Hemolysis in sickle cell mice causes pulmonary hypertension due to global impairment in nitric oxide bioavailability

Abstract

Pulmonary hypertension is a highly prevalent complication of sickle cell disease and is a strong risk factor for early mortality. However, the pathophysiologic mechanisms leading to pulmonary vasculopathy remain unclear. Transgenic mice provide opportunities for mechanistic studies of vascular pathophysiology in an animal model. By microcardiac catheterization, all mice expressing exclusively human sickle hemoglobin had pulmonary hypertension, profound pulmonary and systemic endothelial dysfunction, and vascular instability characterized by diminished responses to authentic nitric oxide (NO), NO donors, and endothelium-dependent vasodilators and enhanced responses to vasoconstrictors. However, endothelium-independent vasodilation in sickle mice was normal. Mechanisms of vasculopathy in sickle mice involve global dysregulation of the NO axis: impaired constitutive nitric oxide synthase activity (NOS) with loss of endothelial NOS (eNOS) dimerization, increased NO scavenging by plasma hemoglobin and superoxide, increased arginase activity, and depleted intravascular nitrite reserves. Light microscopy and computed tomography revealed no plexogenic arterial remodeling or thrombi/ emboli. Transplanting sickle marrow into wild-type mice conferred the same phenotype, and similar pathobiology was observed in a nonsickle mouse model of acute alloimmune hemolysis. Although the time course is shorter than typical pulmonary hypertension in human sickle cell disease, these results demonstrate that hemolytic anemia is sufficient to produce endothelial dysfunction and global dysregulation of NO.

Figure 1

Pulmonary artery pressures were elevated in sickle mice and associated with decreasing cardiac output and right-heart failure with advancing age. (A) Elevated pulmonary artery pressures in younger sickle mice (3 to 5 months old, n = 7) were associated with normal right atrial pressures. (B) Elevated cardiac output in younger sickle mice contrasted with low cardiac output in older sickle mice (13 to 15 months old, n = 4). (C) The Fulton ratio of ventricular weights (right ventricle–left ventricle including septum) indicated disproportionately heavier right ventricles in sickle mice than in hemizygous controls (n = 11 and 16, respectively). Hearts were excised and sectioned using the method of Fulton^, from younger mice not used for other cardiac studies. (D) Thoracic micro-CT of the hemizygous control in vivo demonstrated normal heart size, lung vasculature, and lung aeration. (E) CT in vivo of the sickle mouse at the same axial level showed an enlarged heart. Lungs were very similar to normal, except for dilated pulmonary vessels and mildly increased attenuation with a speckled pattern consistent with perfusion heterogeneity (n = 11). (F) Hemizygous control mouse lung histology at low magnification had normal lung parenchyma and vascularity and (G) at high magnification demonstrated normal thin alveolar capillaries. (H) Sickle mouse lung histology at low magnification confirmed the radiologic findings of dilated pulmonary vessels and patchy vascular congestion. However, pulmonary arterial walls were not significantly thickened, and no plexiform lesions were observed. (I) Histology at high magnification showed engorged alveolar capillaries. No sickle mouse of any age had intra-alveolar edema, fat and/or bone marrow emboli, thromboemboli, pulmonary infarcts, or significant interstitial fibrosis. *P < .05 versus age-matched hemizygous control. ***P < .001 versus age-matched hemizygous control. CT scans (panels D-E) acquired by MicroCAT II (Imtek, Knoxville, TN) were processed by image analysis software (Amira 3.0, TGS Inc, San Diego, CA) and formatted using Adobe Photoshop (Adobe Systems, San Jose, CA). Photomicrographs of lung sections stained with hematoxylin and eosin (panels F-I) were visualized using an Olympus IX70 microscope equipped with UPlanFl 10×/0.30 numerical aperture (NA) and LCPlanFl 40×/0.60 NA PH2 objective lenses (Olympus, Melville, NY); images were acquired by Spot Flex digital camera (Spot Diagnostic Instruments, Sterling Heights, MI) with Spot 3.02 application software, and were formatted using Adobe Photoshop (Adobe Systems, San Jose, CA) and ImageJ software (National Institutes of Health, Bethesda, MD).

