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This study examines the molecular mechanisms by which hypoxia regulates phosphorylated endothelial nitric oxide synthase (eNOS) activity and NO production in porcine coronary artery endothelial cells (PCAEC). Exposure to hypoxia (pO(2)=10 mmHg) for periods up to 3 h resulted in a time-dependent increase in eNOS protein expression and an early (15 min) and sustained increase in eNOS phosphorylation at Ser-1177. Exposure to hypoxia for 30 min led to a doubling in eNOS activity (control=6.2+/-4.4 vs hypoxia=14.1+/-5.0 fmol cGMP/microg protein, P<0.05) and NO release (control=5.9+/-0.8 vs hypoxia=11.8+/-1.2 nM/microg protein, P<0.05). Hypoxia also led to a significant increase in Akt phosphorylation and upregulation of Hsp90 binding to eNOS. Pretreatment of cells with either 1 microg/ml geldanamycin (a specific inhibitor of Hsp90) or 500 nM wortmannin (a specific PI3 kinase inhibitor) suppressed hypoxia-stimulated Akt and eNOS phosphorylation and significantly attenuated hypoxia-stimulated Hsp90 binding to eNOS. Both eNOS activity and NO production were inhibited by geldanamycin and wortmannin. Although hypoxia led to early activation of p42/44 mitogen-activated protein kinases (MAPK), inhibition of their pathway by PD98059 did not suppress hypoxia-stimulated eNOS phosphorylation and eNOS activity. These data demonstrate that hypoxia leads to increased eNOS phosphorylation via stimulated Hsp90 binding to eNOS and activation of the PI3-Akt pathway. We conclude that a coordinated interaction between Hsp90 and PI3-Akt may be an important mechanism by which eNOS activity and NO production is upregulated in hypoxic heart.

Endotoxemia is associated with an early phase of pulmonary hypertension and a later increase in microvascular permeability. These physiologic changes are attended by peripheral blood and lung lymph leukopenia and a rapid accumulation of both granulocytes and lymphocytes in the peripheral lung. In the present study, the numbers of lymphocytes in blood, lung lymph, and lung tissue after infusion of endotoxin were determined by fluorescent labeling of lymphocyte populations with monoclonal antibodies to sheep T1, T4, T8, or leukocyte common antigen and with rabbit anti-sheep immunoglobulins (B cells). Peripheral blood, lung lymph, and lung tissue samples were collected at baseline and 15, 30, 60, 120, 180, and 240 minutes after the start of intravenous infusion of E. coli endotoxin (1.25 micrograms/kg, N = 6) or saline (N = 4) from open-chest anesthetized sheep. Pulmonary artery pressure and lung lymph flow were also monitored at these times. Endotoxin caused marked reductions in the number of T and B lymphocytes in blood and lung lymph. As compared with baseline, total blood leukocytes and granulocytes were significantly reduced below control levels from 30 minutes of endotoxin, and lymphocyte numbers were reduced from 60 minutes. T1, T4, T8 and B lymphocyte subsets contributed to the fall in blood lymphocytes. Endotoxin caused a significant fall in number of lung lymph leukocytes (T1, T4 and T8 cells) from 120 minutes; numbers of B lymphocytes were also reduced. Counts of the number of lymphocytes in the biopsy tissue revealed a significant rise in T lymphocytes sequestered in the lung. We conclude endotoxemia in sheep causes a reduction in lymphocytes in blood and lung lymph and an increase in these cell types in peripheral lung tissue. These findings suggest that lymphocyte subpopulations accumulate in the lungs during periods of pulmonary hypertension and increased permeability and may participate in endotoxin-induced lung injury.

