Potential Modulation of Vascular Function by Nitric Oxide and Reactive Oxygen Species Released From Erythrocytes.

The primary role for erythrocytes is oxygen transport that requires the reversible binding of oxygen to hemoglobin. There are, however, secondary reactions whereby the erythrocyte can generate reactive oxygen species (ROS) and nitric oxide (NO). ROS such as superoxide anion and hydrogen peroxide are generated by the autoxidation of hemoglobin. NO can be generated when nitrite reacts with hemoglobin forming an HbNO+ intermediate. Both of these reactions are dramatically enhanced under hypoxic conditions. Within the erythrocyte, interactions of NO with hemoglobin and enzymatic reactions that neutralize ROS are expected to prevent the release of any generated NO or ROS. We have, however, demonstrated that partially oxygenated hemoglobin has a distinct conformation that enhances hemoglobin-membrane interactions involving Band 3 protein. Autoxidation of the membrane bound partially oxygenated hemoglobin facilitates the release of ROS from the erythrocyte. NO release is made possible when HbNO+, the hemoglobin nitrite-reduced intermediate, which is not neutralized by hemoglobin, is bound to the membrane and releases NO. Some of the released ROS has been shown to be transferred to the vasculature suggesting that some of the released NO may also be transferred to the vasculature. NO is known to have a major effect on the vasculature regulating vascular dilatation. Erythrocyte generated NO may be important when NO production by the vasculature is impaired. Furthermore, the erythrocyte NO released, may play an important role in regulating vascular function under hypoxic conditions when endothelial eNOS is less active. ROS can react with NO and, can thereby modulate the vascular effects of NO. We have also demonstrated an inflammatory response due to erythrocyte ROS. This reflects the ability of ROS to react with various cellular components affecting cellular function.

Late manifestation of alloantibody-associated injury and clinical pulmonary antibody-mediated rejection: Evidence from cell-free DNA analysis.

BACKGROUND: Antibody-mediated rejection (AMR) often progresses to poor health outcomes in lung transplant recipients (LTRs). This, combined with the relatively insensitive clinical tools used for its diagnosis (spirometry, histopathology) led us to determine whether clinical AMR is diagnosed significantly later than its pathologic onset. In this study, we leveraged the high sensitivity of donor-derived cell-free DNA (ddcfDNA), a novel genomic tool, to detect early graft injury after lung transplantation. METHODS: We adjudicated AMR and acute cellular rejection (ACR) in 157 LTRs using the consensus criteria of the International Society for Heart and Lung Transplantation (ISHLT). We assessed the kinetics of allograft injury in relation to ACR or AMR using both clinical criteria (decline in spirometry from baseline) and molecular criteria (ddcfDNA); percent ddcfDNA was quantitated via shotgun sequencing. We used a mixed-linear model to assess the relationship between and ddcfDNA levels and donor-specific antibodies (DSA) in AMR+ LTRs. RESULTS: Compared with ACR, AMR episodes (n = 42) were associated with significantly greater allograft injury when assessed by both spirometric (0.1 liter vs -0.6 liter, p < 0.01) and molecular (ddcfDNA) analysis (1.1% vs 5.4%, p < 0.001). Allograft injury detected by ddcfDNA preceded clinical AMR diagnosis by a median of 2.8 months. Within the same interval, spirometry or histopathology did not reveal findings of allograft injury or dysfunction. Elevated levels of ddcfDNA before clinical diagnosis of AMR were associated with a concurrent rise in DSA levels. CONCLUSION: Diagnosis of clinical AMR in LTRs lags behind DSA-associated molecular allograft injury as assessed by ddcfDNA.

Exhaled nitric oxide in pulmonary arterial hypertension associated with systemic sclerosis.