Figure 2

Sickle mice and mice receiving transplants with marrow from sickle mice (BM-S) exhibit blunted pulmonary vasodilatory responses to NO and endothelium-dependent vasodilators, showing mean ± SEM for 5 or 6 mice per group. Responses to each vasodilator challenge are shown by the magnitude of drop in mean pulmonary artery pressure (PAP). (A) Sickle mice demonstrated less decrease in pulmonary arterial pressure in response to inhaled NO (data shown for 4 ppm). A similarly blunted response was observed at a lower NO dose (0.4 ppm, P < .05, data not shown). There was no systemic response to inhaled NO (not shown), consistent with the very brief half-life of NO in blood. (B) The NO donor sodium nitroprusside (10 μg/kg intravenous bolus) was similar to inhaled NO in the blunted pulmonary vasodilator response in sickle mice and BM-S mice compared with controls. Similar blunted responsiveness was observed at lower doses (3 μg/kg intravenous bolus, P < .05, data not shown). (C) The phosphodiesterase-5 inhibitor sildenafil (30 μg/kg/min), which acts through the cGMP pathway like NO, had blunted pulmonary vasodilatory responses in sickle mice. BM-S mice preserved more vasodilatory responsiveness to sildenafil than did sickle mice but were still significantly less responsive than controls (P < .05). (D) Bradykinin (3 μg/kg intravenous bolus) response was also blunted in the sickle mice. Similar results were observed at lower doses (0.3 and 1μg/kg, P < .05, data not shown). (E) Pulmonary vascular response to another endothelium-dependent vasodilator, adrenomedullin (0.3 μg/kg intravenous bolus), was likewise blunted in sickle mice and in BM-S mice. Similar results were observed at lower doses (0.1 μg/kg intravenous bolus, P < .05, data not shown). (F) Lung homogenate assays demonstrated significantly lower activity of cGMP-dependent protein kinase in mice with circulating sickle erythrocytes compared with wild-type and hemizygous controls (4 to 7 mice per group) using a colorimetric assay (CycLex, Nagano, Japan)^, according to followed manufacturer's specifications. Statistically significant differences, P < .05 by t test, are indicated as follows: *, versus hemizygous controls, #, versus recipients of marrow from wild-type mice (BM-C); **, sickle versus BM-S mice.

Figure 3

Systemic responsiveness to endothelium-dependent vasodilators was blunted in sickle mice and BM-S mice, showing mean ± SEM for 5 or 6 mice per group. (A) NO donor sodium nitroprusside (10 μg/kg intravenous bolus) had blunted systemic vasodilator response in sickle mice and BM-S mice compared with controls. Similar blunted responsiveness was observed at a lower dose (3 μg/kg intravenous bolus, P < .05, data not shown). (B) Sildenafil (30 μg/kg/min) had significantly blunted systemic vasodilatory responses in sickle mice and in BM-S mice compared with controls. (C) Bradykinin had blunted mean arterial pressure response in the sickle mice at 3 μg/kg intravenous bolus. Similar results were observed at lower doses for BM-S (0.3 and 1 μg/kg, P < .05 and <.05, respectively; data not shown). Mixed response was observed at lower doses for sickle mice (0.3 and 1 μg/kg, P = NS and P < .05, respectively; data not shown). (D) Response to another endothelium-dependent vasodilator, adrenomedullin (1 μg/kg intravenous bolus) was likewise blunted in sickle mice but not in BM-S mice. Neither type of mouse was significantly different from controls at a lower dose of adrenomedullin (0.3 μg/kg, P = NS, data not shown). Statistically significant results noted as follows: *P < .05 versus hemizygotes; #P < .05 compared with mice receiving transplants with wild-type marrow (BM-C).

Figure 4

Preserved vasodilator responses to CGRP. CGRP was infused as an intravenous bolus (0.1 or 0.3 nmol/kg) to probe whether endothelium-independent vascular regulation remained normal. The responses of sickle mice and BM-S mice were no different from hemizygous controls. Pulmonary responsiveness (A) was mirrored by systemic responsiveness (B) in 5 to 6 mice per group. Numbers represent mean ± SEM.

Figure 5

Enhanced responses to pulmonary vasoconstrictors. The vasoconstrictor challenges triggered enhanced pulmonary arterial pressure in the sickle mice and BM-S mice. (A) Hypoxic pulmonary vasoconstrictor response. Acute exposure to 10% oxygen increased pulmonary artery pressure in both sickle and mice receiving transplants with marrow from sickle mice compared with hemizygous controls. (B) Norepinephrine (0.3 μg/kg/min) was associated with increased pulmonary artery pressure in both sickle mice and BM-S mice compared with controls. This difference was also observed at other doses of norepinephrine (0.1 and 1.0 μg/kg/min, P < .05 for both, data not shown). (C) Angiotensin II (0.3 μg/kg/min) was also associated with increased pulmonary artery pressure in sickle mice and BM-S mice compared with controls. Similar results were observed at a lower dose (0.1 μg/kg intravenous bolus, P < .05, data not shown). Statistically significant differences from hemizygous controls are noted by *P < .05. Numbers represent mean ± SEM for n = 4-7 per group.

