Wolfgang Steudel

Pulmonary Vasoconstriction and Hypertension in Mice With Targeted Disruption of the Endothelial Nitric Oxide Synthase (NOS 3) Gene

NO, synthesized in endothelial cells by endothelial NO synthase (NOS 3), is believed to be an important endogenous pulmonary vasodilator substance that contributes to the normal low pulmonary vascular resistance. To selectively investigate the role of NOS 3 in the pulmonary circulation, mice with targeted disruption of the NOS 3 gene were studied. Pulmonary hemodynamics were studied by measuring pulmonary artery pressure, left ventricular end-diastolic pressure, and lower thoracic aortic flow by using a novel open-chest technique. Transient partial occlusion of the inferior vena cava was used to assess the pulmonary artery pressure-flow relationship. Tension developed by isolated pulmonary artery segments after acetylcholine stimulation was measured in vitro. The histological appearance of NOS 3-deficient and wild-type murine lungs was compared. NOS 3-deficient mice (n = 27), when compared with wild-type mice (n = 32), had pulmonary hypertension (pulmonary artery pressure, 19.0 +/- 0.8 versus 16.4 +/- 0.6 mm Hg [mean +/- SE]; P < .05) that was due to an increased total pulmonary resistance (62 +/- 6 versus 33 +/- 2 mm Hg.min.g.mL-1; P < .001). In vitro, acetylcholine induced vasodilation in the main pulmonary arteries of wild-type but not NOS 3-deficient mice. The morphology of the lungs of NOS 3-deficient mice did not differ from that of wild-type mice. We conclude that NOS 3 is a key enzyme responsible for providing basal pulmonary NO release. Congenital NOS 3 deficiency produces mild pulmonary hypertension in mice.

Pulmonary hypertension in transgenic mice expressing a dominant-negative BMPRII gene in smooth muscle.

Abstract— Bone morphogenetic peptides (BMPs), a family of cytokines critical to normal development, were recently implicated in the pathogenesis of familial pulmonary arterial hypertension. The type-II receptor (BMPRII) is required for recognition of all BMPs, and targeted deletion of BMPRII in mice results in fetal lethality before gastrulation. To overcome this limitation and study the role of BMP signaling in postnatal vascular disease, we constructed a smooth muscle–specific transgenic mouse expressing a dominant-negative BMPRII under control of the tetracycline gene switch (SM22-tet-BMPRIIdelx4+ mice). When the mutation was activated after birth, mice developed increased pulmonary artery pressure, RV/LV+S ratio, and pulmonary arterial muscularization with no increase in systemic arterial pressure. Studies with SM22-tet-BMPRIIdelx4+ mice support the hypothesis that loss of BMPRII signaling in smooth muscle is sufficient to produce the pulmonary hypertensive phenotype.

Sustained pulmonary hypertension and right ventricular hypertrophy after chronic hypoxia in mice with congenital deficiency of nitric oxide synthase 3.

Chronic hypoxia induces pulmonary hypertension and right ventricular (RV) hypertrophy. Nitric oxide (NO) has been proposed to modulate the pulmonary vascular response to hypoxia. We investigated the effects of congenital deficiency of endothelial NO synthase (NOS3) on the pulmonary vascular responses to breathing 11% oxygen for 3-6 wk. After 3 wk of hypoxia, RV systolic pressure was greater in NOS3-deficient than in wild-type mice (35+/-2 vs 28+/-1 mmHg, x+/-SE, P < 0.001). Pulmonary artery pressure (PPA) and incremental total pulmonary vascular resistance (RPI) were greater in NOS3-deficient than in wild-type mice (PPA 22+/-1 vs 19+/-1 mmHg, P < 0.05 and RPI 92+/-11 vs 55+/-5 mmHg.min.gram.ml-1, P < 0.05). Morphometry revealed that the proportion of muscularized small pulmonary vessels was almost fourfold greater in NOS3-deficient mice than in wild-type mice. After 6 wk of hypoxia, the increase of RV free wall thickness, measured by transesophageal echocardiography, and of RV weight/body weight ratio were more marked in NOS3-deficient mice than in wild-type mice (RV wall thickness 0.67+/-0.05 vs 0.48+/-0.02 mm, P < 0.01 and RV weight/body weight ratio 2.1+/-0.2 vs 1.6+/-0.1 mg. gram-1, P < 0.05). RV hypertrophy produced by chronic hypoxia was prevented by breathing 20 parts per million NO in both genotypes of mice. These results suggest that congenital NOS3 deficiency enhances hypoxic pulmonary vascular remodeling and hypertension, and RV hypertrophy, and that NO production by NOS3 is vital to counterbalance pulmonary vasoconstriction caused by chronic hypoxic stress.