Chronic pulmonary hypertension occurs in several human diseases in which there is evidence of chronic or repeated bouts of pulmonary inflammation. To determine whether prolonged lung inflammation causes persistent pulmonary hypertension Escherichia coli endotoxin was given to seven chronically instrumented awake sheep three times a week for 10 to 14 weeks. Pulmonary artery, left atrial and systemic arterial pressures, cardiac output, arterial blood gases and pH were monitored before starting endotoxin treatment and twice weekly, immediately before endotoxin infusion. Three sheep receiving saline over a similar time period served as controls. Pulmonary vasoreactivity to breathing 12% oxygen and a bolus infusion of an analog of prostaglandin H2 was also assessed. Peripheral lung biopsy tissue was taken at baseline and at periods throughout the experiment to assess pulmonary inflammation. Repeated endotoxin infusions resulted in a significant increase in mean pulmonary artery pressure from the 8th week of treatment and more than a 50% increase from week 10 (baseline = 18.4 cm H2O +/- 1.0 (mean +/- SE); 10 weeks endotoxin = 27.8 +/- 4.3; p less than 0.05). Pulmonary vasoreactivity to both an analog of prostaglandin H2 and 12% oxygen decreased in the period from 4 to 8 weeks of endotoxin treatment. Light microscopic assessment of lung biopsy tissue showed a persistent four-fold increase above baseline in number of peripheral lung granulocytes. Electron microscopy revealed that granulocytes, lymphocytes, and monocytes sequestered in the lungs of these animals, and that structural damage to the endothelium was minimal. Morphometry of lungs obtained at autopsy in which the pulmonary arteries had been distended with barium-gelatin showed extension of muscle into the walls of smaller intra-acinar arteries (than normal) and a reduction in number of filled peripheral arteries. We conclude that repeated infusions of endotoxin into sheep cause persistent lung inflammation, altered pulmonary vasoreactivity, sustained pulmonary hypertension, and some of the structural changes characteristic of this disease. Chronic inflammation may play a role in the pathogenesis of chronic pulmonary hypertension.

Whereas hypoxia is known to cause an increase in thickness of both the medial and adventitial layers of the muscular pulmonary arteries, little is known of its effect on the endothelial cell and rest of the intima. The present study describes the ultrastructural changes in the intima of the hilar intrapulmonary muscular artery of the rat after exposure to hypobaric hypoxia of 380 torr for 10 days and their resolution during recovery periods of between 3 and 70 days in room air. After 10 days of hypoxia, the intima is 3 times thicker than the controls (control = 1.81 micrometers. +/- 0.13 standard error (S.E.); hypoxia = 5.59 micrometers. +/- 1.14 S.E.; p less than 0.05). The thickness of the endothelial cell layer doubles (control = 1.57 +/- 0.12 S.E.; hypoxia = 3.29 micrometer. +/- 0.23 S.E.; p less than 0.001) and point-counting the cytoplasm of these cells reveal significant relative increase in areal proportions, and hence most marked hypertrophy, of ribosomes, rough endoplasmic reticulum, and Golgi apparatus. An increase in number of endothelial cells is also apparent. The increase in thickness of the subendothelial layer is due to appearance of edema. In addition, microfibrillar basement membrane-like material is found focally within the subendothelial layer. In some sections, approximately 50 per cent of the endothelial associated basement membrane is absent. During recovery periods, there is regression of all the hypoxia-induced changes, save that the amount of subendothelial elastin increases to day 28 recovery, thereby altering the structure of the wall. The presence of elastin, collagen fibers, and microfibrils on the luminal side of the endothelial basement membrane, in control and experimental animals, points to their synthesis by the endothelial cell.

Hypoxia is known to cause pulmonary hypertension with an increase in arterial muscle-both hypertrophy of the medial coat of the normally muscular arteries and development of new muscle more peripherally than is normal--and a decrease in the number of small arteries. The present study descirbes the ultrastructural features of these changes. Pulmonary arteries less than 100 microgram. in diameter after distension and including alveolar capillaries and the muscular arter at the hilum were examined after exposure to 380 torr for between 1 and 52 days. The earliest change, detected from day 2, is the appearance of new muscle in the nonmuscular regions of artery. The position of the new muscle internal to the single elastic lamina and immediately subjacent to endothelium distinguished it from normal muscle. Ultrastructural examination shows not an "extension" of muscle along a pathway, but differentiation to form new muscle, by preexisting cells--by the pericyte in the nonmuscular arteries and by the intermediate cell in the partially muscular. The veins are spared. Endothelial cells also change significantly from day 3. In the normally nonmuscular regions of artery and in the alveolar capillaries, they are thickened. In the nonmuscular regions of artery, although the endothelial cells appear edematous, the number of Weibel-Palade bodies is increased and new muscle appears subjacent. Elsewhere the Weibel-Palade bodies show normal distribution. The extreme narrowing of the lumen caused by thickened endothelial cells and by encroachment of new muscle as well as the finding of capillaries that are no longer patent doubtless explain the reduction in small peripheral areteries.

