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Are Pulmonary Capillaries Susceptible to Mechanical Stress?
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Are Pulmonary Capillaries Susceptible to Mechanical Stress?* Odile A Mathieu-Costello, Ph.D.; and john B. West, M.D .. Ph.D.
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he extreme thinness of the pulmonary blood-gas barrier has long been recognized. In fact , large portions of the barrier are so thin that they are at the limit of the power of resolution of the light microscope . It is only with the advent of electron microscopy that the cellular structure of the blood-gas barrier was elucidated. Forty years ago, Frank Low 1 published the first electron micrographs of the lung parenchyma in laboratory animals and humans. and demonstrated the presence of a continuous epithelial lining of the alveolar wall. Since then, the remarkable thinness of the pulmonary bloodgas barrier has mainly been viewed in terms of the characteristics that make it so effective for diffusion . Respiratory gases are transferred by passive diffusion and therefore at a given partial pressure difference across the barrier, increased passage of gases occurs with greater surface area and reduced tissue thickness, according to the Fick law of diffusion . In the human lung, the alveolar surface area is very large (up to 150m 2 ), approximately half of which is on the thin side of the blood-gas barrier with a thickness of only 0.2 to 0.4 J..Lm. 2 As noted elsewhere, 3 A however, there is evidence of diffusion-limitation of 0 . uptake in elite athletes during maximal exercise;U suggesting that the blood-gas barrier could not be any thicker without further limitations of 0 2 uptake during heavy exercise. At the same time, the pulmonary blood-gas barrier must be very strong to maintain its structural integrity in spite of its extreme thinness , and to withstand the high wall stresses during intense activity. The strength of the blood-gas barrier; and the pathophysiologic conditions where it fails because of stress failure of pulmonary capillaries, 3·7·s have not been previously recognized.
or circumferential tension in the capillary wall is caused by the capillary transmural pressure acting on the curved surface. It can be calculated via the Laplace relationship which states that the circumferential tension is the product of transmural pressure and radius of curvature. The second force is the surface tension of the alveolar lining layer, and third is the longitudinal tension in the alveolar wall associated with lung inflation. Surface tension (force 2) is thought to be protective, based on the fact that capillaries bulge into the alveoli at high capillary transmural pressure.3 · 10 On the contrary, the longitudinal tension of the tissue elements in the alveolar wall (force 3) predispose the capillary wall to stress failure. We summarize below experiments from our laboratory showing that the incidence of stress failure of pulmonary capillaries increases at high lung volume . 11 Whether or not the capillary wall fails under the hoop tension (force 1) does not depend on the wall tension per se, but rather on wall stress which is the ratio of wall tension to thickness. In the following section, we summarize our studies in rabbit lung, showing that stress failure of pulmonary capillaries consistently occurs at a capillary transmural pressure of 52.5 em H 20 , ie, about 40 mm Hg. 3· 12 At this transmural pressure of 52.5 em Hp, the wall stress at a nominal capillary radius of curvature of 5 J..Lm and a wall thickness of 0.3 J..Lm is 9 x 104 N/m 2 (or 9 x 103 dynes/cm 2 ), which is an extremely high stress. It is comparable to that in the wall of the human aorta, 3 which is subjected to a much higher transmural pressure (100
STRENGTH OF BLOOD-GAS BARRIER AND FORCES A CTING ON THE ALVEOLAR WALL
There is evidence that the mechanical strength of the pulmonary blood-gas barrier comes from the extracellular matrix.' On the thin side of the blood-gas barrier, the extracellular matrix is mainly reduced to the fused basement membranes of the epithelial and endothelial layers. The electron dense center portion of the basement membrane, or lamina densa, contains collagen IV which confers tensile strength to the matrix.'·9 The three main forces acting on the pulmonary blood gas barrier (Fig 1) have been identified.3 First, the hoop *From the Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, Calif. This study was supported by NIH program project HL 17331 and R01 HL4619ll.
Reprint requests: Dr. Mathieu-Costello, Department of Medicine. University of California , San Diego 92093-0623
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ENDOTHELIUM
EPITHELIUM
FIGURE 1. Pulmonary capillary in an alveolar wall, showing the three main forces acting on the blood gas barrier. (I) Circumferential or hoop tension is given by capillary transmural pressure x radius of curvature; (2) surface tension of the alveolar lining layer is thought to be protective; and (3) longitudinal tension in the alveolar wall associated with lung inflation increases hoop tension and therefore the likelihood of stress failure. (With permission.3 ) 36th Annual Aspen Lung Conference
mm Hg) and has a much greater radius of curvature (o.:l.3 em) but also has a much thicker wall (""2 mm) and
large amounts of collagen and elastin. In other words, the high stress imposed on the pulmonary capillary, in spite of its small radius of curvature, is due to the extreme wall thinness, one of the design requirements for an adequate transfer of gas by passive diffusion. STRESS
F AlLURE
OF PULMONARY CAPILLARIES IN RABBIT
We examined the ultrastructure of the blood-gas barrier in rabbit lungs exposed to increased capillary transmural pressure.3 · 12· 13 Briefly, the following experimental preparation was used to expose the lung to a known capillary transmural pressure and then fix it for electron microscopy at the same pressure. Anesthetized animals were exsanguinated, the chest was opened, the pulmonary artery and left atrium cannulated to control inflow and outflow perfusion pressures, and a cannula was placed in the trachea to control alveolar pressure. The lung was first perfused with the animal's own blood for 1 min, followed by saline solution/dextran (3 min) and then buffered glutaraldehyde fixative ( 10 min) at the same pressure. Pulmonary artery pressures of 20, 40, 60, and 80 em H 20 were used, with pulmonary venous pressures 5 em H 20 below arterial pressure and an alveolar pressure of 5 em H 20. Therefore, capillary transmural pressures , ie, the pressure differences between the inside and outside of the capillary, were 12.5, 32.5, 52.5, and 72.5 ± 2.5 em Hp. Three animals were analyzed at each pressure, except for 32.5 em H 20 transmural pressure (Ptm) where six animals were studied . In the lungs exposed to a Ptm of 52.5 em H 20 and
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above, disruptions of the blood-gas barrier were consistently found. They included disruption of the endothelial layer or the alveolar layer, or sometimes all layers of the wall. 3· 12· 13 A frequent finding was that the capillary endothelial or alveolar epithelial layers were disrupted, but the basement membrane was continuous (Fig 2). Also, platelets or red blood cells were often seen at the site of disruption. in close vicinity of the exposed basement membrane (Fig 28). Quantification of the extent of damage of the blood-gas barrier 12 showed that the number of breaks per millimeter of epithelial and endothelial boundary length increased markedly with increased Ptm. No break was found at a Ptm of 12.5 em H 20 and a few breaks were seen at 32.5 em H.O Ptm, mostly in one animal. From a Ptm of 52.5 to 72~5 em Hp, the number of breaks per millimeter rose from 8.5 ± 3.2(SE) to 27.8 ± 8.6 for endothelium and from 10.4 ± 4.3 to 13.6 ± 1.4 for epithelium. On average, the length of the breaks as seen by transmission electron microscopy of sections, was about 1 to 2 ~m for endothelium and 2 to 3 Jlm for epithelium. break length being significantly greater in epithelium compared to endothelium only at the highest pressure (72.5 em H 20 Ptm). There was no significant difference in break length with increasing pressure either for endothelium or epithelium, suggesting that once disruptions had occurred, the stresses were greatly relieved . The basement membrane was continuous in approximately half of the endothelial and epithelial breaks, at least in the plane of the section, underlining the greater strength of the extracellular matrix compared to the cellular linings. The thickness of the blood-gas barrier increased markedly above 32.5 em H.O Ptm. This was entirely due to the widening of the inte-rstitium as a result of edema, while the thickness of both the en dot he-