Inhaled Nitric Oxide Basic Biology and Clinical Applications

A REMARKABLY exciting field of research has developed since nitric oxide (NO) was identified in 1987 as a key endothelium-derived relaxing factor (EDRF). The awarding of the 1998 Nobel prize in physiology or medicine to three seminal researchers in the field of NO biology provided the most recent evidence for the emerging prominence of this area of study. The understanding of the roles of NO in the cardiovascular, immune, and nervous systems; the isolation and localization of NO synthases (NOS); the manipulation of the genes for NOS, including their cloning and selective transfer or knock-out; and the therapeutic use of inhaled NO gas have revolutionized many fields of physiologic research and are influencing clinical therapy. Many insights into the mechanisms of action of NO have been gained. Since the reported applications of inhaled NO in the laboratory and in adult patients with primary pulmonary hypertension in 1991, hundreds of studies have been conducted to determine the clinical applicability of inhaled NO. In subgroups of severely ill and hypoxic children and adults, inhaled NO improves arterial oxygenation and selectively decreases pulmonary arterial hypertension (PAH). NO inhalation therapy, in combination with conventional or high-frequency oscillatory ventilation, can reduce the need for extracorporeal membrane oxygenation (ECMO), an expensive and invasive procedure in newborn patients with hypoxic respiratory failure. However, it remains uncertain whether NO inhalation improves survival rates in adults or children with severe lung injury. New applications for NO inhalation have been discovered. Recent studies indicate that inhaled NO may decrease intestinal ischemia–reperfusion injury and may be useful to treat thrombotic disorders. By increasing the oxygen affinity of sickle cell hemoglobin, inhaled NO may prevent or treat sickle cell crisis. This article reviews the relevant physiologic effects, therapeutic uses, side-effects, and toxicity of NO inhalation. The first portion of this article concentrates on the chemistry, biochemistry, toxicology, and biology of NO; the second portion summarizes the results of NO inhalation studies to date in experimental settings and the results of clinical studies in newborns, children, and adults.

Quantitative Assessment of Regional Myocardial Function in Mice by Tissue Doppler Imaging Comparison With Hemodynamics and Sonomicrometry

Background—Tissue Doppler imaging (TDI) is a novel echocardiographic method to quantify regional myocardial function. The objective of this study was to assess whether myocardial velocities and strain rate (SR) could be obtained by TDI in mice and whether these indices accurately quantified alterations in left ventricular (LV) systolic function. Methods and Results—TDI was performed in 10 healthy mice to measure endocardial (vendo) and epicardial systolic velocities and SR. In further experiments, TDI indices were compared with dP/dtmax and with sonomicrometer-derived regional velocities, at rest and after administration of dobutamine or esmolol. TDI indices were also studied serially in 8 mice before and 4 and 7 hours after endotoxin challenge. Myocardial velocities and SR were obtained in all mice with low measurement variability. TDI indices increased with administration of dobutamine (vendo from 2.2±0.3 to 3.8±0.2 cm/s [P<0.01]; SR from 12±2 to 20±2 s−1 [P<0.05]) and decreased with administration of esmolol (vendo 1.4±0.2 cm/s [P<0.05]; SR 6±1 s−1 [P<0.01]). Both indices correlated strongly with dP/dtmax (r2=0.79 for SR and r2= 0.69 for vendo; both P<0.0001). SR and shortening fraction were predictors of dP/dtmax even after adjustment for the confounding effect of the other variables. Vendo correlated closely with sonomicrometer-measured velocity (r2=0.71, P<0.0005). After endotoxin challenge, decreases in both vendo and SR were detected before decreases in shortening fraction became manifest. Conclusions—Myocardial velocities and SR can be measured noninvasively in mice with the use of TDI. Both indices are sensitive markers for quantifying LV global and regional function in mice.

