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We examined the longitudinal changes of VEGF levels after percutaneous coronary intervention for predicting major adverse cardiac events (MACE) in coronary artery disease (CAD) patients. VEGF was measured in 94 CAD patients' serum before revascularization, 1-month and 1-year after. Independently of clinical presentation, patients had lower VEGF concentration than a cohort of healthy subjects (median, IQ: 15.9, 9.0–264 pg/mL versus 419, 212–758 pg/mL; P < 0.001) at baseline. VEGF increased to 1-month (median, IQ: 276, 167–498 pg/mL; P < 0.001) and remained steady to 1-year (median, IQ: 320, 173–497 pg/mL; P < 0.001) approaching control levels. Drug eluting stent apposition and previous medication intake produced a less steep VEGF evolution after intervention (P < 0.05). Baseline VEGF concentration <40.8 pg/mL conveyed increased risk for MACE in a 5-year follow-up. Results reflect a positive role of VEGF in recovery and support its importance in CAD prognosis.

Abbreviations: AMI ¼ acute myocardial infarction; APC ¼ allophycocyanin; Asp ¼ aspartate; cTNT ¼ cardiac troponin; CAD ¼ coronary artery disease; CRP ¼ C-reactive protein; CK ¼ creatine kinase; eNOS ¼ endothelial nitric oxide synthase; ELISA ¼ enzyme-linked immunosorbent assay; FITC ¼ fluorescein isothiocyanate; FAU ¼ fluorescence arbitrary units; Glu ¼ glutamic acid; LME ¼ linear mixed effects model; MPs ¼ microparticles; NO ¼ nitric oxide; NT-proBNP ¼ N-terminal pro-brain natriuretic peptide; PCI ¼ percutaneous coronary intervention; PBS ¼ phosphate-buffered saline; PE ¼ Phycoerythrin; CD62P ¼ P-selectin; sCD40L ¼ soluble CD40 ligand; SA ¼ stable angina pectoris; VEGF ¼ vascular endothelial growth factor INTRODUCTION CD40L is a signaling molecule, 1-3 implicated in thrombosis and inflammatory response to vascular injury. [4][5][6] The relationship of CD40L with coronary artery disease (CAD) has been established, 2,7-9 as also its implication in endothelial dysfunction. [10][11][12][13][14] However, whether the soluble CD40 ligand (sCD40L) could also influence endothelial dysfunction after acute myocardial infarction (AMI) injury remains unclear.In vitro studies have shown that sCD40L inhibits angiogenesis and also growth factor-induced human umbilical vein endothelial cell migration, which is achieved by generation of free radicals and inhibition of nitric oxide (NO) production. 10 The authors hypothesized that the sCD40L could inhibit reendothelialization of an injured vessel, thereby affecting the restenosis. 10Research efforts have been directed toward the finding of biomarkers to assess endothelial function and its correlation with AMI. Genetic indicators, such as the polymorphisms of endothelial NO synthase (eNOS) gene, 15,16 may provide insight into endothelial cells function.Vascular endothelial growth factor (VEGF) is a wellknown promoter of angiogenesis and an endogenous regulator of endothelial integrity. [17][18][19] The prognostic information provided by VEGF independently of other markers likely points toward an important role for angiogenesis in regulating myocardial repair and reperfusion after AMI. 17,20 Current opinion suggests a differential role of CD40L (both soluble and membrane-bound forms, which includes microparticles in circulation) 21 at different stages of CAD, contrasting with the traditional view of an unvarying function of the CD40L-CD40-sCD40L system interactions in the disease. 6 In that perspective, no clear indication of the interplay of CD40L with endothelial and vascular function markers and their importance in the pathophysiology of the AMI has been obtained so far in human clinical studies. Therefore, the aim of this study was to evaluate the relationship of sCD40L with markers of platelet activation, endothelial and vascular function during an early recovery period after AMI. To achieve this goal, the time changes over 1 month of sCD40L levels were assessed in AMI patients and correlated with the CD40L expressed on platelets and microparticles, CD62P expression on platele...

We demonstrated that erythrocytes from glaucoma patients have similar availability to release NO both in absence and presence of timolol, and have higher GSNO values in presence of timolol.

In the human erythrocyte, band 3 protein mediates nitric oxide (NO) translocation and its effects are strongly related to phosphorylated/dephosphorylated intracellular states. The metabolism of NO could change in the presence of acetylcholinesterase (AChE). Therefore, the present study was designed to assess the effect of conformational changes in AChE (via N-19 and C-16 antibodies) and enzymatic inhibition/activation of protein kinase C (PKC) in erythrocyte NO mobilization in vitro. Our results show that by inhibiting PKC with cheletrine, impaired erythrocyte NO efflux and s-nitrosoglutathione (GSNO) levels were verified, while PKC's activation by Phorbol 12-myristate 13-acetate had the opposite effect. Those results demonstrate the influence of 4.1R complex and band 3 protein level of phosphorylation on NO efflux and GSNO concentration mediated by PKC inhibition/activation. In addition, the present study shows evidence that conformational changes in AChE promoted by incubation with N-19 and C-16 antibodies alter the enzyme's functional connection to acetylcholine (ACh) (AChE-ACh complex) in an irreversible manner, resulting in impaired GSNO concentration and NO efflux from the erythrocyte. Novel insight into NO metabolism in the erythrocyte is brought with the presented findings allowing new possibilities of modulating NO delivery, possibly involving PKC and AChE conformational alterations in combination.

Glutathione is an abundant molecule inside erythrocyte, originating S-nitrosoglutathione (GSNO) by reacting with nitric oxide (NO). GSNO has been regarded as a store and transporter of NO, with significant interest as a potential therapeutic agent, acting as an NO donor.NO metabolism inside the erythrocyte generates several derivatives, which can be altered by external and internal stimuli such as acetylcholine (ACh), a natural substrate of acetylcholinesterase (AChE). In spite of the knowledge gained in the last decades concerning NO efflux in erythrocytes little is known regarding erythrocyte GSNO efflux, which has also a significant role in microcirculation. Hence, the objective of this research was to evaluate the efflux of GSNO, concomitant with the efflux of NO, after stimulation with AChE effectors. To achieve these goals, the in vitro effect of AChE modulators - ACh and timolol - in erythrocyte NO and GSNO were studied. Timolol is an erythrocyte AChE inhibitor. Venous blood samples were collected from 18 healthy Caucasian men. For each blood sample, erythrocyte suspensions were obtained and incubated in the absence (controls) and presence of ACh and timolol maleate (10 μM final concentration of each modulator). Both timolol and ACh induced significant GSNO efflux in the erythrocyte when compared to the control; however the efflux was lower in the presence of timolol compared to ACh. Although erythrocyte NO efflux in presence of timolol is similar to the control, the efflux decreased when compared to the ACh treatment. The presence of timolol induces significant decrease of intra-erythrocyte GSNO levels, relative to control and ACh treatment. In conclusion, when erythrocytes were stimulated with ACh or timolol, GSNO efflux occurred associated with NO efflux. These new results bring new insight into the metabolism of erythrocyte NO and new possible therapeutic applications for GSNO.

The results of the present study indicate that NO efflux from RBCs and RBCs AChE should be further explored as potential biomarkers for ALS.

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