B
FIGURE 2. Transmission electron micrographs showing disruption of the blood-gas barrier in rabbit lung exposed to a capillary transmural pressure of 52.5 em Hp. A (left) , capillary endothelium is disrupted (arrow), but alveolar epithelium and two basement membranes are continuous in the plane of the section . 8 (right), alveolar epithelium (open arrows) and capillary endothelial layer (solid arrows) are disrupted; note a platelet protruding into the endothelial opening and dosely applied to the exposed basement membrane. Transpulmonary pressure was 5 em H 20 . (With permission .3 )
CHEST I 105 I 31 MARCH, 1994 I Supplement 103$
lium and epithelium was not changed.12 Further insight into the pattern of disruption of the blood-gas barrier was obtained by examination of adjacent samples by scanning electron microscopy (SEM)Y Evidence of disruption of the alveolar surface was consistently found at Ptm of 52.5 em Hp and above. The majority of the breaks were elongated slits, oriented perpendicular to the capillary axis (Fig 3A ). Most breaks were complete disruptions of all layers of the blood-gas barrier. Figure 38 shows a red blood cell protruding into the alveolus . While several breaks were in close vicinity to intercellular junctions (Fig 3A and B), almost no break was seen at the junction itself, suggesting greater mechanical strength at the junction. The dimensions of the elongated slits of the blood-gas barrier were about 7j.lm in length for a width of about 1.5 J.lm. They varied little with pressure, confirming our previous observation by transmission electron microscopy (TEM) and suggesting the relief of stresses once disruption has occurred. Further striking similarities with our previous TEM studies 3· 12 were the extent of disruption of the blood-gas barrier as a function of Ptm, the SEM appearance of the breaks, and the close morphometric estimates of their fractional density compared with TEM . 13 This indicated that similar ruptures were examined by the two techniques, even after greatly different tissue processing and preparation procedures. The effect of fixation on the incidence of breaks in the blood-gas barrier was investigated by raising Ptm to 52.5 em Hp for blood perfusion ( 1 min) and then reducing the capillary pressure for either perfusion with saline solution and glutaraldehyde fixative at lower pressure, 14 or instillation of fixative via the airways. 15 Perfusion with saline solution and then fixative at reduced Ptm (12.5 em H.O) resulted in a lesser number of both endothelial and epithelial breaks compared with the perfusions with saline solution and fixative at high pressure. Interestingly, the breaks no longer seen 14 were those that were initially small and were associated with a continuous basement membrane in the previous study. 12 We attributed this to the rapid reversibility of some breaks after the pressure was lowered, 14 consistent with functional studies which documented the rapid reversibility of the increased capillary permeability observed in the lung after short periods of high vascular pressure . 16 · 1 ~ After fixation by airway instillation immediately after 1 min blood perfusion at high pressure, 15 the number of endothelial breaks was similar to that seen in the previous study with perfusionfixation at high pressure. 12 This indicated that stress failure of pulmonary capillaries occurred within the first minute of perfusion with blood. Also, it ruled out a significant effect of the glutaraldehyde perfusion at high pressure in causing capillary stress failure in the previous study, 12 since as great a number of endothelial breaks was found after fixation by airways instillation, ie, without vascular perfusion fixation at high pressure. Another interesting finding was that the incidence of disruptions of the epithelium was lower in the lungs with instillation immediately after l min blood perfusion than in those perfused with saline solution (3 min) and then glutaraldehyde fixative at high pressure. This suggested that addi104S
FIGURE 3. Scanning electron micrographs showing disruptions of the blood-gas barrier in rabbit lungs exposed to a Ptm of 72.5 (A) and 52.5 (B) cmH 20 . A (top ), elongated slit of the blood-gas barrier (solid arrow) very close (about 0.41!m) to an intercellular junction (white arrow) . B (bottom), disruption of the blood gas barrier crossing an intercellular junction (white arrow) and showing a red blood cell (asterisk) protruding into the alveolus. Note abundance of proteinaceous material (A and B) and red blood cell ( A) on the alveolar surface. Transpulmo nary pressure was 5 em H 20. (With permission . 11 )
tiona! time may be necessary for epithelial rupture to occur after stress failure of pulmonary capillaries, depending on the accumulation of fluid in the interstitium . 1; EHECT OF HIGH LUNG VoLUME
At the same capillary Ptm of 32.5 or 52.5 em Hp, the incidence of capillary stress failure was greatly increased when rabbit lungs were perfused at high lung volume, ie, while inflated to a transpulmonary pressure of 20 em Hp, compared to the low lung volume (5 em H.O transpulmonary pressure) used in our previous studies 11 (Fig 4) . Interestingly, the increase in the frequency of both endothelial and epithelial breaks was about the same for approximately the same rise in transpulmonary pressure ( 15 em H 20, from 5 to 20 em H 20; Fig 4, left panel) or Ptm (20 em H 20 , from 32.5 to 52.5 em H 2 0 ; open columns, Fig 4) . It suggests an equivalent effect of the 36th Annual Aspen Lung Conference
increase in transpulmonary pressure or Ptm in these conditions. Measurement of blood-gas barrier thickness revealed a thickening of the interstitium at high lung volume, consistent with the presence of edema. Disruptions of the endothelium with red blood cells protruding into the opening were found both on the thin and thick sides of the blood-gas barrier. As in our previous study at low lung volume,l 2 the basement membrane was continuous in about half of the endothelial and epithelial breaks. Examination by scanning electron microscopy revealed that at high lung volume, the breaks tended to be of similar length but narrower than at low lung volume, for a similar Ptm. Fewer breaks were oriented perpendicular to the capillary axis while breaks with multiple segments of different orientation, not seen at low lung volume, were found. They were possibly due to the increased wall stress in all directions with the uniform stretching of the alveolar wall at high lung volume. Comparison of measurements by SEM and TEM indicated that similar ruptures were analyzed by the two techniques . 11 The increased vulnerability of the pulmonary capillaries to stress failure at high degree of inflation provides a physiologic mechanism for studies showing increased pulmonary microvascular permeability \vith overinflation. 1'· 21 It has important implications in the critical care setting where great care should be taken to prevent damage to the lung due to overdistension during mechanical ventilation. As pointed out elsewhere, 7 this is often a catch-22 situation because high inflation pressures and high levels of positive end-expiratory pressure are necessary to maintain arterial Po 2 at an acceptable level. However, the increased stress on the blood-gas barrier because of the high inflation makes it vulnerable to rupture at a lower capillary Ptm, a process further accelerated if pathologic changes have occurred, ie, the blood-gas barrier or collagen IV are weakened.