Hypoxic pulmonary blood flow redistribution and arterial oxygenation in endotoxin-challenged NOS2-deficient mice

Sepsis and endotoxemia impair hypoxic pulmonary vasoconstriction (HPV), thereby reducing arterial oxygenation and enhancing hypoxemia. Endotoxin induces nitric oxide (NO) production by NO synthase 2 (NOS2). To assess the role of NO and NOS2 in the impairment of HPV during endotoxemia, we measured in vivo the distribution of total pulmonary blood flow (QPA) between the right (QRPA) and left (QLPA) pulmonary arteries before and after left mainstem bronchus occlusion (LMBO) in mice with and without a congenital deficiency of NOS2. LMBO reduced QLPA/QPA equally in saline-treated wild-type and NOS2-deficient mice. However, prior challenge with Escherichia coli endotoxin markedly impaired the ability of LMBO to reduce QLPA/QPA in wild-type, but not in NOS2-deficient, mice. After endotoxin challenge and LMBO, systemic oxygenation was impaired to a greater extent in wild-type than in NOS2-deficient mice. When administered shortly after endotoxin treatment, the selective NOS2 inhibitor L-NIL preserved HPV in wild-type mice. High concentrations of inhaled NO attenuated HPV in NOS2-deficient mice challenged with endotoxin. These findings demonstrate that increased pulmonary NO levels (produced by NOS2 or inhaled at high levels from exogenous sources) are necessary during the septic process to impair HPV, ventilation/perfusion matching and arterial oxygenation in a murine sepsis model.

Echocardiographic determination of risk area size in a murine model of myocardial ischemia

Genetically altered mice are useful to understand cardiac physiology. Myocardial contrast echocardiography (MCE) assesses myocardial perfusion in humans. We hypothesized it could evaluate murine myocardial perfusion before and after acute coronary ligation. MCE was performed before and after this experimental myocardial infarction (MI) in anesthetized mice by intravenous injection of contrast microbubbles and transthoracic echo imaging. Time-video intensity curves were obtained for the anterior, lateral, and septal myocardial walls. After MI, MCE defects were compared with the area of no perfusion measured by Evans blue staining. In healthy animals, intramyocardial contrast was visualized in all the cardiac walls. The anterior wall had a higher baseline video intensity (53 ± 17 arbitrary units) than the lateral (34 ± 13) and septal (27 ± 13) walls ( P < 0.001) and a lower increase in video intensity after contrast injection [50 ± 17 vs. 60 ± 24 (lateral) and 65 ± 29 (septum), P < 0.01]. After MI, left ventricular (LV) dimensions were enlarged, and the shortening fraction was decreased. A perfusion defect was imaged with MCE in every mouse, with a correlation between MCE perfusion defect size (35 ± 13%) and the nonperfused area by Evans blue (37 ± 16%, y = 0.77 x + 6.1, r = 0.93, P < 0.001). Transthoracic MCE is feasible in the mouse and can accurately detect coronary occlusions and quantitate nonperfused myocardium.Genetically altered mice are useful to understand cardiac physiology. Myocardial contrast echocardiography (MCE) assesses myocardial perfusion in humans. We hypothesized it could evaluate murine myocardial perfusion before and after acute coronary ligation. MCE was performed before and after this experimental myocardial infarction (MI) in anesthetized mice by intravenous injection of contrast microbubbles and transthoracic echo imaging. Time-video intensity curves were obtained for the anterior, lateral, and septal myocardial walls. After MI, MCE defects were compared with the area of no perfusion measured by Evans blue staining. In healthy animals, intramyocardial contrast was visualized in all the cardiac walls. The anterior wall had a higher baseline video intensity (53 +/- 17 arbitrary units) than the lateral (34 +/- 13) and septal (27 +/- 13) walls (P < 0.001) and a lower increase in video intensity after contrast injection [50 +/- 17 vs. 60 +/- 24 (lateral) and 65 +/- 29 (septum), P < 0.01]. After MI, left ventricular (LV) dimensions were enlarged, and the shortening fraction was decreased. A perfusion defect was imaged with MCE in every mouse, with a correlation between MCE perfusion defect size (35 +/- 13%) and the nonperfused area by Evans blue (37 +/- 16%, y = 0.77x + 6.1, r = 0.93, P < 0. 001). Transthoracic MCE is feasible in the mouse and can accurately detect coronary occlusions and quantitate nonperfused myocardium.

