Mechanisms of lung vascular injury and edema after pulmonary microembolism

REVIEWS

Mechanisms of Lung Vascular Injury and Edema After Pulmonary Microembolism Thomas J. Ferro and Asrar B. Malik

N THIS ARTICLE, we review recent studies of the mechanisms of pulmonary vascular injury and edema induced by pulmonary embolism. Several models of embolism-induced pulmonary edema have been studied.’ The focus of this review will be the microembolism induced by the intravenous (IV) infusion of a-thrombin in sheep. The term “microembolism” describes the diffuse deposition of fibrin-platelet thrombi in the small blood vessels (~500 mm in diamater) of the lung, as opposed to the large vessel embolism which may result from deep venous thrombosis. We will discuss the roles of blood components (eg, fibrinogen), inflammatory cells (eg, macrophages and neutrophils), and pulmonary hemodynamic factors in mediating the lung vascular injury and pulmonary edema.

I

EFFECTS OF THROMBIN ON THE PULMONARY MICROVASCULATURE Morphologic

Alterations

Fibrin is evenly distributed in pulmonary vessels up to 500 pm in diameter after the induction of pulmonary intravascular coagulation by IV infusion of a-thrombin (the native enzyme). The amount of intravascular fibrin in the lungs decreases within three hours following thrombin infusion, reflecting the activation of fibrinolysis. Interstitial fibrin is also observed in dogs infused with thrombin and in patients with acute lung injury.lA4 Interstitial fibrin is an important factor in edema formation, since it can reduce the clearance of extravascular fluid by the lung lymphatic system.4 Platelets and neutrophils are also trapped in pulmonary vessels and are found From the Departments of Medicine and Physiology, The Albany Medical College of Union University, Albany, NY. Supported by grant no. PO1 HL 32418 from the National Institutes of Health and the Veterans Administration Research Service. Address reprint requests to Asrar B. Malik, PhD, Department of Physiology, The Albany Medical College. 47 New Scotland Ave. Albany, NY. 0 I989 by W.B. Saunders Company. 0883-9441/89~0402-0008$05.00j0 118

in various stages of degranulation and activation.’ Discrete endothelial lesions near sites of microthrombi are present in the dog lung, but they are absent after thrombin-induced pulmonary microembolism in the sheep.la4 Severe pulmonary edema present as early as 30 minutes after pulmonary microembolism in sheep may result from increased endothelial-junction “pore” dimensions, rather than the denudation type of endothelial injury seen in dog lungs.“4 The variable pattern of endothelial injury reflects differences in species in the resistance of endothelial cells to injury (eg, as a result of varying intracellular concentration of anti-oxidant enzymes’), differences in activation of humoral pathways mediating the injury, and the presence of intravascular macrophages in the pulmonary circulation of some species, such as the sheep.6 Intravascular Coagulation Vascular Injury

is Required

for Lung

Intravascular coagulation is necessary for the induction of lung vascular injury after cr-thrombin challenge.’ Gamma-thrombin, formed in vivo by autoproteolysis or in vitro by the limited trypsin digestion of a-thrombin,* lacks clotting activity as well as the fibrinogen recognition site. The other cellular thrombin-ascribed activities are retained by y-thrombin, although platelet aggregation and activation are an order of magnitude less than with cr-thrombin.8 The absence of fibrinogen clotting in y-thrombin is attributed to the loss of fibrinogen recognition sites on the “fibrin side” of catalytic sites.* However, both thrombin forms are able to cleave synthetic substrates and are inactivated by anti-thrombin III.* The differing properties of LY-and y-thrombin’ are reflected in their responses.’ Arterial fibrinogen concentration decreased markedly after cY-thrombin infusion, whereas the decrease was small after y-thrombin.’ In contrast, pulmonary intravascular fibrin is present after a-thrombin challenge and fibrin degradation products are Journal

of Critical Care, Vol 4, No 2 (June).