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PATHOPHYSIOLOGIC Co!\SEQlJD iCES OF STRESS F .\ I I.UIIE OF PULMOSARY CAPJI.I.ARJES
The pathophysiologic conditions in which stress failure of pulmonary capillaries likely plays a role have been reviewed elsewhere.~-' They indude conditions causing high-permeability edema (for example, neurogenic pulmonary edema, high altitude pulmonary edema, and some cases of adult respiratory distress syndronw for example following trauma causing catecholamine release and transient rise in pulmonary vascular pressure) , frank hemorrhage (as seen in exercise-induced pulmonary hemorrhage) or both (eg, in mitral stenosis) . A particularly interesting condition is high altitude pulmonary edema where increased capillary pressure, probably due to uneven hypoxic pulmonary vasoconstriction, 22 causes failure of pulmonary capillaries and a high permeability type of edema. High molecular weight proteins, blood cells, and the inflammatory markers leukotriene B4 and complement fragment C5a are found in lung lavage fluid of patients with high altitude pulmonary edema (HAPE)Y Both the high permeability type of edema and the presence of inflammatory markers are consistent with pulmonary capillary stress failure as the mechanism of HAPE, the disruption of the endothelial and epithelial lining causing the high permeability type of edema, and the exposure of basement membranes triggering platelet and white blood cell activation and inflammatory processes.' The presence of inflammatory marker (leukotriene B) in the lavage fluid of rabbit lungs perfused at high pressure was recently demonstrated.24 The fact that ultrastructural appearances of the blood-gas barrier in rats \vith HAPE are remarkably consistent with those seen in rabbit lungs after perfusion at high pressure suggests that stress failure of pulmonary capillaries is the mechanism of HAPE.' Other pathologic conditions where pulmonary capillary stress failure likely plays a role indude situa-
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Fl<: l i RF: 4 . Histogram of average number of breaks per millimeter boundary length of endothelium and epithelium at 20 (high lung volume) and 5 em 11,0 transpulmonary pres.s ure (low lung volume). Asterisk, significantly greater (p<0.05) at high compared to low lung volume at the same capillary Ptm . (With permission.")
CHEST I 1051 3 I MARCH, 1994 I Supplement 105S
tions where the extracellular matrix weakened, for example in Goodpasture's syndrome where autoantibodies attack type IV collagen ,25 causing bleeding in the lung and also glomeruli. We recently obtained evidence that exercise-induced pulmonary hemorrhage (EIPH) in thoroughbred racehorses is caused by stress failure of pulmonary capillaries.26 This study was done in collaboration with Drs. Eric Birks, James Jones, John Pascoe, and Walter Tyler of the School of Veterinary Medicine, University of California, Davis. The lungs of three thoroughbred horses with known EIPH were removed and fixed for electron microscopy after the horse had galloped at high speed ( 13 to 16 m/s) on a treadmill. Evidence of intrapulmonary bleeding was confirmed by bronchoscopy in one animal. Tissue fixation was either by airways instillation (one animal) or vascular perfusion of glutaraldehyde solution at S 30 ± 5 em Hp Ptm (two animals). Ultrastructural evidence of stress failure of pulmonary capillaries was the presence of a large number of red blood cells in the interstitium, disruption of the capillary endothelial or alveolar epithelial layers, proteineous material and red blood cells in the alveolar spaces, interstitial edema, and fluid-filled protrusions of the endothelium into the capillary lumen .26 These are the same appearances we have found in rabbit lungs exposed to high capillary transmural pressure. 3•12•15 Pulmonary vascular pressures are extremely high in thoroughbred horses during galloping. Jones and coworkers27 measured pulmonary arterial and left atrium pressures of up to 120 mm Hg and 70 mm Hg, respectively, in thoroughbreds galloping at speeds up to 10 m/s, 27 indicating that capillary pressures must also be very high. These very high vascular pressures are associated with the extremely high cardiac outputs ( > 750 ml!minlkg) achieved in these animals, which require very high left atrial pressure for adequate ventricular filling.26 At the same time, the extremely high V0 2 max achieved during maximal exercise ( 180 ml!minlkg) requires that the bloodgas barrier be extremely thin. However, exercise-induced hypoxemia in thoroughbreds during heavy exercise was shown to be the result of diffusion limitation, 26 indicating that the barrier could not be any thicker without further limitation to V0 2 max. In other words, selection for increased aerobic performance imposes contradictory requirements on the heart-lung system. In thoroughbreds, those requirements have reached the point of failure of the blood-gas barrier. The selective breeding of thoroughbreds for a large number of generations has provided animals with enormous 0 2 uptake rates, which necessitate not only a very thin pulmonary blood-gas barrier but also extremely high cardiac outputs, which in turn require pulmonary pressures to be so high that they exceed the mechanical strength of the thin blood-gas barrier, causing failure of pulmonary capillaries and alveolar hemorrhage.26 Interestingly, while a large number of red blood cells were found in the alveoli and interstitium in areas showing clear abnormalities macroscopically, actual endothelial and epithelial breaks were difficult to find . This was probably due to the reversibility of ruptures once the 106S
vascular pressure was reduced, from the end of the gallop to tissue fiXation 60 to 70 min later. In addition, platelets and cytoplasmic extension of leukocytes were seen in close vicinity to the exposed basement membrane at the site of disruption of the capillary wall, suggesting platelet and white blood cell activation and the initiation of an inflammatory process.26 As mentioned earlier, inflammatory markers were found in lavage fluid of rabbit lungs perfused at high pressure , 2~ as well as in bronchoalveolar fluid of patients with HAPE,23 suggesting that stress failure of pulmonary capillaries is the mechanism of HAPE . In summary, pulmonary capillaries are subjected to high mechanical stress in spite of their small radius of curvature, because of the extreme thinness of the alveolar wall, which in turn is required for the adequate transfer of gases by passive diffusion. The contradictory design requirements imposed on the blood-gas barrier, which must be thin enough not to limit diffusion but at the same time very strong to withstand high stresses across such a thin barrier, have only recently been recognized. Stress failure of pulmonary capillaries plays a role in several pathophysiologic conditions causing high-permeability edema and pulmonary hemorrhage. Adramatic example is exercise-induced pulmonary hemorrhage in thoroughbred horses where selective breeding for extreme aerobic performance over a large number of generations has allowed the cardiovascular system to develop beyond the mechanical strength of the pulmonary blood-gas barrier. ACKNOWLEDGMENTS: This work was done in collaboration with Michael Costello, Ann Elliott, Zhenxing Fu, Sanli Kurdak, Yasuo Namba, Koichi Tsukimoto, and Renato Prediletto. REFERENCES
2 3 4 5 6 7 8 9
