Upregulation of nitric oxide synthase in mice with severe hypoxia-induced pulmonary hypertension.

BACKGROUND: The importance of nitric oxide (NO) in hypoxic pulmonary hypertension has been demonstrated using nitric oxide synthase (NOS) knockout mice. In that model NO from endothelial NOS (eNOS) plays a central role in modulating pulmonary vascular tone and attenuating hypoxic pulmonary hypertension. However, the normal regulation of NOS expression in mice following hypoxia is uncertain. Because genetically engineered mice are often utilized in studies of NO, we conducted the present study to determine how hypoxia alters NOS expression in wild-type mice. METHOD: Mice were exposed to sea level, ambient conditions (5280 feet) or severe altitude (17,000 feet) for 6 weeks from birth, and hemodynamics and lung NOS expression were assessed. RESULTS: Hypoxic mice developed severe pulmonary hypertension (right ventricular systolic pressure [RVsP] 60 mmHg) as compared with normoxic mice (27 mmHg). Using quantitative reverse-transcription PCR, it was found that expressions of eNOS and inducible NOS (iNOS) increased 1.5-fold and 3.5-fold, respectively, in the lung. In addition, the level of lung eNOS protein was increased, neuronal NOS (nNOS) protein was unchanged, and iNOS was below the limit of detection. Immunohistochemistry demonstrated no change in lung iNOS or nNOS staining in either central or peripheral areas, but suggested increased eNOS in the periphery following hypoxia. CONCLUSION: In mice, hypoxia is associated with increases in lung eNOS, possibly in iNOS, but not in nNOS; this suggests that the pattern of lung NOS expression following hypoxia must be considered in studies using genetically engineered mice.

p27(Kip1) is important in modulating pulmonary artery smooth muscle cell proliferation.

Vascular remodeling due to pulmonary arterial smooth muscle cell (PASMC) proliferation is central to the development of pulmonary hypertension. Cell proliferation requires the coordinated interaction of cyclins and cyclin-dependent kinases (cdk) to drive cells through the cell cycle. Cdk inhibitors can bind cyclin-cdk complexes and cause G(1) arrest. To determine the importance of the cdk inhibitor p27(Kip1) in PASMC proliferation we studied [(3)H]thymidine incorporation, changes in cell cycle, cell proliferation, and protein expression of p27(Kip1) following serum stimulation in early passage rat PASMC. p27(Kip1) expression decreased to 40% of baseline after serum stimulation, which was associated with an increase in both [(3)H]thymidine incorporation and the percent of cells in S phase. p27(Kip1) binding to cyclin E decreased at 24 h, and this correlated with an increase in phosphorylation of retinoblastoma both in vivo and in vitro. Overexpression of p27(Kip1) decreased [(3)H]thymidine incorporation and reduced cell counts at 5 d compared with controls. PASMC obtained from p27(Kip1-/-) mice showed a 2-fold increase in [(3)H]thymidine incorporation (at 24 h) and cell proliferation compared with p27(Kip1+/+) PASMC when cultured in 10% fetal bovine serum (FBS). These results suggest an important role for p27(Kip1) in regulating PASMC mitogenesis and proliferation.

Hypoxemia explained 36 years later.

A 37-year-old man who had an atrial septal defect (ASD) corrected as an infant was found to be hypoxemic with a 22% shunt. An MRI scan revealed that the patient's inferior vena cava drained into his left rather than his right atrium, a previously undetected complication of his ASD repair 36 years before.

Relative contributions of endothelial, inducible, and neuronal NOS to tone in the murine pulmonary circulation.