1989:

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generated, but not after y-thrombin.’ We have exploited this difference to delineate how a!thrombin induces pulmonary vascular injury and edema.’ Gamma-thrombin did not increase pulmonary lymph flow (Qlym) and transvascular protein clearance, as is the case with cY-thrombin, indicating that intravascular coagulation is required to increase lung vascular permeability. Intravascular clotting also contributes to the hemodynamic changes induced by thrombin since pulmonary arterial pressure and pulmonary vascular resistance (PVR) increased after cythrombin but not after y-thrombin.’ Pulmonary

Hemodynamic

Alterations

The increases in pulmonary arterial pressure and PVR after a-thrombin are associated with thromboxane generation and are blunted by thromboxane synthetase inhibitors’; therefore, thromboxane generation secondary to pulmonary intravascular coagulation contributes to the increases in pulmonary arterial pressure and PVR. The increase in pulmonary arterial pressure, and a consequent increase in the pulmonary capillary hydrostatic pressure, is likely an important factor in the formation of pulmonary edema. Since lung vascular permeability to protein is increased after pulmonary microembolism, even a small increase in the capillary hydrostatic pressure produces a marked increase in transcapillary fluid filtration.’ Thrombin-Induced Release From the Endothelium

of Mediators

Thrombin induces the generation of endothelium-derived mediators, which modulate pulmonary vascular permeability and hemodynamic alterations. The lipid mediators platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3and the prostacyclin phosphorylcholine)’ (PGI,)” are generated by endothelial cells exposed to thrombin. PAF remains cell-associated’ and PGI, is released into the media.” PAF increases lung vascular permeability and PVR,” whereas PGI, is a pulmonary vasodilator and may decrease lung vascular permeability.’ The release of these factors with opposing agent can, therefore, modulate the degree of pulmonary edema. Thrombin also augments the release of tissue plasminogen activator and tissue plasminogen

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activator inhibitor by endothelial cells.” The release of plasminogen activator or inhibitor may contribute to increased endothelial permeability by the local generation of fibrin degradation products, which increase endothelial permeabiliiy.

3.13

Thrombin-Endothelial

Interactions

Direct Efect of Thrombin on Endothelial Permeability Studies have shown that a-thrombin ( 10-8mol/L) increases endothelial permeability to albumin in vitro.14 Transendothelial permeability of ‘251-labeled albumin was studied using bovine pulmonary endothelial monolayers grown on membranes consisting of gelatinized micropore filters.14 The thrombin effect was direct since no fibrin or blood cells were present in the system. Incubation of bovine endothelial monolayers with thrombin ( 10e8 mol/L) resulted in no significant release of lactate dehydrogenase, and removal of the thrombin reversed the permeability increase, indicating that cell damage did not occur.‘4 Endothelial cytoskeletal changes may explain this effect of thrombin since rhodamine phalloidin staining revealed alteration in cellular f-actin.14 Thrombin induces alterations in endothelial cell morphology, including changes in shape, size, and cell contracture.““’ The shape change is prevented by metabolic inhibitors but not by calcium channel blockers or agents that disrupt microfilaments or microtubules.” Binding of thrombin to endothelial cells via thrombomodulin’* and membrane-bound heparin sulfate” may be required for the effect of thrombin on endothelial permeability. Efect of Thrombin on Endothelial Adhesiveness Neutrophil adherence to endothelium is a requisite for the development of lung vascular lung injury.‘9,2’ An interaction is determined by expression of endothelial adhesion sites and the neutrophil CD 18 adhesion glycoprotein complex.20 Thrombin induces neutrophil adherence to the endothelium.‘9~2’ The adherence response is dependent on the catalytic site of thrombin, since hirudin or anti-thrombin III-heparin inhibit the adherence.” The endothelium is required for thrombin-induced neutrophil adherence.” PAF generated by the endothelium may

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mediate a part of the adherence response.*’ In addition, adhesive sites appear on the endothelium within 30 minutes after exposure to thrombin.” We observed that neutrophils treated with monoclonal antibody directed against the neutrophi1 adhesion complex do not adhere to the endothelium made adhesive by the action of thrombin,” indicating that the endothelial adhesive sites expressed on the endothelium interact with the glycoprotein adherence sites on neutrophils. ROLE OF FIBRIN IN LUNG VASCULAR