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Low FN. The pulmonary alveolar epithelium of laboratory mammals and man. Anat Rec 1953; ll7:24l-63 Gehr P, Bachofen M, Weibel ER. The normal human lung: ultrastructure and morphometric estimation of diffusion capacity. Respir Physiol 1978; 32:121-40 West JB, Tsukimoto K, Mathieu-Costello 0 , et al. Stress failure in pulmonary capillaries. JAppl Physiol1991 ; 70:173142 West JB. Mathieu-Costello 0 . Strength of the pulmonary blood-gas barrier. Respir Physiol 1992; 88:141-48 Dempsey JA. Hanson PG, Henderson KS. Exercise-induced alveolar hypoxemia in healthy human subjects at sea-level. J Physiol (Lond) 1984; 355:161-75 Wagner PD, Gale GE, Moon RE, et al. Pulmonary gas exchange in humans exercising at sea level and simulated altitude. J Appl Physiol 1986; 61:260-70 West JB, Mathieu-Costello 0 . Stress failure of pulmonary capillaries in the intensive care setting. Schweiz Med Wschr 1992; 122:751-57 West JB . Mathieu-Costello 0 . Stress failure of pulmonary capillaries: role in lung and heart disease. Lancet 1992; 340:762-67 Crouch EC, Martin GR, Brody JS. Basement membranes. In: Crystal RG, West JB, Barnes PJ, et al, eds. The lung: scientific foundations. New York: Raven Press Ltd, 1991; 421-37 Glazier JB, Hughes JMB, Maloney JE, et al. Measurements of capillary dimensions and blood volume in rapidly frozen lungs. J Appl Physiol 1969; 26:65-76 36th Annual Aspen Lung Conference
11 Fu Z, Costello ML, Tsukimoto K, et al. High lung volume increases stress failure in pulmonary capillaries. J Appl Physiol 1992; 73:123-33 12 Tsukimoto K, Mathieu-Costello 0, Prediletto R, et al. Ultrastructural appearances of pulmonary capillaries at high transmural pressures. J Appl Physiol 1991; 71 :573-82 13 Costello ML, Mathieu-Costello 0 , West JB. Stress failure of alveolar epithelial cells studied by scanning electron microscopy. Am Rev Respir Dis 1992; 145:1446-55 14 Elliott AR, Fu Z, Tsukimoto K, et al. Short-term reversibility of ultrastructural changes in pulmonary capillaries caused by stress failure. J Appl Physiol 1992; 73:1150-58 15 Fu Z, Kurdak S, Namba Y, et al. Effect of fixation by airway instillation on the incidence of stress failure in pulmonary capillaries [abstract] . Am Rev Respir Dis 1993; 147:A40 16 Rippe B, Townsley M, Thigpen J, et al. Effects of vascular pressure on the pulmonary microvasculature in isolated dog lungs. J Appl Physiol 1984; 57:233-39 17 Nicolaysen G, Waaler BA, Aarseth P. On the existence of stretchable pores in the exchange vessels of the isolated rabbit lung preparation. Lymphology 1979; 12:201-07 18 Egan EA. Lung inflation, lung solute permeability, and alveolar edema. J Appl Physiol 1982; 53:121-25 19 Parker JC, Townsley MI, Rippe B. et al. Increased microvascular permeability in dog lungs due to high peak airway pressures. J Appl Physiol 1984; 57:1809-16 20 Dreyfuss D, Basset G, Soler P, et al. Intermittent positivepressure hyperventilation with high inflation pressures produces pulmonary microvascular injury in rats. Am Rev Respir Dis 1985; 132:880-84 21 Dreyfuss D, Saumon G. Lung overinflation: physiologic and anatomic alterations leading to pulmonary edema. In : Zapol WM, Lemaire F, eds. Adult respiratory distress syndrome. New York: Marcel Dekker Inc, 1991 ; 433-49 22 Hultgren H. High altitude pulmonary edema. In: Hegnauer A, ed. Biomedicine of high terrestrial elevations. Nattick, Mass: US Army Research Institute of Environmental Medicine, 1969; 131-41 23 Schoene RB, Hackett PH, Henderson WR, et al. Highaltitude pulmonary edema: characteristics of lung lavage fluid. JAMA 1986; 256:63-9 24 Tsukimoto K, Yoshimura N, lchioka M, et al. Protein, cell, and leukotriene B. concentrations of the lung edema fluid produced by high capillary pressures in rabbit. J Appl Physiol 1994 (in press) 25 Wieslander J, HeinegArd D . The involvement of type IV collagen in Goodpasture's syndrome. Ann NY Acad Sci 1985; 460:363-74 26 West JB. Mathieu-Costello 0, Jones JH. et al. Stress failure of pulmonary capillaries in racehorses with exercise-induced pulmonary hemorrhage. J Appl Physiol 1993; 75:1097-1109 27 Jones JH, Smith BL, Birks EK, et al. Left atrial and pulmonary arterial pressures in exercising horses [abstract] . FASEB J 1992; 6:A2020 28 Wagner PD. Gillespie JR. Landgren GL, et al. Mechanism of exercise-induced hypoxemia in horses. J Appl Physiol 1989; 66:1227-33
Centripetal Tension and Endothelial Retraction* Ala11 B. Mvy. M.D.; Rebecca Sheldo11, B.A.; Kathy Li11dsley. B.A.; Sa11dra Shasby. B.A.; a11d D. Michael Shasby, M.D.
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nflammatory molecules such as histamine increase endothelial permeability by initiating retraction of adjacent endothelial cells. We recently reported that in human umbilical vein endothelial (HUVE) cells histamine increased phosphorylation of the light chain of myosin (MLC) by 0.18 ± 0.02 moles phosphate per mole MLC (moiP/moiMLC). This is consistent with the hypothesis that histamine initiates retraction by increasing centripetal tension within endothelial cells. However, chelation of extracellular calcium which interrupts cellcell and cell-substrate binding also increases endothelial permeability and causes endothelial cell retraction both i11 situ in perfused lungs and in vitro with cultured cells. The effects of calcium chelation are consistent with the hypothesis that release of forces tethering endothelial cells in place allows expression of a constitutive centripetal tension . In our recent report, we found constitutive phosphorylation of MLC at 0.20 ± 0.02 molP/molMLC, consistent with a constitutive actomyosin-mediated tension. We asked if reduction of MLC phosphorylation would prevent the response to histamine or calcium chelation, and conversely, if increased constitutive MLC phosphorylation would cause cell retraction . In control and histamine-stimulated HUVE cells and in control porcine pulmonary artery endothelial (PPAE) cells tryptic peptide maps indicated that MLC was phosphorylated by the calcium calmodulin-dependent myosin light chain kinase . Pretreatment of HUVE cells with cAMP prevented the histamine-stimulated increase in MLC phosphorylation (-0.08 ± 0.02 molP/moiMLC) and a histamine initiated increase in HUVE cell monolayer permeability (-12 ±I percent). Pretreatment of HUVE cells with the calcium-calmodulin inhibitor, ML-9, also prevented the histamine stimulated increase in MLC phosphorylation (-o.29 ± 0.04 molP/molMLC) and the increase in HUVE cell monolayer permeability (14 ± 4 percent of the control response) . Similarly, pretreatment of PPAE cells with cAMP reduced basal MLC phosphorylation (-0.16 ± 0.03molP/molMLC) and reduced the increase in monolayer permeability following calcium chelation (21 ± 14 percent of the control response). Pretreatment of PPAE cells with ML-9 also reduced basal MLC phosphorylation (-0.13 ± 0.02 moiP/molMLC) and reduced the increase in monolayer permeability following calcium chelation (47 ± 5 percent of control response). The basal level of MLC phosphorylation likely represents constitutive kinase and phosphatase activity. Okadaic acid increased MLC phosphorylation in PPAE cells (I J..LM, 0.06 ± 0.04moiP/molMLC and 10 J..LM. 0.17 ± 0.05 molP/molMLC ) in a dose-dependent manner, and 10 J..LM (289 ± 32 percent) but not 1 J..LM (4 ± 5 percent) okadaic acid increased PPAE cell monolayer permeabil*From the University of Iowa College of Medicine. Iowa City. CHEST I 1051 3 I MARCH, 1994 I Supplement 1075
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Are Pulmonary Capillaries Susceptible to Mechanical Stress?* Odile A Mathieu-Costello, Ph.D.; and john B. West, M.D .. Ph.D.