The fractional exhaled concentration of nitric oxide (FENO) has been shown to be reduced in idiopathic pulmonary arterial hypertension (PAH) but has not been adequately studied in PAH associated with systemic sclerosis (SSc). We measured FENO at an expiratory flow rate of 50 mL/s in 21 treatment-naive patients with SSc-associated PAH (SSc-PAH), 94 subjects with SSc without pulmonary involvement, and 84 healthy volunteers. Measurements of FENO at additional flow rates of 100, 150, and 250 mL/s were obtained to derive the flow-independent nitric oxide exchange parameters of maximal airway flux (J'awNO) and steady-state alveolar concentration (CANO). FENO at 50 mL/s was similar (P = 0.22) in the SSc-PAH group (19 ± 12 parts per billion [ppb]) compared with the SSc group (17 ± 12 ppb) and healthy control group (21 ± 11 ppb). No change was observed after 4 months of targeted PAH therapy in 14 SSc-PAH group patients (P = 0.9). J'awNO was modestly reduced in SSc group subjects without lung disease (1.2 ± 0.5 nl/s) compared with healthy controls (1.64 ± 0.9; P < 0.05) but was similar to that in the SSc-PAH group. CANO was elevated in individuals with SSc-PAH (4.8 ± 2.6 ppb) compared with controls with SSc (3.3 ± 1.4 ppb) and healthy subjects (2.6 ± 1.5 ppb; P < 0.001 for both). However, after adjustment for the diffusing capacity of CO, there was no significant difference in CANO between individuals with SSc-PAH and controls with SSc. We conclude that FENO is not useful for the diagnosis of PAH in SSc. Increased alveolar nitric oxide in SSc-PAH likely represents impaired diffusion into pulmonary capillary blood.

Red blood cell membrane-facilitated release of nitrite-derived nitric oxide bioactivity.

The reduction of nitrite by deoxyhemoglobin to nitric oxide (NO) has been proposed as a mechanism for the transfer of NO bioactivity from the red blood cell (RBC) to the vasculature. This transfer can increase vascular dilatation. The major challenge to this hypothesis is the very efficient scavenging of NO by hemoglobin, which prevents the release of NO from RBCs. Previous studies indicate that the reaction of nitrite with deoxyhemoglobin produces two metastable intermediates involving nitrite bound to deoxyhemoglobin and a hybrid intermediate [Hb(II)NO(+) ↔ Hb(III)NO] where the nitrite is reduced, but unavailable to react with hemoglobin. We have now shown how unique properties of these intermediates provide a pathway for the release of NO bioactivity from RBCs. The high membrane affinity of these intermediates (>100-fold greater than that of deoxyhemoglobin) places these intermediates on the membrane. Furthermore, membrane-induced conformational changes of the nitrite-reacted intermediates facilitate the release of NO from the hybrid intermediate and nitrite from the nitrite-bound intermediate. Increased membrane affinity, coupled with facilitated dissociation of NO and nitrite from the membrane-bound intermediates, provides the first realistic mechanism for the potential release of NO and nitrite from the RBC and their potential transfer to the vasculature.

Nitrite enhances RBC hypoxic ATP synthesis and the release of ATP into the vasculature: a new mechanism for nitrite-induced vasodilation.

A role for nitric oxide (NO) produced during the reduction of nitrite by deoxygenated red blood cells (RBCs) in regulating vascular dilation has been proposed. It has not, however, been satisfactorily explained how this NO is released from the RBC without first reacting with the large pools of oxyhemoglobin and deoxyhemoglobin in the cell. In this study, we have delineated a mechanism for nitrite-induced RBC vasodilation that does not require that NO be released from the cell. Instead, we show that nitrite enhances the ATP release from RBCs, which is known to produce vasodilation by several different methods including the interaction with purinergic receptors on the endothelium that stimulate the synthesis of NO by endothelial NO synthase. This mechanism was established in vivo by measuring the decrease in blood pressure when injecting nitrite-reacted RBCs into rats. The observed decrease in blood pressure was not observed if endothelial NO synthase was inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME) or when any released ATP was degraded by apyrase. The nitrite-enhanced ATP release was shown to involve an increased binding of nitrite-modified hemoglobin to the RBC membrane that displaces glycolytic enzymes from the membrane, resulting in the formation of a pool of ATP that is released from the RBC. These results thus provide a new mechanism to explain nitrite-induced vasodilation.

Nitrite-induced improved blood circulation associated with an increase in a pool of RBC-NO with no bioactivity.

The reduction of nitrite by RBCs producing NO can play a role in regulating vascular tone. This hypothesis was investigated in rats by measuring the effect of nitrite infusion on mean arterial blood pressure (MAP), cerebral blood flow (CBF) and cerebrovascular resistance (CVR) in conjunction with the accumulation of RBC-NO. The nitrite infusion reversed the increase in MAP and decrease in CBF produced by L-NAME inhibition of e-NOS. At the same time there was a dramatic increase in RBC-NO. Correlations of RBC-NO for individual rats support a role for the regulation of vascular tone by this pool of NO. Furthermore, data obtained prior to treatment with L-NAME or nitrite are consistent with a contribution of RBC reduced nitrite in regulating vascular tone even under normal conditions. The role of the RBC in delivering NO to the vasculature was explained by the accumulation of a pool of bioactive NO in the RBC when nitrite is reduced by deoxygenated hemoglobin chains. A comparison of R and T state hemoglobin demonstrated a potential mechanism for the release of this NO in the T-state present at reduced oxygen pressures when blood enters the microcirculation. Coupled with enhanced hemoglobin binding to the membrane under these conditions the NO can be released to the vasculature.