INJURY

The contribution of fibrin has been examined by inducing fibrinogen depletion using Ancrod, a purified fraction of Malayan pit viper venom.22*23 Ancrod cleaves fibrinogen and releases fibrinopeptide A,** so fibrinogen is not able to form fibrin. Ancrod administration resulted in plasma fibrinogen concentrations close to zero.23 Defibrinogenation prevented the decrease in leukocyte count,24 indicating that leukocyte consumption is secondary to activation of the coagulation cascade, and that activation of coagulation and fibrinolysis releases leukocyte-aggregating agent(s). Pulmonary hypertension and increased PVR occurring normally after thrombin challenge are probably also related to intravascular coagulation since pulmonary arterial pressure and PVR did not increase significantly after a-thrombin infusion in the defibrinogenated animals. 24 Intravascular clots induce pulmonary hemodynamic changes by uneven obstruction of the pulmonary vascular bed with thrombi as well as by inducing the generation of vasoactive humoral mediators (particularly thromboxane A*), lung macrophages and neutrophils and platelets sequestered in the pulmonary circulation.3*25 Defibrinogenation attenuated the thrombininduced increase in lung vascular permeability.24 This study indicates that fibrin entrapment is the initiating event that leads to the increase in lung vascular permeability. This does not, however, rule out an independent effect of thrombin in increasing pulmonary endothelial permeability at higher thrombin concentrations (ie, >lO-* mol/L).14 Several effects of fibrin on endothelial cells have been reported which support the concept that fibrin has a permeability increasing effect. Exposure of cultured endothelium to a fibrin clot

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resulted in endothelium becoming disorganized,26927whereas fibrinogen does not have this effect.27 The cellular disorganization was reversible since fibrin clots increase permeability of endothelial monolayers grown on micropore filters, and permeability returns to normal after removal of the fibrin.** It is unclear, however, whether fibrin, or fibrin degradation products, are responsible for this effect. Both plasminderived and elastase-derived low molecular weight fibrin degradation products27,29 and fragment D (also a fibrin degradation product)30 have been postulated as the mediators by which fibrin induces lung vascular injury. ROLE OF NEUTROPHILS IN LUNG VASCULAR INJURY

Several studies have shown that neutrophils are the effector cells mediating lung vascular injury after pulmonary microembolism. Neutrophils are sequestered in the lung after thrombin infusion3* and are also capable of producing factors which in turn induce or promote lung vascular injury and edema and contribute to the increases in pulmonary arterial pressure and PVR.31 The following material discusses the data and mechanisms by which neutrophils induce lung vascular injury and edema. Neutrophil

Depletion

Studies

Removal of circulating neutrophils blunts the permeability increase induced by thrombin.3’*33 Infusion of a-thrombin into sheep made neutropenic (neutrophil count was ~200 cells/pL) using hydroxyurea or anti-neutrophil serum attenuated the increase in Qlym. Moreover, the increased olym was associated with a decrease in L/P ratio, indicating that the increased fluid filtration is solely the result of elevated P,. Neutrophils have also been shown to mediate the increase in lung vascular permeability after fat embolism-induced intravascular coagulation34 and glass bead embolism.35 Pulmonary

Neutrophil

Kinetics

Pulmonary microembolism results in the rapid uptake in the lung of “‘Iridium-labeled neutrophils.32 The duration of neutrophil entrapment is related to activation of fibrinolysis since suppression of fibrinolysis and increased fibrin deposition prolonged neutrophil entrapment.30 Therefore, the fibrin-dependent neutrophil entrapment

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plays an important role in the modulation of lung vascular injury.30

such as elastase may contribute vascular injury.l

Mechanisms of Neutrophil and Activation

Activation of Plasminogen and the Complement System: Role in Fibrin-Neutrophil Interactions