T
he extreme thinness of the pulmonary blood-gas barrier has long been recognized. In fact , large portions of the barrier are so thin that they are at the limit of the power of resolution of the light microscope . It is only with the advent of electron microscopy that the cellular structure of the blood-gas barrier was elucidated. Forty years ago, Frank Low 1 published the first electron micrographs of the lung parenchyma in laboratory animals and humans. and demonstrated the presence of a continuous epithelial lining of the alveolar wall. Since then, the remarkable thinness of the pulmonary bloodgas barrier has mainly been viewed in terms of the characteristics that make it so effective for diffusion . Respiratory gases are transferred by passive diffusion and therefore at a given partial pressure difference across the barrier, increased passage of gases occurs with greater surface area and reduced tissue thickness, according to the Fick law of diffusion . In the human lung, the alveolar surface area is very large (up to 150m 2 ), approximately half of which is on the thin side of the blood-gas barrier with a thickness of only 0.2 to 0.4 J..Lm. 2 As noted elsewhere, 3 A however, there is evidence of diffusion-limitation of 0 . uptake in elite athletes during maximal exercise;U suggesting that the blood-gas barrier could not be any thicker without further limitations of 0 2 uptake during heavy exercise. At the same time, the pulmonary blood-gas barrier must be very strong to maintain its structural integrity in spite of its extreme thinness , and to withstand the high wall stresses during intense activity. The strength of the blood-gas barrier; and the pathophysiologic conditions where it fails because of stress failure of pulmonary capillaries, 3·7·s have not been previously recognized.
or circumferential tension in the capillary wall is caused by the capillary transmural pressure acting on the curved surface. It can be calculated via the Laplace relationship which states that the circumferential tension is the product of transmural pressure and radius of curvature. The second force is the surface tension of the alveolar lining layer, and third is the longitudinal tension in the alveolar wall associated with lung inflation. Surface tension (force 2) is thought to be protective, based on the fact that capillaries bulge into the alveoli at high capillary transmural pressure.3 · 10 On the contrary, the longitudinal tension of the tissue elements in the alveolar wall (force 3) predispose the capillary wall to stress failure. We summarize below experiments from our laboratory showing that the incidence of stress failure of pulmonary capillaries increases at high lung volume . 11 Whether or not the capillary wall fails under the hoop tension (force 1) does not depend on the wall tension per se, but rather on wall stress which is the ratio of wall tension to thickness. In the following section, we summarize our studies in rabbit lung, showing that stress failure of pulmonary capillaries consistently occurs at a capillary transmural pressure of 52.5 em H 20 , ie, about 40 mm Hg. 3· 12 At this transmural pressure of 52.5 em Hp, the wall stress at a nominal capillary radius of curvature of 5 J..Lm and a wall thickness of 0.3 J..Lm is 9 x 104 N/m 2 (or 9 x 103 dynes/cm 2 ), which is an extremely high stress. It is comparable to that in the wall of the human aorta, 3 which is subjected to a much higher transmural pressure (100
STRENGTH OF BLOOD-GAS BARRIER AND FORCES A CTING ON THE ALVEOLAR WALL
There is evidence that the mechanical strength of the pulmonary blood-gas barrier comes from the extracellular matrix.' On the thin side of the blood-gas barrier, the extracellular matrix is mainly reduced to the fused basement membranes of the epithelial and endothelial layers. The electron dense center portion of the basement membrane, or lamina densa, contains collagen IV which confers tensile strength to the matrix.'·9 The three main forces acting on the pulmonary blood gas barrier (Fig 1) have been identified.3 First, the hoop *From the Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, Calif. This study was supported by NIH program project HL 17331 and R01 HL4619ll.
Reprint requests: Dr. Mathieu-Costello, Department of Medicine. University of California , San Diego 92093-0623
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ENDOTHELIUM
EPITHELIUM
FIGURE 1. Pulmonary capillary in an alveolar wall, showing the three main forces acting on the blood gas barrier. (I) Circumferential or hoop tension is given by capillary transmural pressure x radius of curvature; (2) surface tension of the alveolar lining layer is thought to be protective; and (3) longitudinal tension in the alveolar wall associated with lung inflation increases hoop tension and therefore the likelihood of stress failure. (With permission.3 ) 36th Annual Aspen Lung Conference
mm Hg) and has a much greater radius of curvature (o.:l.3 em) but also has a much thicker wall (""2 mm) and
large amounts of collagen and elastin. In other words, the high stress imposed on the pulmonary capillary, in spite of its small radius of curvature, is due to the extreme wall thinness, one of the design requirements for an adequate transfer of gas by passive diffusion. STRESS
F AlLURE
OF PULMONARY CAPILLARIES IN RABBIT
We examined the ultrastructure of the blood-gas barrier in rabbit lungs exposed to increased capillary transmural pressure.3 · 12· 13 Briefly, the following experimental preparation was used to expose the lung to a known capillary transmural pressure and then fix it for electron microscopy at the same pressure. Anesthetized animals were exsanguinated, the chest was opened, the pulmonary artery and left atrium cannulated to control inflow and outflow perfusion pressures, and a cannula was placed in the trachea to control alveolar pressure. The lung was first perfused with the animal's own blood for 1 min, followed by saline solution/dextran (3 min) and then buffered glutaraldehyde fixative ( 10 min) at the same pressure. Pulmonary artery pressures of 20, 40, 60, and 80 em H 20 were used, with pulmonary venous pressures 5 em H 20 below arterial pressure and an alveolar pressure of 5 em H 20. Therefore, capillary transmural pressures , ie, the pressure differences between the inside and outside of the capillary, were 12.5, 32.5, 52.5, and 72.5 ± 2.5 em Hp. Three animals were analyzed at each pressure, except for 32.5 em H 20 transmural pressure (Ptm) where six animals were studied . In the lungs exposed to a Ptm of 52.5 em H 20 and
-o.s"
above, disruptions of the blood-gas barrier were consistently found. They included disruption of the endothelial layer or the alveolar layer, or sometimes all layers of the wall. 3· 12· 13 A frequent finding was that the capillary endothelial or alveolar epithelial layers were disrupted, but the basement membrane was continuous (Fig 2). Also, platelets or red blood cells were often seen at the site of disruption. in close vicinity of the exposed basement membrane (Fig 28). Quantification of the extent of damage of the blood-gas barrier 12 showed that the number of breaks per millimeter of epithelial and endothelial boundary length increased markedly with increased Ptm. No break was found at a Ptm of 12.5 em H 20 and a few breaks were seen at 32.5 em H.O Ptm, mostly in one animal. From a Ptm of 52.5 to 72~5 em Hp, the number of breaks per millimeter rose from 8.5 ± 3.2(SE) to 27.8 ± 8.6 for endothelium and from 10.4 ± 4.3 to 13.6 ± 1.4 for epithelium. On average, the length of the breaks as seen by transmission electron microscopy of sections, was about 1 to 2 ~m for endothelium and 2 to 3 Jlm for epithelium. break length being significantly greater in epithelium compared to endothelium only at the highest pressure (72.5 em H 20 Ptm). There was no significant difference in break length with increasing pressure either for endothelium or epithelium, suggesting that once disruptions had occurred, the stresses were greatly relieved . The basement membrane was continuous in approximately half of the endothelial and epithelial breaks, at least in the plane of the section, underlining the greater strength of the extracellular matrix compared to the cellular linings. The thickness of the blood-gas barrier increased markedly above 32.5 em H.O Ptm. This was entirely due to the widening of the inte-rstitium as a result of edema, while the thickness of both the en dot he-