The present morphometric and electron microscopic study follows the development of the hilar intrapulmonary artery of rat form 12 hours to 36 days of age and its structural adaptation to hypobaric hypoxia (380 torr) when exposed for periods of 3, 7, 14, and 28 days starting at 8 days of age. In control animals a striking increase in medial thickness is seen at 11 and 15 days of age and is accompanied, at 11 days, by an increase in the organelles of smooth muscle contraction-the myofilaments-and at 15 days, by an increase in rough sarcoplasmic reticulum. From 15 days, a gradual increase in collagen fibrils and elastin occurs in the extracellular ground substance. With exposure to hypoxia, from day 14 medial thickness increases more than the age-matched controls due to an increase in extracellular ground substance and to thickening of the elastic laminae. At all times studied, smooth muscle diameter is similar to the age-matched controls, but the normal fluctuations in volume density of the contractile elements and the rough sarcoplasmic reticulum are blunted. In the extracellular ground substance, the matrix is increased and collagen fibrils reduced, both in concentration and diameter. Thus, in young hypoxic rats, the narrowed lumen and external diameter of the large muscular arteries is associated with abnormal collagen formation. The hypoxia-induced changes in the young rat are different from those reported in the adult in which medial thickening is the result of increased extracellular ground substance with a normal concentration of collagen fibrils and hypertrophy of the smooth muscle cells. In the young lung, the structural changes have more far-reaching effects than the adult since they also interfere with lung growth.

Infusion of Escherichia coli endotoxin into sheep causes a syndrome analogous to the adult respiratory distress syndrome. Physiologic measurements show an initial phase of marked pulmonary hypertension followed by a phase characterized by the production of large quantities of protein-rich lung lymph. The present study relates the structural changes that occur during endotoxemia to concomitant functional changes. In five anesthetized open-chest sheep, we monitored pulmonary and systemic artery pressure for a 1 hour baseline period and for 4 hours after the start of E. coli endotoxin infusion (1.25 microgram/kg, intravenously). We also measured cardiac output, arterial blood gases and pH, and number of circulating leukocytes. In addition, we sequentially biopsied random lobes from the lungs of each sheep at baseline and at 15, 30, 60, 120, 180, and 240 minutes after the start of endotoxin. Five control sheep were treated identically except that they received saline instead of endotoxin. By 15 minutes after the start of endotoxin infusion, light microscopy revealed margination and accumulation of leukocytes in the lungs' microcirculation. Counts of the number of peripheral lung granulocytes in biopsy specimens showed a 3-fold increase above baseline by 15 minutes and a 6-fold increase by 4 hours. By electron microscopy, the leukocytes were identified as both granulocytes and lymphocytes, present in approximately equal numbers. Some granulocytes were fragmented, and specific granules were found free in the vessel lumen. By 30 minutes, some leukocytes were migrating into the interstitium. By 60 minutes, interstitial edema was seen, and there was focal endothelial cell damage. Correlation of the structural with the physiologic changes shows that the initial accumulation of leukocytes in the microcirculation occurs when pulmonary hypertension develops. The migration of leukocytes into the interstitium and endothelial cell damage precedes the physiologic changes that we interpret as increased pulmonary vascular permeability. Since gram negative septicemia is a frequent occurrence in the adult respiratory distress syndrome the changes described here may be similar to the alterations that occur early in the development of the syndrome in man.