Effect of pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, on ischemia-reperfusion injury in rats.

Two groups of rats were used to examine the effect of pioglitazone, a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, on rat hearts using an in vivo model of ischemia-reperfusion (I/R) to elucidate potential mechanisms. One group was the 30-min reperfusion group, which was further subdivided into sham (n=5), vehicle (n=6) and pioglitazone (3 mg x kg(-1), n=7) treatment groups with 30 min ischemia followed by 30 min reperfusion to detect data related to cardiac function and the area of myocardial infarction. The other group was the 120-min reperfusion group, subdivided into sham (n=5), vehicle (n=6), and pioglitazone 0.3 mg x kg(-1) (n=6), 1 mg x kg(-1) (n=7) and 3 mg x kg(-1) (n=6) treatment groups. Immunohistochemistry, in situ hybridization, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and DNA agarose gel electrophoresis were performed to detect apoptosis and expressions of Bax, Bcl-2, caspase 3, MMP-2 and PPARgamma protein, and MMP-2 and PPARgamma mRNA. We found that, after acute treatment with pioglitazone, the ratio of necrosis to area at risk decreased by 28% (p<0.01) and that of necrosis to left ventricle was reduced by 32% (p<0.01), compared with the vehicle group. Heart rate and +dp/dt(max), representing the cardiac systolic function, as well as -dp/dt(max), the indicator of cardiac diastolic function, improved significantly at 1 and 30 min after reperfusion (p<0.05-0.01). Furthermore, myocardial apoptosis was significantly suppressed by acute treatment with pioglitazone as evidenced by the decreased number of TUNEL-positive myocytes and DNA ladder, enhanced Bcl-2 protein expression, reduced Bax and caspase 3 protein expression in a dose-dependent manner compared with vehicle-treated rats. In addition, acute treatment with pioglitazone dose-dependently increased PPARgamma expression and decreased MMP-2 expression at protein and mRNA levels. Our findings demonstrate that a PPARgamma agonist may protect the heart from I/R injury. The protective effect is likely to occur by reducing cardiomyocyte apoptosis and inhibiting MMP-2.

[The effect of pioglitazone on apoptotic cardiomyocytes for ischemia reperfusion].

OBJECTIVE: This study was to investigate the effect of pioglitazone on apoptotic cardiomyocytes with the model of ischemia-reperfusion at rat heart in vivo. METHODS: Sprague-Dawley rats were randomly divided into two groups. One was 30 min reperfusion group, which was subdivided into sham (n = 5), model (vehicle, n = 6) and pioglitazone 3 mg/kg (n = 7) with 30 min ischemia followed by 30 min reperfusion to detect the area of myocardial infarction (MI). Another was 2 h reperfusion group, which was further subdivided into sham (n = 5), model (vehicle, n = 6), and pioglitazone 0.3 mg/kg (n = 6), 1 mg/kg (n = 7) and 3 mg/kg (n = 6). Apart from the sham, pioglitazone and vehicle were administered intravenously 30 min before occlusion. Then hearts were excised, paraffined and cut into 4 microm thick. Immunohistochemistry, in situ hybridization, TUNEL and DNA agarose gel electrophoresis were performed to detect the expression of Bax, Bcl-2, Caspase-3 and PPARgamma protein and PPARgamma mRNA. RESULTS: (1) Compared with model, nec/aar of pioglitazone decreased by 28% (P < 0.01). The nec/lv ratio reduced by 32% (P < 0.01). (2) In a dose-dependent manner, the expressions of Bax and Caspase-3 were depressed, while the expression of Bcl-2, PPARgamma protein and PPARgamma mRNA were enhanced by pioglitazone. (3) The apoptotic index of subgroups injected pioglitazone reduced significantly by TUNEL compared with model (P < 0.05). Agarose gel electrophoresis demonstrated that DNA ladder existed in model, pioglitazone 0.3 mg/kg and pioglitazone 1 mg/kg, but not pioglitazone 3 mg/kg. CONCLUSIONS: Pioglitazone could protect the heart from I/R injury evidenced by the improvement in the expression of PPARgamma at the levels of protein and mRNA after pioglitazone administrated, and by the decrease in the apoptotic cardiomyocytes.