B
FIGURE 2. Transmission electron micrographs showing disruption of the blood-gas barrier in rabbit lung exposed to a capillary transmural pressure of 52.5 em Hp. A (left) , capillary endothelium is disrupted (arrow), but alveolar epithelium and two basement membranes are continuous in the plane of the section . 8 (right), alveolar epithelium (open arrows) and capillary endothelial layer (solid arrows) are disrupted; note a platelet protruding into the endothelial opening and dosely applied to the exposed basement membrane. Transpulmonary pressure was 5 em H 20 . (With permission .3 )
CHEST I 105 I 31 MARCH, 1994 I Supplement 103$
lium and epithelium was not changed.12 Further insight into the pattern of disruption of the blood-gas barrier was obtained by examination of adjacent samples by scanning electron microscopy (SEM)Y Evidence of disruption of the alveolar surface was consistently found at Ptm of 52.5 em Hp and above. The majority of the breaks were elongated slits, oriented perpendicular to the capillary axis (Fig 3A ). Most breaks were complete disruptions of all layers of the blood-gas barrier. Figure 38 shows a red blood cell protruding into the alveolus . While several breaks were in close vicinity to intercellular junctions (Fig 3A and B), almost no break was seen at the junction itself, suggesting greater mechanical strength at the junction. The dimensions of the elongated slits of the blood-gas barrier were about 7j.lm in length for a width of about 1.5 J.lm. They varied little with pressure, confirming our previous observation by transmission electron microscopy (TEM) and suggesting the relief of stresses once disruption has occurred. Further striking similarities with our previous TEM studies 3· 12 were the extent of disruption of the blood-gas barrier as a function of Ptm, the SEM appearance of the breaks, and the close morphometric estimates of their fractional density compared with TEM . 13 This indicated that similar ruptures were examined by the two techniques, even after greatly different tissue processing and preparation procedures. The effect of fixation on the incidence of breaks in the blood-gas barrier was investigated by raising Ptm to 52.5 em Hp for blood perfusion ( 1 min) and then reducing the capillary pressure for either perfusion with saline solution and glutaraldehyde fixative at lower pressure, 14 or instillation of fixative via the airways. 15 Perfusion with saline solution and then fixative at reduced Ptm (12.5 em H.O) resulted in a lesser number of both endothelial and epithelial breaks compared with the perfusions with saline solution and fixative at high pressure. Interestingly, the breaks no longer seen 14 were those that were initially small and were associated with a continuous basement membrane in the previous study. 12 We attributed this to the rapid reversibility of some breaks after the pressure was lowered, 14 consistent with functional studies which documented the rapid reversibility of the increased capillary permeability observed in the lung after short periods of high vascular pressure . 16 · 1 ~ After fixation by airway instillation immediately after 1 min blood perfusion at high pressure, 15 the number of endothelial breaks was similar to that seen in the previous study with perfusionfixation at high pressure. 12 This indicated that stress failure of pulmonary capillaries occurred within the first minute of perfusion with blood. Also, it ruled out a significant effect of the glutaraldehyde perfusion at high pressure in causing capillary stress failure in the previous study, 12 since as great a number of endothelial breaks was found after fixation by airways instillation, ie, without vascular perfusion fixation at high pressure. Another interesting finding was that the incidence of disruptions of the epithelium was lower in the lungs with instillation immediately after l min blood perfusion than in those perfused with saline solution (3 min) and then glutaraldehyde fixative at high pressure. This suggested that addi104S
FIGURE 3. Scanning electron micrographs showing disruptions of the blood-gas barrier in rabbit lungs exposed to a Ptm of 72.5 (A) and 52.5 (B) cmH 20 . A (top ), elongated slit of the blood-gas barrier (solid arrow) very close (about 0.41!m) to an intercellular junction (white arrow) . B (bottom), disruption of the blood gas barrier crossing an intercellular junction (white arrow) and showing a red blood cell (asterisk) protruding into the alveolus. Note abundance of proteinaceous material (A and B) and red blood cell ( A) on the alveolar surface. Transpulmo nary pressure was 5 em H 20. (With permission . 11 )
tiona! time may be necessary for epithelial rupture to occur after stress failure of pulmonary capillaries, depending on the accumulation of fluid in the interstitium . 1; EHECT OF HIGH LUNG VoLUME
At the same capillary Ptm of 32.5 or 52.5 em Hp, the incidence of capillary stress failure was greatly increased when rabbit lungs were perfused at high lung volume, ie, while inflated to a transpulmonary pressure of 20 em Hp, compared to the low lung volume (5 em H.O transpulmonary pressure) used in our previous studies 11 (Fig 4) . Interestingly, the increase in the frequency of both endothelial and epithelial breaks was about the same for approximately the same rise in transpulmonary pressure ( 15 em H 20, from 5 to 20 em H 20; Fig 4, left panel) or Ptm (20 em H 20 , from 32.5 to 52.5 em H 2 0 ; open columns, Fig 4) . It suggests an equivalent effect of the 36th Annual Aspen Lung Conference
increase in transpulmonary pressure or Ptm in these conditions. Measurement of blood-gas barrier thickness revealed a thickening of the interstitium at high lung volume, consistent with the presence of edema. Disruptions of the endothelium with red blood cells protruding into the opening were found both on the thin and thick sides of the blood-gas barrier. As in our previous study at low lung volume,l 2 the basement membrane was continuous in about half of the endothelial and epithelial breaks. Examination by scanning electron microscopy revealed that at high lung volume, the breaks tended to be of similar length but narrower than at low lung volume, for a similar Ptm. Fewer breaks were oriented perpendicular to the capillary axis while breaks with multiple segments of different orientation, not seen at low lung volume, were found. They were possibly due to the increased wall stress in all directions with the uniform stretching of the alveolar wall at high lung volume. Comparison of measurements by SEM and TEM indicated that similar ruptures were analyzed by the two techniques . 11 The increased vulnerability of the pulmonary capillaries to stress failure at high degree of inflation provides a physiologic mechanism for studies showing increased pulmonary microvascular permeability \vith overinflation. 1'· 21 It has important implications in the critical care setting where great care should be taken to prevent damage to the lung due to overdistension during mechanical ventilation. As pointed out elsewhere, 7 this is often a catch-22 situation because high inflation pressures and high levels of positive end-expiratory pressure are necessary to maintain arterial Po 2 at an acceptable level. However, the increased stress on the blood-gas barrier because of the high inflation makes it vulnerable to rupture at a lower capillary Ptm, a process further accelerated if pathologic changes have occurred, ie, the blood-gas barrier or collagen IV are weakened.