Neutrophil

Sequestration

Adherence to the Endothelium

Neutrophil adherence to the endothelium is required for the development of microvascular lung injury.‘9-2’ Neutrophil adhesion occurs as a result of interaction between endothelial adhesion sites and neutrophil CD18 adhesion glycoprotein complex.20 Thrombin,‘922’ interleukin- I ,36 lipopolysaccharide,36 phorbol myristate acetate,36 tumor necrosis factor,37 C5a,38 fibrin3’ and plasmin3’ have been shown to induce neutrophi1 adherence to endothehal monolayers. The increased adherence is either the result of the action of these factors on the endothelium (eg, tumor necrosis factor), the neutrophils (eg, plasmin), or both (eg, phorbol myristate acetate). Thrombin induces neutrophil adherence to the endothelium by making the endothelium adhesive to neutrophils.‘9~2’

Role of Fibrin in Neutrophil and Activation

Sequestration

The duration of neutrophil entrapment in the lungs after thrombin infusion is dependent on the duration of fibrin sequestration in pulmonary vessels.30Extending the duration of fibrin entrapment by inhibiting fibrinolysis resulted in a sustained neutrophil sequestration.30 Studies indicate that fibrin possesses a neutrophil adherent property that is independent of its effect on the endothelium.30 Neutrophils may also be drawn into the microthrombi by the generation of local chemotaxins, such as complement-derived peptides (eg, C5a) and fibrin degradation products, during intravascular coagulation and fibrinolysis. Fibrin clots are known reservoirs of neutroPhil-attracting factors such as thrombin and c,,, I,3.40 Neutrophils are often seen adherent to the endothelium and in the end-stages of degranulation after thrombin infusion.’ Fibrin may promote neutrophil activation since adherence is associated with activation (ie, superoxide generation and elastase release).‘9,2’ Moreover, since neutrophil elastase induces fibrinolysis,4’ the generation of fibrin degradation products subsequent to the release of neutrophil secretagogues

to the lung

Complement activation has been proposed as a key factor in the induction of lung vascular injury.‘,42 Plasmin generation is associated with the cleavage of complement proteins and the formation of the complement-derived chemotactic and leukocyte-aggregating peptides, C3a and C5a.40.43The role of the complement system was examined by reducing the hemolytic complement activity through the administration of phospholipase A,-free cobra venom factor fraction for several days.42,44The infusion of a-thrombin in this preparation prevented the increase in lung vascular permeability, a response similar to neutropenic sheep. The basis for leukocyte aggregation and activation after pulmonary microembolism is probably more complex than complement activation. The reason for this is that complement activation in vivo does not produce a sustained increase in lung vascular permeability.45 Fibrin degradation products3 leukotriene B4 (LTB4),46.47 and PAF,48 are also likely to be involved in the leukocyte aggregation and activation. Plasmin has effects independent of complement activation. Plasmin generation releases thrombin trapped in fibrin clots.49 The release of entrapped thrombin forms may further promote injury, since both cu-thrombin and y-thrombins are known to have factor D activity in the alternate complement pathway.50 Plasmin remains bound to fibrin until dissolution of the Clot,40 and can thus also promote fibrin-neutrophi1 interaction. The plasmin in immediate contact with fibrin reaches high concentrations and would not be inactivated by circulating inhibitors. Plasmin induces neutrophils to adhere to the endothelium and other surfaces.39 The effect is independent of the proteolytic action of plasmin.39

Role of Arachidonic Acid Metabolites Sequestration and Activation Generation of metabolites. Thrombin-in-

in Neutrophil

duced intravascular coagulation results in the generation of arachidonic acid metabolites capable of neutrophil chemotaxis, aggregation, and superoxide anion generation.” The release of

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these factors parallels the uptake of labeled neutrophils after thrombin challenge. LTB, is a major candidate since it is a known neutrophil chemotactic and aggregating agent.*,‘** 53 Arachidonic acid metabolites were also detected in bronchoalveolar lavage (BAL) fluid.5’ Pulmonary microembolism induced by thrombin causes neutrophil alveolitis that is temporally related to the neutrophil chemotactic activity detected in the organic phase of etherextracted BAL fluid.” Analysis of the organic phase of the BAL fluid by RP-HPLC revealed a peak that corresponded to LTB4 and correlated in appearance with BAL fluid neutrophil chemotactic activity. 2o The problems with invoking LTB4 as the sole mediator of lung vascular injury are that it does not induce an increase in lung vascular permeabilitys4 and it is a poor activator of neutrophils.s4 Since other lipoxygenase products (such as 5-HETE and 15HETE) generated with LTB, enhance the neutrophil activation induced by LTB,, 54this may be a mo re important process by which these lipoxygenase metabolites promote neutrophil-dependent lung injury.54 Efects of prostaglandin E, in inhibiting injury. Administration of prostaglandin E, (30