Figure 6

Mechanisms of abnormal NO synthase activity. Lung homogenate analysis is shown for 4 to 7 mice per group. (A) Lung NO synthase activity in the presence of calcium (constitutive NOS activity in the lung, the vast majority of which is eNOS) was decreased in sickle mice. (B) Calcium-independent citrulline formation (iNOS activity) did not differ between groups. (C) Western blot under nondenaturing conditions demonstrated 280 kDa eNOS dimer (active form) and 140 kDa eNOS monomer, which is inactive.^, Hemizygous mice had slightly more eNOS dimer than monomer, but sickle mice had almost completely lost dimerized eNOS. Positive controls show eNOS dissociated completely to monomeric form by boiling. Images were acquired by scanning the gel (Microtek [Carson, CA] i700 flatbed scanner with a transparency adapter) and saved without processing as a “tiff” image and then converted to “jpg” using Adobe Photoshop CS. (D) Lung nitrotyrosine, evidence of NO scavenging by reaction with superoxide or nitrite myeloperoxidase, was elevated in sickle mice and nitrotyrosine when measured by colorimetric assay (Oxis International) following the manufacturer's specifications. Statistically significant results indicated as follows: *P <.05 versus hemizygote; #P < .05 versus mice receiving transplants with wild-type marrow (BM-C). Numbers represent mean ± SEM.

Figure 7

Laboratory indicators of hemolytic anemia. Mean ± SEM are shown for 6 to 10 animals of each genotype. (A) Hemoglobin levels of sickle mice were lower than hemizygotes, but both were below wild-type, consistent with previous reports.^, The hemizygous mouse is anemic but does not have erythrocyte sickling. (B) Elevated reticulocyte fraction indicated markedly increased erythropoiesis in sickle mice. (C) Spleen as a fraction of body weight was elevated in sickle mice. The spleen is a normal site of erythropoiesis in adult mice and greatly increases in conditions of increased erythropoiesis. (D) Indirect bilirubin was elevated in sickle mouse plasma. (E) Lactate dehydrogenase in plasma was elevated in sickle mice as a nonspecific indicator of hemolysis. (F) Plasma NO consumption in blood processed under low shear to avoid artifactual hemolysis was measured using the gas-phase chemiluminescent NO analyzer (Sievers, Boulder, CO) as previously described. Nitric oxide consumption measurement was elevated in sickle mice (n = 21) compared with hemizygotes (n = 11) or wild-type controls (n = 6) by Kruskal-Wallis test. Absorbance spectrophotometry in the Soret band determined that plasma hemoglobin concentration correlated with NO consumption in mice (r² = 0.96, n = 49, data not shown). The wide variability in NO consumption in the sickle mice was similar to the variability of plasma NO consumption in humans with sickle cell disease. (G) Whole blood nitrite levels were obtained by mixing fresh blood immediately with nitrite preservation solution and then assaying by chemiluminescence (Sievers, Boulder, CO) as an indirect measure of NO synthase activity. Whole blood nitrite levels were lower in sickle mice than hemizygous and wild-type controls by the Mann-Whitney test (n = 4 to 7 per group). Nitrite in the BM-S mice (n = 9) was lower than the nitrite in the controls receiving transplants with wild-type marrow (BM-C, n = 4). (H) Lung homogenate assays demonstrated significantly higher arginase activity in sickle mice and in BM-S mice (n = 4 to 7 mice per group). Statistically significant results are indicated as follows: *P < .05 versus hemizygous controls; #P < .05 versus BM-C controls.

Figure 8

Pulmonary vascular responsiveness and lung homogenate activity of alloimmune hemolytic mice were similar to sickle mice and not to hemizygous sickle controls (mean ± SEM for n = 4 to 6 per group). A mouse model of acute hemolytic transfusion reaction was studied during alloimmune hemolysis by microcardiac catheterization. Because mice studied on the third day of hemolysis did not appear to have greater abnormality in the parameters measured than mice studied on the first or second days of hemolysis, data were pooled together. (A) Vasodilation to inhaled nitric oxide at 4 ppm was significantly blunted in alloimmune hemolytic mice compared with hemizygous controls (P < .05). (B) Vasodilation to bradykinin infusion was significantly blunted at 3 μg/kg in alloimmune hemolytic mice (P < .05). (C) The endothelium-independent agent CGRP (0.3 nmol/kg intravenous bolus) produced the same vasodilation response in alloimmune hemolytic mice and sickle mice as in hemizygous controls (P = NS). (D) eNOS assay of lung homogenate was significantly lower in alloimmune hemolytic mice than in hemizygous controls (P < .05), similar to sickle mice. (E) Arginase assay of plasma and lung homogenate was significantly elevated in both alloimmune hemolytic mice and sickle mice compared with hemizygous controls (P < .05). (F) Luminol assay of lung homogenate indicated significantly elevated reactive oxygen species in alloimmune hemolytic mice and sickle mice compared with hemizygous controls (P < .05). Statistically significant results are indicated by an asterisk (P < .05 versus hemizygous controls).