Transport and peripheral bioactivities of nitrogen oxides carried by red blood cell hemoglobin: role in oxygen delivery.

The biology of NO (nitric oxide) is poorly explained by the activity of the free radical NO ((.)NO) itself. Although (.)NO acts in an autocrine and paracrine manner, it is also in chemical equilibrium with other NO species that constitute stable stores of NO bioactivity. Among these species, S-nitrosylated hemoglobin (S-nitrosohemoglobin; SNO-Hb) is an evolved transducer of NO bioactivity that acts in a responsive and exquisitely regulated manner to control cardiopulmonary and vascular homeostasis. In SNO-Hb, O(2) sensing is dynamically coupled to formation and release of vasodilating SNOs, endowing the red blood cell (RBC) with the capacity to regulate its own principal function, O(2) delivery, via regulation of blood flow. Analogous, physiological actions of RBC SNO-Hb also contribute to central nervous responses to blood hypoxia, the uptake of O(2) from the lung to blood, and baroreceptor-mediated control of the systemic flow of blood. Dysregulation of the formation, export, or actions of RBC-derived SNOs has been implicated in human diseases including sepsis, sickle cell anemia, pulmonary arterial hypertension, and diabetes mellitus. Delivery of SNOs by the RBC can be harnessed for therapeutic gain, and early results support the logic of this approach in the treatment of diseases as varied as cancer and neonatal pulmonary hypertension

Vascular caveolin deficiency supports the angiogenic effects of nitrite, a major end product of nitric oxide metabolism in tumors

The biological status of nitrite recently evolved from an inactive end product of nitric oxide (NO) metabolism to a major intravascular and tissue storage of NO. Several enzymes and proteins may indeed work as nitrite reductases. The endothelial NO synthase (eNOS) is proposed to be one of them, particularly when oxygen is lacking. Here, we examined whether the lack of caveolin, a scaffold protein known to limit eNOS activity under basal conditions and to be down-regulated in tumor vessels, could favor the reconversion of nitrite into NO and thereby promote angiogenesis. We found that nitrite-rich serum from caveolin-deficient mice and exogenous nitrite exert proangiogenic effects on aortic explants cultured in a three-dimensional collagen matrix. We identified a higher intrinsic capacity of caveolin-deficient vessels and endothelial cells to convert nitrite into bioactive NO. These effects did occur under moderate hypoxia and were abolished on exposure to a NO scavenger. Evidence for eNOS acting as a nitrite reductase derived from the failure to reproduce the proangiogenic effects of nitrite on eNOS-deficient aorta rings and endothelial cells. Finally, in a mouse tumor model, we documented the higher nitrite content in hypoxic tumors and identified inducible NO synthase as the major source of nitrite. Altogether, these data identify the lack of caveolin observed in the tumor vasculature as a favorable ground for nitrite-driven formation of endothelial tubes in the hypoxic tumor microenvironment. This work also strengthens the therapeutic value of the modulation of caveolin expression to interfere with tumor angiogenesis. (Mol Cancer Res 2009;7(7):OF1-8)

Nitrosylated hemoglobin levels in human venous erythrocytes correlate with vascular endothelial function measured by digital reactive hyperemia

Impaired nitric oxide (NO)-dependent endothelial function is associated with the development of cardiovascular diseases. We hypothesized that erythrocyte levels of nitrosylated hemoglobin (HbNO-heme) may reflect vascular endothelial function in vivo. We developed a modified subtraction method using Electron Paramagnetic Resonance (EPR) spectroscopy to identify the 5-coordinate α-HbNO (HbNO) concentration in human erythrocytes and examined its correlation with endothelial function assessed by peripheral arterial tonometry (PAT). Changes in digital pulse amplitude were measured by PAT during reactive hyperemia following brachial arterial occlusion in a group of healthy volunteers (50 subjects). Erythrocyte HbNO levels were measured at baseline and at the peak of hyperemia. We digitally subtracted an individual model EPR signal of erythrocyte free radicals from the whole EPR spectrum to unmask and quantitate the HbNO EPR signals. RESULTS: Mean erythrocyte HbNO concentration at baseline was 219+/-12 nmol/L (n = 50). HbNO levels and reactive hyperemia (RH) indexes were higher in female (free of contraceptive pills) than male subjects. We observed a dynamic increase of HbNO levels in erythrocytes isolated at 1-2 min of post-occlusion hyperemia (120+/-8% of basal levels); post-occlusion HbNO levels were correlated with basal levels. Both basal and post-occlusion HbNO levels were significantly correlated with reactive hyperemia (RH) indexes (r = 0.58; P<0.0001 for basal HbNO). CONCLUSION: The study demonstrates quantitative measurements of 5-coordinate α-HbNO in human venous erythrocytes, its dynamic physiologic regulation and correlation with endothelial function measured by tonometry during hyperemia. This opens the way to further understanding of in vivo determinants of NO bioavailability in human circulation

Endothelial dysfunction induced by hydroxyl radicals – the hidden face of biodegradable Fe-based materials for coronary stents

Fe-based materials are currently considered for manufacturing biodegradable coronary stents. Here we show that Fe has a strong potential to generate hydroxyl radicals (HO·) during corrosion. This HO· generation, but not corrosion, can be inhibited by catalase. Oxidative stress was observed (increased HO-1 expression) in aortic rings after direct exposure to Fe, but not in the presence of catalase or after indirect exposure. This oxidative stress response induced an uncoupling of eNOS in, and a consequent reduced NO production by endothelial cells exposed to Fe. In isolated rat aortic rings NO production was also reduced by HO· generated during Fe corrosion, as indicated by the protective role of catalase. Finally, all these mechanisms contributed to impaired endothelium- dependent relaxation in aortic rings caused by HO· generated during the direct contact with Fe. This deleterious impact of Fe corrosion on the endothelial function should be integrated when considering the use of biodegradable Fe-based alloys for vascular implants

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

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

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