Quantification and regulation of erythrocyte HbNO as an index of vascular NO using EPR spectroscopy

Reduced nitric oxide (NO) bioavailability is a major feature of endothelial dysfunction characteristic of cardiovascular diseases (CVDs). However, the short half-life of NO precludes an accurate quantification of vascular NO. In red blood cells (RBCs), NO reacts with hemoglobin to form an iron-nitrosyl complex (HbNO), quantifiable by EPR spectroscopy in venous blood ex vivo. We hypothesized that HbNO reflects NO bioavailability and correlates with endothelial function in vivo. We showed that an eNOS in RBC marginally contributes to HbNO, highlighting vascular NO as predominant source for the HbNO complex steady-state level in vivo. We characterized the influence of pO2, T°, pH, reactive oxygen species and RBC antioxidant system on HbNO stability. We identified aquaporin-1 as a peroxiporin that facilitates the H2O2 transport into RBCs. Finally, HbNO correlates with endothelial dysfunction before any clinical manifestation of CVD and inversely with vascular oxidative stress. Our study opens the way to further development of HbNO as a biomarker for early diagnosis and targeted treatment of CVDs with NO-dependent endothelial dysfunction.(SP - Sciences de la santé publique) -- UCL, 201

Redox regulation of nitrosyl-hemoglobin in human erythrocytes.

Oxidative stress perturbs vascular homeostasis leading to endothelial dysfunction and cardiovascular diseases. Vascular reactive oxygen species (ROS) reduce nitric oxide (NO) bioactivity, a hallmark of cardiovascular and metabolic diseases. We measured steady-state vascular NO levels through the quantification of heme nitrosylated hemoglobin (5-coordinate-α-HbNO) in venous erythrocytes of healthy human subjects using electron paramagnetic resonance (EPR) spectroscopy. To examine how ROS may influence HbNO complex formation and stability, we identified the pro- and anti-oxidant enzymatic sources in human erythrocytes and their relative impact on intracellular redox state and steady-state HbNO levels. We demonstrated that pro-oxidant enzymes such as NADPH oxidases are expressed and produce a significant amount of ROS at the membrane of healthy erythrocytes. In addition, the steady-state levels of HbNO were preserved when NOX (e.g. NOX1 and NOX2) activity was inhibited. We next evaluated the impact of selective antioxidant enzymatic systems on HbNO stability. Peroxiredoxin 2 and catalase, in particular, played an important role in endogenous and exogenous HO degradation, respectively. Accordingly, inhibitors of peroxiredoxin 2 and catalase significantly decreased erythrocyte HbNO concentration. Conversely, steady-state levels of HbNO were preserved upon supplying erythrocytes with exogenous catalase. These findings support HbNO measurements as indicators of vascular oxidant stress and of NO bioavailability and potentially, as useful biomarkers of early endothelial dysfunction

Biological Assessment of the NO-Dependent Endothelial Function.

Nitric oxide (NO) is implicated in numerous physiological processes, including vascular homeostasis. Reduced NO bioavailability is a hallmark of endothelial dysfunction, a prequel to many cardiovascular diseases. Biomarkers of an early NO-dependent endothelial dysfunction obtained from routine venous blood sampling would be of great interest but are currently lacking. The direct measurement of circulating NO remains a challenge due by its high reactivity and short half-life. The current techniques measure stable products from the NO signaling pathway or metabolic end products of NO that do not accurately represent its bioavailability and, therefore, endothelial function per se. In this review, we will concentrate on an original technique of low temperature electron paramagnetic resonance spectroscopy capable to directly measure the 5-α-coordinated heme nitrosyl-hemoglobin in the T (tense) state (5-α-nitrosyl-hemoglobin or HbNO) obtained from fresh venous human erythrocytes. In humans, HbNO reflects the bioavailability of NO formed in the vasculature from vascular endothelial NOS or exogenous NO donors with minor contribution from erythrocyte NOS. The HbNO signal is directly correlated with the vascular endothelial function and inversely correlated with vascular oxidative stress. Pilot studies support the validity of HbNO measurements both for the detection of endothelial dysfunction in asymptomatic subjects and for the monitoring of such dysfunction in patients with known cardiovascular disease. The impact of therapies or the severity of diseases such as COVID-19 infection involving the endothelium could also be monitored and their incumbent risk of complications better predicted through serial measurements of HbNO

