Nagato Sato

Inhibition of c-Jun NH2-Terminal Kinase Activity Improves Ischemia/Reperfusion Injury in Rat Lungs

Although c-Jun NH2-terminal kinase (JNK) has been implicated in the pathogenesis of transplantation-induced ischemia/reperfusion (I/R) injury in various organs, its significance in lung transplantation has not been conclusively elucidated. We therefore attempted to measure the transitional changes in JNK and AP-1 activities in I/R-injured lungs. Subsequently, we assessed the effects of JNK inhibition by the three agents including SP600125 on the degree of lung injury assessed by means of various biological markers in bronchoalveolar lavage fluid and histological examination including detection of apoptosis. In addition, we evaluated the changes in p38, extracellular signal-regulated kinase, and NF-κB-DNA binding activity. I/R injury was established in the isolated rat lung preserved in modified Euro-Collins solution at 4°C for 4 h followed by reperfusion at 37°C for 3 h. We found that AP-1 was transiently activated during ischemia but showed sustained activation during reperfusion, leading to significant lung injury and apoptosis. The change in AP-1 was generally in parallel with that of JNK, which was activated in epithelial cells (bronchial and alveolar), alveolar macrophages, and smooth muscle cells (bronchial and vascular) on immunohistochemical examination. The change in NF-κB qualitatively differed from that of AP-1. Protein leakage, release of lactate dehydrogenase and TNF-α into bronchoalveolar lavage fluid, and lung injury were improved, and apoptosis was suppressed by JNK inhibition. In conclusion, JNK plays a pivotal role in mediating lung injury caused by I/R. Therefore, inhibition of JNK activity has potential as an effective therapeutic strategy for preventing I/R injury during lung transplantation.

Response of Intra-acinar Pulmonary Microvessels to Hypoxia, Hypercapnic Acidosis, and Isocapnic Acidosis

To elucidate the differential reactivity of pulmonary microvessels in the acini to hypoxia, excessive CO2, and increased H+, we investigated changes in the diameter of precapillary arterioles, postcapillary venules, and capillaries in isolated rat lungs on exposure to normocapnic hypoxia (2% O2), normoxic hypercapnia (15% CO2), and isocapnic acidosis (0.01 mol/L HCl). Microvascular diameters were precisely examined using a real-time confocal laser scanning luminescence microscope coupled to a high-sensitivity camera with an image intensifier. Measurements were made under conditions with and without indomethacin or N(omega)-nitro-L-arginine methyl ester to assess the importance of vasoactive substances produced by cyclooxygenase (COX) or NO synthase (NOS) as it relates to the reactivity of pulmonary microvessels to physiological stimuli. We found that acute hypoxia contracted precapillary arterioles that had diameters of 20 to 30 microm but did not constrict postcapillary venules of similar size. COX- and NOS-related vasoactive substances did not modulate hypoxia-elicited arteriolar constriction. Hypercapnia induced a distinct venular dilatation closely associated with vasodilators produced by COX but not by NOS. Arterioles were appreciably constricted in isocapnic acidosis when NOS, but not COX, was suppressed, whereas venules showed no constrictive response even when both enzymes were inhibited. Capillaries were neither constricted nor dilated under any experimental conditions. These findings suggest that reactivity to hypoxia, CO2, and H+ is not qualitatively similar among intra-acinar microvessels, in which COX- and NOS-associated vasoactive substances function differently.

Nitric oxide differentially attenuates microvessel response to hypoxia and hypercapnia in injured lungs