Nitric oxide plays an important role in modulating pulmonary vascular tone. All three isoforms of nitric oxide synthase (NOS), neuronal (nNOS, NOS I), inducible (iNOS, NOS II), and endothelial (eNOS, NOS III), are expressed in the lung. Recent reports have suggested an important role for eNOS in the modulation of pulmonary vascular tone chronically; however, the relative contribution of the three isoforms to acute modulation of pulmonary vascular tone is uncertain. We therefore tested the effect of targeted disruption of each isoform on pulmonary vascular reactivity in transgenic mice. Isolated perfused mouse lungs were used to evaluate the effect of selective loss of pulmonary nNOS, iNOS, and eNOS with respect to hypoxic pulmonary vasoconstriction (HPV) and endothelium-dependent and -independent vasodilation. eNOS null mice had augmented HPV (225 +/- 65% control, P < 0.02, mean +/- SE) and absent endothelium-dependent vasodilation, whereas endothelium-independent vasodilation was preserved. HPV was minimally elevated in iNOS null mice and normal in nNOS null mice. Both nNOS and iNOS null mice had normal endothelium-dependent vasodilation. In wild-type lungs, nonselective NOS inhibition doubled HPV, whereas selective iNOS inhibition had no detectable effect. In intact, lightly sedated mice, right ventricular systolic pressure was elevated in eNOS-deficient (42.3 +/- 1.2 mmHg, P < 0.001) and, to a lesser extent, in iNOS-deficient (37.2 +/- 0.8 mmHg, P < 0.001) mice, whereas it was normal in nNOS-deficient mice (30.9 +/- 0.7 mmHg, P = not significant) compared with wild-type controls (31.3 +/- 0.7 mmHg). We conclude that in the normal murine pulmonary circulation 1) nNOS does not modulate tone, 2) eNOS-derived nitric oxide is the principle mediator of endothelium-dependent vasodilation in the pulmonary circulation, and 3) both eNOS and iNOS play a role in modulating basal tone chronically.

The pulmonary circulation of homozygous or heterozygous eNOS-null mice is hyperresponsive to mild hypoxia.

Acute hypoxic vasoconstriction and development of hypoxic pulmonary hypertension (PHTN) are unique properties of the pulmonary circulation. The pulmonary endothelium produces vasoactive factors, including nitric oxide (NO), that modify these phenomena. We tested the hypothesis that NO produced by endothelial nitric oxide synthase (eNOS) modulates pulmonary vascular responses to hypoxia using mice with targeted disruption of the eNOS gene (eNOS-/-). Marked PHTN was found in eNOS-/- mice raised in mild hypoxia when compared with either controls or eNOS-/- mice raised in conditions simulating sea level. We found an approximate twofold increase in partially and fully muscularized distal pulmonary arteries in eNOS-/- mice compared with controls. Consistent with vasoconstriction being the primary mechanism of PHTN, however, acute inhalation of 25 ppm NO resulted in normalization of RV pressure in eNOS-/- mice. In addition to studies of eNOS-/- mice, the dose-effect of eNOS was tested using heterozygous eNOS+/- mice. Although the lungs of eNOS+/- mice had 50% of normal eNOS protein, the response to hypoxia was indistinguishable from that of eNOS-/- mice. We conclude that eNOS-derived NO is an important modulator of the pulmonary vascular response to chronic hypoxia and that more than 50% of eNOS expression is required to maintain normal pulmonary vascular tone.

Dilatation of bronchial stenoses due to sarcoidosis using a flexible fiberoptic bronchoscope.

Stenosis of the trachea and bronchi can complicate many diseases and lead to significant pulmonary complaints. Unfortunately, steroids rarely yield satisfactory results in reversing symptoms. We describe six patients with symptomatic airway stenosis from sarcoidosis, all of whom were refractory to steroid therapy. By using a Fogarty embolectomy catheter inserted through the inner channel of a flexible bronchoscope, we were able to dilate the stenotic areas under direct vision. Patients had significant subjective improvement following dilatation and no significant complications occurred. We believe this technique represents an improvement on previously described methods because it can easily access the upper lobes and more distal segments and can be performed at the bedside.

Role of neutrophils in generalized reperfusion injury associated with resuscitation from shock.

Recent studies suggest that neutrophils are an important factor in the organ injury associated with ischemia and shock. Increased neutrophil-endothelial adhesiveness is essential for neutrophil-mediated vascular injury. To examine the role of neutrophils and neutrophil adhesiveness in the development of injury after hypovolemic shock, and to determine whether this injury is a consequence of reperfusion, we used the monoclonal antibody (MAb) 60.3 (directed to the primary human neutrophil adherence glycoprotein, CD18) to block neutrophil adherence functions at the time of resuscitation in a rabbit model of hemorrhagic shock. None of the unanesthetized control animals subjected to 2 hours of shock (cardiac output, 30% of baseline) followed by resuscitation survived 5 days. All had gross and histologic evidence of injury to lungs, liver, and gastrointestinal mucosa. In contrast, 71% of the animals that received MAb 60.3 immediately before resuscitation survived 5 days (p less than 0.005), and visceral organ injury was absent or markedly attenuated. We conclude that a significant proportion of injury resulting from shock and resuscitation occurs after the ischemic insult and that increased neutrophil adhesiveness plays an important role in the development of multiple organ injury and death following shock and resuscitation (in this model). This injury may be significantly reduced by blocking neutrophil adherence functions with the MAb 60.3--even if administration is delayed until resuscitation.