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PATHOPHYSIOLOGIC Co!\SEQlJD iCES OF STRESS F .\ I I.UIIE OF PULMOSARY CAPJI.I.ARJES
The pathophysiologic conditions in which stress failure of pulmonary capillaries likely plays a role have been reviewed elsewhere.~-' They indude conditions causing high-permeability edema (for example, neurogenic pulmonary edema, high altitude pulmonary edema, and some cases of adult respiratory distress syndronw for example following trauma causing catecholamine release and transient rise in pulmonary vascular pressure) , frank hemorrhage (as seen in exercise-induced pulmonary hemorrhage) or both (eg, in mitral stenosis) . A particularly interesting condition is high altitude pulmonary edema where increased capillary pressure, probably due to uneven hypoxic pulmonary vasoconstriction, 22 causes failure of pulmonary capillaries and a high permeability type of edema. High molecular weight proteins, blood cells, and the inflammatory markers leukotriene B4 and complement fragment C5a are found in lung lavage fluid of patients with high altitude pulmonary edema (HAPE)Y Both the high permeability type of edema and the presence of inflammatory markers are consistent with pulmonary capillary stress failure as the mechanism of HAPE, the disruption of the endothelial and epithelial lining causing the high permeability type of edema, and the exposure of basement membranes triggering platelet and white blood cell activation and inflammatory processes.' The presence of inflammatory marker (leukotriene B) in the lavage fluid of rabbit lungs perfused at high pressure was recently demonstrated.24 The fact that ultrastructural appearances of the blood-gas barrier in rats \vith HAPE are remarkably consistent with those seen in rabbit lungs after perfusion at high pressure suggests that stress failure of pulmonary capillaries is the mechanism of HAPE.' Other pathologic conditions where pulmonary capillary stress failure likely plays a role indude situa-
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Fl<: l i RF: 4 . Histogram of average number of breaks per millimeter boundary length of endothelium and epithelium at 20 (high lung volume) and 5 em 11,0 transpulmonary pres.s ure (low lung volume). Asterisk, significantly greater (p<0.05) at high compared to low lung volume at the same capillary Ptm . (With permission.")
CHEST I 1051 3 I MARCH, 1994 I Supplement 105S
tions where the extracellular matrix weakened, for example in Goodpasture's syndrome where autoantibodies attack type IV collagen ,25 causing bleeding in the lung and also glomeruli. We recently obtained evidence that exercise-induced pulmonary hemorrhage (EIPH) in thoroughbred racehorses is caused by stress failure of pulmonary capillaries.26 This study was done in collaboration with Drs. Eric Birks, James Jones, John Pascoe, and Walter Tyler of the School of Veterinary Medicine, University of California, Davis. The lungs of three thoroughbred horses with known EIPH were removed and fixed for electron microscopy after the horse had galloped at high speed ( 13 to 16 m/s) on a treadmill. Evidence of intrapulmonary bleeding was confirmed by bronchoscopy in one animal. Tissue fixation was either by airways instillation (one animal) or vascular perfusion of glutaraldehyde solution at S 30 ± 5 em Hp Ptm (two animals). Ultrastructural evidence of stress failure of pulmonary capillaries was the presence of a large number of red blood cells in the interstitium, disruption of the capillary endothelial or alveolar epithelial layers, proteineous material and red blood cells in the alveolar spaces, interstitial edema, and fluid-filled protrusions of the endothelium into the capillary lumen .26 These are the same appearances we have found in rabbit lungs exposed to high capillary transmural pressure. 3•12•15 Pulmonary vascular pressures are extremely high in thoroughbred horses during galloping. Jones and coworkers27 measured pulmonary arterial and left atrium pressures of up to 120 mm Hg and 70 mm Hg, respectively, in thoroughbreds galloping at speeds up to 10 m/s, 27 indicating that capillary pressures must also be very high. These very high vascular pressures are associated with the extremely high cardiac outputs ( > 750 ml!minlkg) achieved in these animals, which require very high left atrial pressure for adequate ventricular filling.26 At the same time, the extremely high V0 2 max achieved during maximal exercise ( 180 ml!minlkg) requires that the bloodgas barrier be extremely thin. However, exercise-induced hypoxemia in thoroughbreds during heavy exercise was shown to be the result of diffusion limitation, 26 indicating that the barrier could not be any thicker without further limitation to V0 2 max. In other words, selection for increased aerobic performance imposes contradictory requirements on the heart-lung system. In thoroughbreds, those requirements have reached the point of failure of the blood-gas barrier. The selective breeding of thoroughbreds for a large number of generations has provided animals with enormous 0 2 uptake rates, which necessitate not only a very thin pulmonary blood-gas barrier but also extremely high cardiac outputs, which in turn require pulmonary pressures to be so high that they exceed the mechanical strength of the thin blood-gas barrier, causing failure of pulmonary capillaries and alveolar hemorrhage.26 Interestingly, while a large number of red blood cells were found in the alveoli and interstitium in areas showing clear abnormalities macroscopically, actual endothelial and epithelial breaks were difficult to find . This was probably due to the reversibility of ruptures once the 106S
vascular pressure was reduced, from the end of the gallop to tissue fiXation 60 to 70 min later. In addition, platelets and cytoplasmic extension of leukocytes were seen in close vicinity to the exposed basement membrane at the site of disruption of the capillary wall, suggesting platelet and white blood cell activation and the initiation of an inflammatory process.26 As mentioned earlier, inflammatory markers were found in lavage fluid of rabbit lungs perfused at high pressure , 2~ as well as in bronchoalveolar fluid of patients with HAPE,23 suggesting that stress failure of pulmonary capillaries is the mechanism of HAPE . In summary, pulmonary capillaries are subjected to high mechanical stress in spite of their small radius of curvature, because of the extreme thinness of the alveolar wall, which in turn is required for the adequate transfer of gases by passive diffusion. The contradictory design requirements imposed on the blood-gas barrier, which must be thin enough not to limit diffusion but at the same time very strong to withstand high stresses across such a thin barrier, have only recently been recognized. Stress failure of pulmonary capillaries plays a role in several pathophysiologic conditions causing high-permeability edema and pulmonary hemorrhage. Adramatic example is exercise-induced pulmonary hemorrhage in thoroughbred horses where selective breeding for extreme aerobic performance over a large number of generations has allowed the cardiovascular system to develop beyond the mechanical strength of the pulmonary blood-gas barrier. ACKNOWLEDGMENTS: This work was done in collaboration with Michael Costello, Ann Elliott, Zhenxing Fu, Sanli Kurdak, Yasuo Namba, Koichi Tsukimoto, and Renato Prediletto. REFERENCES