Nitrosyl-hemoglobin formation in rodent and human venous erythrocytes reflects NO formation from the vasculature in vivo

<div><p>Reduced bioavailability of nitric oxide (NO) is a major feature of endothelial dysfunction characteristic of cardiovascular and metabolic diseases but the short half-life of NO precludes its easy quantification in circulating blood for early diagnosis. In erythrocytes, NO can react with hemoglobin to form an iron-nitrosyl complex (5-coordinate-α-HbNO) directly quantifiable by Electron Paramagnetic Resonance spectroscopy (EPR) in mouse, rat and human venous blood <i>ex vivo</i>. However, the sources of the nitrosylating species <i>in vivo</i> and optimal conditions of HbNO preservation for diagnostic use in human erythrocytes are unknown. Using EPR spectroscopy, we found that HbNO stability was significantly higher under hypoxia (equivalent to venous pO₂; 12.0±0.2% degradation of HbNO at 30 minutes) than at room air (47.7±0.2% degradation) in intact erythrocytes; at 20°C (15.2±0.3% degradation after 30 min versus 29.6±0.1% at 37°C) and under acidic pH (31.7±0.8% versus 62.2±0.4% degradation after 30 min at physiological pH) at 50% of haematocrit. We next examined the relative contribution of NO synthase (NOS) from the vasculature or in erythrocytes themselves as a source of nitrosylating NO. We detected a NOS activity (and eNOS expression) in human red blood cells (RBC), and in RBCs from eNOS^(+/+) (but not eNOS^(-/-)) mice, as measured by HbNO formation and nitrite/nitrate accumulation. NO formation was increased after inhibition of arginase but abrogated upon NOS inhibition in human RBC and in RBCs from eNOS^(+/+) (but not eNOS^(-/-)) mice. However, the HbNO signal from freshly drawn venous RBCs was minimally sensitive to the inhibitors <i>ex vivo</i>, while it was enhanced upon caveolin-1 deletion <i>in vivo</i>, suggesting a minor contribution of erythrocyte NOS to HbNO complex formation compared with vascular endothelial NOS or other paracrine NO sources. We conclude that HbNO formation in rodent and human venous erythrocytes is mainly influenced by vascular NO sources despite the erythrocyte NOS activity, so that its measurement by EPR could serve as a surrogate for NO-dependent endothelial function.</p></div

Temperature and pH influence on HbNO stability.

<p>A. Stability of HbNO complex accumulated in human RBCs pre-incubated with Spermine-NONOate (100 μmol/L, 60 minutes) under venous O₂ level, and subsequently incubated at 20°C (circle) or 37°C (triangle) during 50 minutes under 21% of O₂. B. Effect of pH on HbNO stability in human RBCs pre-incubated with NO-donor and reconstituted at 50% haematocrit with isotonic buffer at acidic pH (6.2–4.7) or at physiological pH (7.4–7.2) at 21% of O₂ and 37°C. Data are shown as mean values ± SEM; * P < 0.05; n = 4 different RBC preparations.</p

Detection of erythrocyte eNOS activity by EPR spectroscopy and by nitrite and nitrate colorimetric assay.

<p>A. Typical procedure used for subtraction analysis: the EPR spectrum of RBC sample before (a) and after (b) subtraction of PFR EPR spectrum (c) obtained as described in Material and Methods. Typical HbNO EPR signal from immediately frozen mouse whole blood (d); the hyperfine structure (hfs) of HbNO is shown by the arrows. (B-F). Quantification of erythrocyte HbNO EPR signals (B) and products of NO oxidation, nitrite and nitrate formed in isolated RBCs of eNOS^(+/+)and eNOS^(-/-) mice (C and D) and in human erythrocytes (E and F), after incubation <i>ex vivo</i> with norNOHA and/or L-NAME as described in Materials and Methods. Data are shown as mean values ± SD; *<i>P</i> < 0.05, ^$P<0.01, ^§ P < 0.0001; n = 3–7 different mouse RBC preparations and n = 10 different human preparations.</p

Human RBCs express eNOS proteins.