ng/min/kg for 90 minutes) prevented the increase in Qlym seen after infusion of zymosanactivated plasma in sheep.55 This protective effect occurred despite the persistence of neutrophi1 sequestration in the lung. In addition, PGE, inhibited the adherence of sheep neutrophils stimulated by zymosan-treated serum, thrombin, or thrombin-generated serum to sheep pulmonary artery endothelial cells.25 These data indicate that there are “protective” arachidonate metabolites such as PGE, and that their salutary effect is related to inhibition of neutrophil adherence. ROLE OF FIBRINOLYSIS IN LUNG VASCULAR INJURY

Plasmin activation promotes lung vascular injury by the generation of fibrin degradation products (FDPs) subsequent to clot lysis. FDPs independently increase lung vascular permeability.3 In vitro studies show that FDPs induce “Cr release from cultured endothelial cells.56 FDPs also increase the capillary hydrostatic pressure.” Infusion of FDPs increased Glym in the presence

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of thrombin (ie, following thrombin-induced pulmonary microembolism), and the increase in hlym was associated with a decrease in the L/P ratio,57 indicating that most FDPs increased fluid filtration by increasing the hydrostatic pressure. The residence time of fibrin microthrombi in the pulmonary circulation is a crucial determinant of the increase in lung vascular permeability. Pretreatment with tranexamic acid (100 mg/sheep) to depresss fibrinolysis resulted in sustained increases in Qlym and lymph protein clearance.” Therefore, increased duration of fibrin microemboli and neutrophil residence in the microcirculation augments lung vascular injury by enhancing the endothelial-fibrin and endothelial-neutrophil contact. ROLE OF MACROPHAGES IN LUNG VASCULAR INJURY

Macrophages are the predominant resident inflammatory cells of the lung.‘* They are the only inflammatory cell present in great numbers in the airspaces shortly after thrombin-induced pulmonary microembolism.s9~60 Macrophages can cause endothelial cell injury59*60and contribute to pulmonary leukostasis by releasing neutrophi1 chemotaxins.14 Effects

of Macrophage

Activation

Alveolar macrophages (AM) obtained after thrombin-induced pulmonary microembolism were examined to determine their effects on the permeability of ‘251-albumin across ovine pulmonary artery endothelial monolayers. Postthrombin AM (but not AM obtained before thrombin infusion) increased transendothelial permeability to albumin.59 AM activation was the result of thrombin-dependent processes in vivo since AM incubated directly with thrombin in vitro did not increase endothelial permeability. The increase in permeability was reduced by the addition of superoxide dismutase or catalase, implicating the local production of the oxygen radicals, superoxide and hydrogen peroxide. Oxygen radical production is likely the result of macrophage-endothelial cell interactions, since postthrombin AM did not produce greater amounts of superoxide than resting macrophages.60

