENW significantly increased NO production in rats treated with pENW at 4 or 2 mg/kg body weight. Furthermore, the production of NO from the cultured vascular endothelial cells was increased with the treatment of 10^–4 M and 10^–5 M pENW; pENW also enhanced eNOS expression and activity both in vivo and in vitro, and elicited a concentration-dependent vasorelaxation which was significantly inhibited by L-NAME. Notably, pENW inhibited ADP-induced platelet aggregation, and the inhibition was more significant in the presence of NO. The inhibition of platelet aggregation by pENW was significantly abolished by L-NAME.
The in-vivo antiplatelet and antithrombotic effects of pENW are at least partly mediated by the increased production of endogenous NO via up-regulation and stimulation of eNOS. The findings suggest that pENW could potentially be developed as a novel therapeutic agent in the treatment of platelet-driven disorders.

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Homocysteine decreases platelet NO level via protein kinase C activation

Abstract

Section snippets

Reagents

Effect of homocysteine on NO and cGMP basal level

Discussion

Acknowledgments

Thromb. Res.

Atherosclerosis

J. Thromb. Haemost.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Free Radic. Biol. Med.

Trends Biochem. Sci.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Pharmacol.

Methods Enzymol.

Blood

Atherosclerosis

Atherosclerosis

Biochem. Pharmacol.

Thromb. Res.

FEBS Lett.

Blood

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Atherosclerosis

FEBS Lett.

Homocysteine upregulates vascular cell adhesion molecule-1 expression in cultured human aortic endothelial cells and enhances monocyte adhesion

Arterioscler. Thromb. Vasc. Biol.

Effects of homocysteine and related compounds on prostacyclin production by cultured human vascular endothelial cells

Thromb. Haemost.

Induction of oxidative stress by homocyst(e)ine impairs endothelial function

J. Cell. Biochem.

Endothelial dysfunction and atherothrombosis in mild hyperhomocysteinaemia

Vasc. Med.