<p>A. Representative flow cytometric two parameter dot plot of isolated RBCs from a healthy subject co-stained with primary anti-Aquaporin-1 and secondary Alexa Fluor-488-conjugated anti-IgG antibodies; and primary anti-eNOS and secondary Alexa Fluor-647-conjugated anti-IgG antibodies. Panel on the left shows co-staining only with conjugated secondary antibodies, panel in the middle shows staining only with mouse isotype IgG1,k Alexa-647-conjugated (negative controls). Panel on the right shows the identification of RBCs as eNOS–positive and Aquaporin1-positive events in the upper right quadrant. The percentage of RBCs double positive is indicated in the upper right quadrant. (B-E) Representative images of eNOS (B) and AQP1 (D) detection in healthy human RBCs using immunofluorescence microscopy. As negative controls, RBCs were stained only with conjugated secondary antibodies (Alexa Fluor-488 anti-mouse IgG for eNOS (C) and Alexa Fluor-568 anti-rabbit IgG for AQP1 (D)).</p

Effect of oxygen on HbNO (5-coordinate alpha-nitrosyl-hemoglobin) formation in human RBCs.

<p>A. Typical EPR spectra recorded in human erythrocyte samples exposed to 1% (a, c) or 21% (b) of O₂ and treated with 50 μmol/L of Spermine-NONOate for 1 hour (a, b); or with a NO-generating mixture (4 μmol/L of NO₂^- and 50 mmol/L of Na₂S₂O₄) (c). Spectra were acquired as described in Materials and Methods. Arrows point to the triplet hyperfine structures (hfs). A(I) corresponds to the amplitude of the first hf component used for HbNO quantification. B. The concentrations of HbNO complex accumulated in human reconstituted RBCs (50% of haematocrit in isotonic buffer) under 21% (square) or 1% (triangle) of O₂ after addition of graded concentrations of Spermine-NONOate. Data are shown as mean values ± SEM; ^$ P < 0.01 ^§P < 0.0001; n = 4 different RBC preparations.</p

Sensitivity of blood HbNO formation to <i>in vivo</i> administered L-NAME or a nitrovasodilator.

<p>A. Concentration of HbNO in venous mouse RBCs after seven days of treatment with L-NAME or vehicle as described in Materials and Methods. B. Typical EPR spectra of HbNO recorded from isolated RBCs after one week administration of vehicle (a) or L-NAME (b) in drinking water. C. Kinetics of relative <i>in vivo</i> changes of HbNO EPR signal in venous rat blood after <i>in vivo</i> isosorbide dinitrate (ISDN) or vehicle injection. D. Typical HbNO EPR spectra after 5 and 10 minutes i.v. administration of vehicle (a-b) or isosorbide dinitrate (ISDN) (c-d). Data are shown as mean values ± SD; ^* P < 0.05 and ^$ P < 0.01; n = 4 different RBC preparations.</p

Effect of oxygen on stability of HbNO formed <i>ex vivo</i>.

<p>A. Typical EPR spectra, and B. concentrations of HbNO complex accumulated in human RBCs pre-incubated with Spermine-NONOate (100 μmol/L, for 60 minutes, under venous O₂ level) and subsequently exposed for 60 minutes to 21% (square) or 1% (triangle) of O₂ as described in Methods. Arrows in (A) point to the triplet hyperfine structures (hfs); A(I) indicates the amplitude of the first hf component. Data in B are shown as mean values ± SEM; ^* P < 0.05; n = 4 different RBC preparations. C-D. Correlation between HbNO concentrations in RBCs and percentage of oxy/deoxy hemoglobin in venous blood during incubation at 21% (C); and 1% of O₂ (D).</p