The issue of whether the acinar microvessel response to alveolar hypoxia and hypercapnia is impaired in injured lungs has not been vigorously addressed, despite the importance of knowing whether it is or not when treating patients with serious lung injury in terms of permissive hypercapnia. Applying a real-time laser confocal luminescence microscope, we studied hypoxia- and hypercapnia-induced changes in the diameter of the intra-acinar arterioles, venules, and capillaries of isolated rat lungs harvested from animals exposed for 48 h to 21% O2 ( group N) or 90% O2 ( group H). Measurements were made with and without inhibition of nitric oxide (NO) synthase (NOS) by N ω-nitro-l-arginine methyl ester or of cyclooxygenase (COX) by indomethacin at different basal vascular tones evoked by thromboxane A2(TXA2) analog. Hypoxia in the absence of TXA2 contracted arterioles in group N but not in group H. Attenuated hypoxia-induced arteriole constriction was restored almost fully by inhibiting NOS and partially by inhibiting COX. Hypercapnia induced venule dilation in group N, but did not dilate venules in group H, irrespective of TXA2. NOS inhibition in hypercapnia unexpectedly enhanced venule and arteriole dilation in group H. These responses no longer occurred when NOS and COX were inhibited simultaneously. In conclusion, microvessel reactions to hypoxia and hypercapnia are abnormal in hyperoxia-injured acini, in which NO directly attenuates hypoxia-induced arteriole constriction, whereas COX inhibited by excessive NO impedes hypercapnia-induced microvessel dilation.The issue of whether the acinar microvessel response to alveolar hypoxia and hypercapnia is impaired in injured lungs has not been vigorously addressed, despite the importance of knowing whether it is or not when treating patients with serious lung injury in terms of permissive hypercapnia. Applying a real-time laser confocal luminescence microscope, we studied hypoxia- and hypercapnia-induced changes in the diameter of the intra-acinar arterioles, venules, and capillaries of isolated rat lungs harvested from animals exposed for 48 h to 21% O(2) (group N) or 90% O(2) (group H). Measurements were made with and without inhibition of nitric oxide (NO) synthase (NOS) by N(omega)-nitro-L-arginine methyl ester or of cyclooxygenase (COX) by indomethacin at different basal vascular tones evoked by thromboxane A(2) (TXA(2)) analog. Hypoxia in the absence of TXA(2) contracted arterioles in group N but not in group H. Attenuated hypoxia-induced arteriole constriction was restored almost fully by inhibiting NOS and partially by inhibiting COX. Hypercapnia induced venule dilation in group N, but did not dilate venules in group H, irrespective of TXA(2). NOS inhibition in hypercapnia unexpectedly enhanced venule and arteriole dilation in group H. These responses no longer occurred when NOS and COX were inhibited simultaneously. In conclusion, microvessel reactions to hypoxia and hypercapnia are abnormal in hyperoxia-injured acini, in which NO directly attenuates hypoxia-induced arteriole constriction, whereas COX inhibited by excessive NO impedes hypercapnia-induced microvessel dilation.

Hyperoxia and Hypercapnic Acidosis Differentially Alter Nuclear Factor-κB Activation in Human Pulmonary Artery Endothelial Cells

Although ventilator-associated pulmonary injury, such as oxygen toxicity or barotrauma, has given rise to critical problems for mechanically ventilated patients with serious lung diseases, mechanical ventilation is an essential procedure to maintain appropriate tissue oxygenation, pH, and Pco2 in patients with respiratory distress (Dreyfuss and Saumon, 1998). In order to reduce the possibility of ventilator-associated pulmonary barotrauma, permissive hypercapnia has been applied as appropriate ventilatory support to acute respiratory distress syndrome (ARDS) (Feihl and Perret, 1994). In spite of the fact that there are some favorable prospective trials reporting that permissive hypercapnia improved mortality in patients with ARDS induced by heterogeneous background disorders (Hickling et al., 1994, Amato et al., 1998), efficacy and indication of this newly developed ventilator strategy are still controversial (Hudson, 1998). Permissive hypercapnia requires higher inspired oxygen concentration to maintain arterial oxygen tension than conventional mechanical ventilation mode and induces systemic hypercapnic acidosis for at least 48 hours (Carvahlo et al., 1997), thus arising the question whether hyperoxia and hypercapnic acidosis would exert a significant influence on sustained inflammation including ARDS.

Effects of active vasoconstriction and total flow on perfusion distribution in the rabbit lung

We analyzed the effects of hypoxic vasoconstriction and total flow on the distribution of pulmonary perfusion in 38 isolated left rabbit lungs perfused under zone 3 conditions. Lungs were suspended in an upright position, oriented to the apicobasal line. Distributions of regional perfusion rates (RPR) along the vertical and horizontal axes were determined using nonradioactive microspheres labeled with heavy metal elements, which were detectable with X-ray fluorescence spectrometry. Changing the O2 concentration of a respirator and an extracorporeal membrane oxygenator independently, respective influences of active vasoconstriction induced by alveolar hypoxia and pulmonary artery hypoxia (PA hypoxia) on the RPR distribution were examined at a flow rate of 0.4 ml x min(-1) x g wet lung tissue(-1). To analyze the effects of changes in total flow, we investigated the RPR distribution at a perfusion rate of 1.2 ml x min(-1) x g wet lung tissue(-1). The RPR distribution in the absence of hypoxia was inhomogeneous and was augmented in the lower lung fields, whereas alveolar hypoxia shifted the RPR upward and significantly diminished the RPR in the lung base. RPR distributions along the horizontal axes under alveolar hypoxia conditions demonstrated that remarkable hypoxic pulmonary vasoconstriction (HPV) takes place in medial regions at the lung base. PA hypoxia altered the RPR distribution in qualitatively the same manner as alveolar hypoxia. Increased flow rate augmented the RPR in the lung, except in the dorsobasal region. These results suggest that the occurrence of HPV and the vascular conductance are not uniform throughout the lung.