2 3 4 5 6 7 8 9
10
Low FN. The pulmonary alveolar epithelium of laboratory mammals and man. Anat Rec 1953; ll7:24l-63 Gehr P, Bachofen M, Weibel ER. The normal human lung: ultrastructure and morphometric estimation of diffusion capacity. Respir Physiol 1978; 32:121-40 West JB, Tsukimoto K, Mathieu-Costello 0 , et al. Stress failure in pulmonary capillaries. JAppl Physiol1991 ; 70:173142 West JB. Mathieu-Costello 0 . Strength of the pulmonary blood-gas barrier. Respir Physiol 1992; 88:141-48 Dempsey JA. Hanson PG, Henderson KS. Exercise-induced alveolar hypoxemia in healthy human subjects at sea-level. J Physiol (Lond) 1984; 355:161-75 Wagner PD, Gale GE, Moon RE, et al. Pulmonary gas exchange in humans exercising at sea level and simulated altitude. J Appl Physiol 1986; 61:260-70 West JB, Mathieu-Costello 0 . Stress failure of pulmonary capillaries in the intensive care setting. Schweiz Med Wschr 1992; 122:751-57 West JB . Mathieu-Costello 0 . Stress failure of pulmonary capillaries: role in lung and heart disease. Lancet 1992; 340:762-67 Crouch EC, Martin GR, Brody JS. Basement membranes. In: Crystal RG, West JB, Barnes PJ, et al, eds. The lung: scientific foundations. New York: Raven Press Ltd, 1991; 421-37 Glazier JB, Hughes JMB, Maloney JE, et al. Measurements of capillary dimensions and blood volume in rapidly frozen lungs. J Appl Physiol 1969; 26:65-76 36th Annual Aspen Lung Conference
11 Fu Z, Costello ML, Tsukimoto K, et al. High lung volume increases stress failure in pulmonary capillaries. J Appl Physiol 1992; 73:123-33 12 Tsukimoto K, Mathieu-Costello 0, Prediletto R, et al. Ultrastructural appearances of pulmonary capillaries at high transmural pressures. J Appl Physiol 1991; 71 :573-82 13 Costello ML, Mathieu-Costello 0 , West JB. Stress failure of alveolar epithelial cells studied by scanning electron microscopy. Am Rev Respir Dis 1992; 145:1446-55 14 Elliott AR, Fu Z, Tsukimoto K, et al. Short-term reversibility of ultrastructural changes in pulmonary capillaries caused by stress failure. J Appl Physiol 1992; 73:1150-58 15 Fu Z, Kurdak S, Namba Y, et al. Effect of fixation by airway instillation on the incidence of stress failure in pulmonary capillaries [abstract] . Am Rev Respir Dis 1993; 147:A40 16 Rippe B, Townsley M, Thigpen J, et al. Effects of vascular pressure on the pulmonary microvasculature in isolated dog lungs. J Appl Physiol 1984; 57:233-39 17 Nicolaysen G, Waaler BA, Aarseth P. On the existence of stretchable pores in the exchange vessels of the isolated rabbit lung preparation. Lymphology 1979; 12:201-07 18 Egan EA. Lung inflation, lung solute permeability, and alveolar edema. J Appl Physiol 1982; 53:121-25 19 Parker JC, Townsley MI, Rippe B. et al. Increased microvascular permeability in dog lungs due to high peak airway pressures. J Appl Physiol 1984; 57:1809-16 20 Dreyfuss D, Basset G, Soler P, et al. Intermittent positivepressure hyperventilation with high inflation pressures produces pulmonary microvascular injury in rats. Am Rev Respir Dis 1985; 132:880-84 21 Dreyfuss D, Saumon G. Lung overinflation: physiologic and anatomic alterations leading to pulmonary edema. In : Zapol WM, Lemaire F, eds. Adult respiratory distress syndrome. New York: Marcel Dekker Inc, 1991 ; 433-49 22 Hultgren H. High altitude pulmonary edema. In: Hegnauer A, ed. Biomedicine of high terrestrial elevations. Nattick, Mass: US Army Research Institute of Environmental Medicine, 1969; 131-41 23 Schoene RB, Hackett PH, Henderson WR, et al. Highaltitude pulmonary edema: characteristics of lung lavage fluid. JAMA 1986; 256:63-9 24 Tsukimoto K, Yoshimura N, lchioka M, et al. Protein, cell, and leukotriene B. concentrations of the lung edema fluid produced by high capillary pressures in rabbit. J Appl Physiol 1994 (in press) 25 Wieslander J, HeinegArd D . The involvement of type IV collagen in Goodpasture's syndrome. Ann NY Acad Sci 1985; 460:363-74 26 West JB. Mathieu-Costello 0, Jones JH. et al. Stress failure of pulmonary capillaries in racehorses with exercise-induced pulmonary hemorrhage. J Appl Physiol 1993; 75:1097-1109 27 Jones JH, Smith BL, Birks EK, et al. Left atrial and pulmonary arterial pressures in exercising horses [abstract] . FASEB J 1992; 6:A2020 28 Wagner PD. Gillespie JR. Landgren GL, et al. Mechanism of exercise-induced hypoxemia in horses. J Appl Physiol 1989; 66:1227-33
Centripetal Tension and Endothelial Retraction* Ala11 B. Mvy. M.D.; Rebecca Sheldo11, B.A.; Kathy Li11dsley. B.A.; Sa11dra Shasby. B.A.; a11d D. Michael Shasby, M.D.
I
nflammatory molecules such as histamine increase endothelial permeability by initiating retraction of adjacent endothelial cells. We recently reported that in human umbilical vein endothelial (HUVE) cells histamine increased phosphorylation of the light chain of myosin (MLC) by 0.18 ± 0.02 moles phosphate per mole MLC (moiP/moiMLC). This is consistent with the hypothesis that histamine initiates retraction by increasing centripetal tension within endothelial cells. However, chelation of extracellular calcium which interrupts cellcell and cell-substrate binding also increases endothelial permeability and causes endothelial cell retraction both i11 situ in perfused lungs and in vitro with cultured cells. The effects of calcium chelation are consistent with the hypothesis that release of forces tethering endothelial cells in place allows expression of a constitutive centripetal tension . In our recent report, we found constitutive phosphorylation of MLC at 0.20 ± 0.02 molP/molMLC, consistent with a constitutive actomyosin-mediated tension. We asked if reduction of MLC phosphorylation would prevent the response to histamine or calcium chelation, and conversely, if increased constitutive MLC phosphorylation would cause cell retraction . In control and histamine-stimulated HUVE cells and in control porcine pulmonary artery endothelial (PPAE) cells tryptic peptide maps indicated that MLC was phosphorylated by the calcium calmodulin-dependent myosin light chain kinase . Pretreatment of HUVE cells with cAMP prevented the histamine-stimulated increase in MLC phosphorylation (-0.08 ± 0.02 molP/moiMLC) and a histamine initiated increase in HUVE cell monolayer permeability (-12 ±I percent). Pretreatment of HUVE cells with the calcium-calmodulin inhibitor, ML-9, also prevented the histamine stimulated increase in MLC phosphorylation (-o.29 ± 0.04 molP/molMLC) and the increase in HUVE cell monolayer permeability (14 ± 4 percent of the control response) . Similarly, pretreatment of PPAE cells with cAMP reduced basal MLC phosphorylation (-0.16 ± 0.03molP/molMLC) and reduced the increase in monolayer permeability following calcium chelation (21 ± 14 percent of the control response). Pretreatment of PPAE cells with ML-9 also reduced basal MLC phosphorylation (-0.13 ± 0.02 moiP/molMLC) and reduced the increase in monolayer permeability following calcium chelation (47 ± 5 percent of control response). The basal level of MLC phosphorylation likely represents constitutive kinase and phosphatase activity. Okadaic acid increased MLC phosphorylation in PPAE cells (I J..LM, 0.06 ± 0.04moiP/molMLC and 10 J..LM. 0.17 ± 0.05 molP/molMLC ) in a dose-dependent manner, and 10 J..LM (289 ± 32 percent) but not 1 J..LM (4 ± 5 percent) okadaic acid increased PPAE cell monolayer permeabil*From the University of Iowa College of Medicine. Iowa City. CHEST I 1051 3 I MARCH, 1994 I Supplement 1075