MECHANISMS

OF PULMONARY

Monokines

and Lung Vascular

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Injury

Macrophages produce the monokines, interleukin-1 (IL-1),36,6’,62 tumor necrosis factor (TNF),37.62,63 and interferon-y ( IFN-T),~~ which may promote lung vascular injury. The functions of IL-I relevant to the pathogenesis of lung vascular injury include the increase in endothelial thrombotic activity,64 the expression of endothelial adhesion sites,62 the induction of neutrophil and lymphocyte adherence to endothelium,36 and the activation and recruitment of neutrophils and lymphocytes.6’ In addition, recombinant human IL-l (10 to 100 p/mL) independently increases the permeability of ‘?-albumin across sheep pulmonary artery endothelial monolayers by 50%. IL-l induces lung edema without an increase in pulmonary vascular resistance in the isolated-perfused rat lung,65 indicating a permeability-increasing effect of IL- 1. TNF stimulates macrophages to produce IL1,(j6 promotes endothelial thrombotic activity,” increases neutrophil adherence to endothelial cells by increasing surface expression of the C3bi receptor/adherence glycoprotein,37 and augments the phagocytic and cytotoxic activities of neutrophils.” Recombinant human TNF (10 to 100 y/mL) increases the permeability of 12’1albumin across sheep pulmonary artery endothelial monolayers by 70’24, indicating that it is directly injurious to endothelial cells. The macrophage-dependent increase in endothelial monolayer permeability’* discussed above may be mediated by the release of IL-l and TNF. PLATELETS DO NOT MEDIATE VASCULAR INJURY

LUNG

The permeability increase observed after cu-thrombin infusion in sheep is not prevented by antiserum-induced thrombocytopenia. Platelet depletion did, however, result in smaller increases in Qlym;67 this may be due to the lack of release of the platelet-derived pulmonary vasoactive mediator, thromboxane AZ, in the platelet-depleted animals.67 Platelets have been shown to decrease the permeability of the endothelial monolayer;68 thus, platelets may have a “protective” effect on endothelial monolayer permeability. Platelet aggregation would, however, contribute to pulmonary edema formation by the

release of pulmonary vasoactive mediators such as thromboxane. OXYGEN

RADICALS AS MEDIATORS VASCULAR INJURY

OF LUNG

Toxic oxygen-derived metabolites superoxide anion (O,-), hydrogen peroxide (H202). and hydroxyl radical (OH. ) appear to be players in the genesis of lung vascular injury, although their role remains controversial since proteases released from neutrophils and macrophages have also been implicated.‘~s8~69There is ample potential for the production of both oxidants and proteases during lung inflammation by neutroPhil?’ and macrophages.60 The thrombin response was studied in sheep infused with superoxide dismutase (which removes 0,) bound to Ficoll to prolong the circulating life of the enzyme.70 Increases in olym and transvascular protein clearance were blunted in these animals in a manner similar to that in neutropenic sheep.3’ The O2 generation (presumably generated from neutrophils and macrophages) is an important mediator of the increase in lung vascular permeability after thrombin. Since the toxic metabolite OH -6g is formed by combination of H202 and Oz~ (the Haber-Weiss reaction), the protective effect observed with superoxide dismutase may be due to the removal of O,- substrate from this reaction. CONCLUSIONS

Pulmonary vascular injury and edema after pulmonary microembolism are multifactorial processes. The factors involved in the development of lung vascular injury after pulmonary microembolism are shown in Fig 1. Alphathrombin proteolytically converts the clottable fibrinogen to fibrin and deposits fibrin microthrombi in pulmonary vessels. The clots act as a “sink” for the residual a-thrombin and other clotting factors. Fibrin-induced plasminogen activation activates the complement system, resulting in the formation of neutrophil-activating peptides (C3a and C5a), which induce neutrophil sequestration in pulmonary microvessels. Plasminogen activation also releases thrombin entrapped in clots, which augments complement activation. In addition, cw-thrombin promotes

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neutrophil-endothelial adherence by effects on neutrophils and the endothelium. Macrophages (both alveolar and intravascular) may promote increased vascular permeability by the secretion of monokines, and thereby the recruitment and activation of neutrophils. The sequestration of neutrophils in the lung after pulmonary microembolization and generation of toxic metabolites, oxygen radicals, and proteases lead to endothelial cell injury and increased permeability. Pulmonary edema is the result of both increased vascular permeability and capillary hydrostatic pressure.

ENDOTHELIAL PERNEABILITY LUNG

AND

/

EY” AND EDEMA

ACKNOWLEDGMENT An overview Fig 1. lar injury and edema. cated by arrows; other the text.

of the pathogenesis of lung vascuThe major steps involved are indimechanisms involved are outlined in

The authors wish to thank Lynn preparation of the manuscript and careful preparation of the figure.

McCarthy for skillful Nancy Gertzberg for

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