Biological Impediment to Oxygen Sensing in Injured Pulmonary Microcirculation Exposed to a High-Oxygen Environment

To resolve the issue whether the O2-sensing mechanism of pulmonary circulation is impaired in lungs exposed to hyperoxia, we prepared isolated perfused lungs from rats exposed to either a normoxic or hyperoxic environment and estimated hypoxia-elicited pressor changes in the pulmonary arteries as well as diameter changes in microvessels in the acini. To assess the important role of vasodilator prostaglandins as well as nitric oxide (NO) in blunting hypoxic pulmonary vasoconstriction (HPV) in hyperoxia-exposed lungs, we examined the effects of inhibition of constitutive and inducible forms of cyclooxygenase (COX-1 and COX-2) and of constitutive and inducible forms of NO synthase (eNOS and iNOS). Indomethacin was used as the inhibitor of both COX-1 and COX-2, while NS-398 was applied as a selective inhibitor of COX-2. Simultaneous restraint of eNOS and iNOS was attained by N ω-nitro-L-arginine methyl ester (L-NAME); restraint of iNOS was achieved by aminoguanidine. By addition of fluorescein isothiocyanate-dextran to the perfusion circuit, the architecture and diameter of the intraacinar microvessel were precisely determined in terms of a real-time confocal laser luminescence microscope. The results derived therefrom are (1) exposure to hyperoxia causes overall HPV and contractility of precapillary arterioles responding to hypoxia to be attenuated; (2) the attenuated overall HPV was significantly restored by adding L-NAME but not by adding aminoguanidine, indomethacin, or NS-398; and (3) hypoxia-induced constriction of precapillary arterioles was ameliorated by either L-NAME, aminoguanidine, or indomethacin but not by NS-398. The extent of recovery of arteriolar constriction in the presence of L-NAME was greater than that in the presence of aminoguanidine. We concluded that overall HPV would be blunted by the enhanced eNOS expression along the arteries rather than those in the acini, while contractility of intraacinar precapillary arterioles would be impaired by augmentation of eNOS and iNOS as well as COX-1 expression.

Modulation of Adhesion Molecule Expression in Pulmonary Vascular Endothelium by Oxygen

Pulmonary oxygen toxicity (POT) is an important clinical problem that occurs in patients on long-term mechanical ventilation requiring a high inspired-oxygen concentration. Although the histological evidence of POT is pulmonary edema with neutrophil infiltration into the lung parenchyma, the pathogenesis of POT is not fully understood. To elucidate the mechanism of the development of POT, we investigated the effect of hyperoxia (90% O2, 5% CO2) on adhesion molecule expression in cultured human endothelial cells. The level of intercellular adhesion molecule-1 (ICAM-1) expression had increased in hyperoxia-exposed endothelial cells at 48h and at 72h as compared with normoxic control (21% O2, 5% CO2). In contrast, the levels of P-selectin and E-selectin expression were unchanged during hyperoxic exposure. These hyperoxia-induced ICAM-1 expressions were dose dependently attenuated by a protein kinase C inhibitor (H-7). The levels of ICAM-1 mRNA and the numbers of adherent neutrophils were increased at 48h and at 72 h of hyperoxia-exposed endothelial cells. These results suggest that increased ICAM-1 expression in endothelial cells plays an important role in neutrophil accumulation during POT.

Sequential Multistep Mechanisms for Leukocyte Adhesion: Applicable to Lung Microcirculation?

This study was designed to examine how leukocyte traffic occurs in the pulmonary microcirculation under physiological shear rates. The leukocyte-endothelium interaction was visualized in perfused rat lungs using an intravital highspeed confocal laser video microscope by injecting fluorescence-tagged isolated leukocytes. In the control lung, transient cessation of leukocyte movement was observed in pulmonary capillaries. The percentage of leukocytes displaying such behavior increased in response to prestimulation by chemoattractants. As a consequence, leukocyte velocity decreased and the density of adherent leukocytes in pulmonary microvessels was markedly elevated. In contrast to observations in the mesenteric microcirculation, a major population of the adherent cells was observed in alveolar capillaries rather than in postcapillary venules. These results suggest that the pulmonary microvascular system is characterized by specific adhesive mechanisms for circulating leukocytes distinct from those previously reported in the mesenteric microcirculation.