Three-Dimensional Echocardiographic Assessment of Left Ventricular Wall Motion Abnormalities in Mouse Myocardial Infarction

We applied 3-dimensional echocardiographic reconstruction to assess left ventricular (LV) volumes, function, and the extent of wall motion abnormalities in a murine model of myocardial infarction (MI). Consecutive parasternal short-axis planes were obtained at 1-mm intervals with a 13-MHz linear array probe. End-diastolic and end-systolic LV volumes were calculated by Simpsons rule, and the ejection fraction and cardiac output were derived. Echocardiography-derived cardiac output was validated by an aortic flow probe in 6 mice. Echocardiography was then performed in 9 mice before and after the left anterior descending coronary artery was ligated. Wall motion was assessed, and the ratio of the abnormally to normally contracting myocardium was calculated. After MI occurred, LV end-diastolic volume and LV end-systolic volume increased (33 +/- 10 vs 24 +/- 6 microL, P <.05 and 24 +/- 9 vs 10 +/- 4 microL, P <.001), whereas cardiac output decreased (4.2 +/- 1.5 mL/min vs 6.6 +/- 2.3 mL/min, P <.01). Forty percent of the myocardium was normokinetic, 24% was hypokinetic, and 36% was akinetic. Echocardiography can measure LV volumes and regional and global function in a murine model of myocardial infarction, thereby providing the potential to quantitate and compare the responses of various transgenic mice to MI and its therapies.

Inaccuracies of Nitric Oxide Delivery Systems during Adult Mechanical Ventilation

Background Various systems to administer inhaled nitric oxide (NO) have been used in patients and experimental animals. We used a lung model to evaluate five NO delivery systems during mechanical ventilation with various ventilatory patterns. Methods An adult mechanical ventilator was attached to a test lung configured to separate inspired and expired gases. Four injection systems were evaluated with NO injected either into the inspiratory circuit 90 cm proximal to the Y piece or directly at the Y piece and delivered either continuously or only during the inspiratory phase. Alternatively, NO was mixed with air using a blender and delivered to the high‐pressure air inlet of the ventilator. Nitric oxide concentration was measured from the inspiratory limb of the ventilator circuit and the tracheal level using rapid‐ and slow‐response chemiluminescence analyzers. The ventilator was set for constant‐flow volume control ventilation, pressure control ventilation, pressure support ventilation, or synchronized intermittent mandatory ventilation. Tidal volumes of 0.5 l and 1 l were evaluated with inspiratory times of 1 s and 2 s. Results The system that premixed NO proximal to the ventilator was the only one that maintained constant NO delivery regardless of ventilatory pattern. The other systems delivered variable NO concentration during pressure control ventilation and spontaneous breathing modes. Systems that injected a continuous flow of NO delivered peak NO concentrations greater than the calculated dose. These variations were not apparent when a slow‐response chemiluminescence analyzer was used. Conclusions NO delivery systems that inject NO at a constant rate, either continuously or during inspiration only, into the inspiratory limb of the ventilator circuit produce highly variable and unpredictable NO delivery when inspiratory flow is not constant. Such systems may deliver a very high NO concentration to the lungs, which is not accurately reflected by measurements performed with slow‐response analyzers.

Determination of Right Ventricular Structure and Function in Normoxic and Hypoxic Mice A Transesophageal Echocardiographic Study

BACKGROUND Noninvasive cardiac evaluation is of great importance in transgenic mice. Transthoracic echocardiography can visualize the left ventricle well but has not been as successful for the right ventricle (RV). We developed a method of transesophageal echocardiography (TEE) to evaluate murine RV size and function. METHODS AND RESULTS Normoxic and chronically hypoxic mice (F(IO2)=0.11, 3 weeks) and agarose RV casts were scanned with a rotating 3.5F/30-MHz intravascular ultrasound probe. In vivo, the probe was inserted in the mouse esophagus and withdrawn to obtain contiguous horizontal planes at 1-mm intervals. In vitro, the probe was withdrawn along the left ventricular posterior wall of excised hearts. The borders of the RV were traced on each plane, allowing calculation of diastolic and systolic volumes, RV mass, RV ejection fraction, stroke volume, and cardiac output. RV wall thickness was measured. Echo volumes obtained in vitro were compared with cast volumes. Echo-derived cardiac output was compared with measurements of an ascending aortic Doppler flow probe. Echo-derived RV free wall mass was compared with true RV free wall weight. There was excellent agreement between cast and TEE volumes (y=0.82x+6.03, r=0.88, P<0.01) and flow-probe and echo cardiac output (y=1.00x+0.45, r=0.99, P<0.0001). Although echo-derived RV mass and wall thickness were well correlated with true RV weight, echo-derived RV mass underestimated true weight (y=0.53x+2.29, r=0.81, P<0.0001). RV mass and wall thickness were greater in hypoxic mice than in normoxic mice (0.78+/-0.19 versus 0.51+/-0.14 mg/g, P<0.03, 0.50+/-0.03 versus 0.38+/-0.03 mm, P<0.04). CONCLUSIONS TEE with an intravascular ultrasound catheter is a simple, accurate, and reproducible method to study RV size and function in mice.