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Molecular and Immune Biomarkers in Acute Respiratory Distress Syndrome: A Perspective From Members of the Pulmonary Pathology Society.

Effective gas exchange in the lung requires thin alveolar septa with a minimum distance between the alveolar epithelium and the endothelium of the microvasculature to facilitate simple diffusion of those gases. (1) When the endothelial-epithelial barrier is injured, interstitial and alveolar edema may develop, which, in turn, has a fundamental role in the development of acute respiratory distress syndrome (ARDS). (2) Acute respiratory distress syndrome is a multifactorial syndrome with high morbidity and mortality rates characterized by a deficiency in gas exchange and lung mechanics, which leads to hypoxemia, dyspnea, and respiratory failure. (3) Because of overlapping criteria applied to the definition of acute lung injury (ALI) and ARDS, the Berlin definition of ARDS emphasizes 3 ARDS categories--mild, moderate, and severe--based on the degree of hypoxemia. (4) Per the Berlin definition, ARDS is defined by an acute hypoxemia, a ratio of the Pa[O.sub.2] to the Fi[O.sub.2] of 300 mm Hg or less on positive end-expiratory pressure (PEEP) of 5 cm [H.sub.2]O or greater, together with bilateral infiltration on radiology that is not otherwise fully explained by fluid overload or cardiac failure. (4) Genetic risk factors and various comorbidities, including infection, ventilator-associated lung injury, transfusion-related ALI, and fluid overload, seem to predispose or contribute to ARDS. The injury process in ARDS involves a complex interaction of numerous factors, including inflammatory cytokines, epithelial and endothelial damage, fibrogenesis, and abnormal lung mechanics. Despite decades of intensive research efforts, including several clinical trials, pharmacologic intervention has been unsuccessful in reducing mortality. (1-5) That failure during the years may be due, at least in part, to our incomplete understanding of the biology of ARDS, but recent advances have broadened our perspective on this process and have suggested potential molecular biomarkers that may be prognostically meaningful. (1-5)

Causes of ARDS can be broadly divided into pulmonary (direct) and extrapulmonary (indirect) injuries (Table 1). Commonly, direct injuries to lung parenchyma arise from infection, aspiration, autoimmune inflammatory injury, and adverse drug reactions. Less-common causes include pulmonary contusion, fat emboli, near-drowning events, toxic inhalational injury, reperfusion edema following lung transplantation, and pulmonary thromboembolism. Common causes of indirect lung injury include sepsis and shock (often related to severe trauma requiring multiple transfusions). Less-common causes include cardiopulmonary bypass, blood-product transfusions, drug overdoses, and acute pancreatitis. Regardless of the cause of injury, epithelial damage stimulates macrophage activation and an inflammatory response. (3) The consequence of the disruption of endothelial and epithelial integrity is the flooding of the alveolar airspaces by edema fluid from the plasma. (3)

BIOMARKERS OF ARDS

Studies of biomarkers in lung tissue, plasma, and bronchoalveolar lavage fluid (BAL) from patients and animal models have provided insight into the pathogenesis of ARDS and suggest novel therapeutic targets. (6-12)

After the development of biomarkers, they may also be useful in increasing risk stratification once ARDS criteria are present. Biomarkers may also have a key role in the strategy of future clinical-trial protocols through the identification of patients at high risk of death, thus decreasing the required sample size needed to show a therapeutic benefit. (13)

Recently, biomarkers have also been proven useful in evaluating the response to therapy. (14)

The status of the development and validation of different types of biomarkers have been investigated, with the goal of their incorporation into routine clinical practice. The immunologic and molecular biomarkers have been described in the context of ARDS pathogenesis and their major biologic activity on inflammation, endothelial and epithelial cell damage, fibrogenesis, and abnormal lung mechanics. (9,15,16)

The injury process in ARDS involves a complex interaction of numerous factors, including the release of proinflammatory cytokines, epithelial and endothelial cell damage, fibrogenic mediators, and abnormal lung mechanics. Shortly after lung injury and damage to alveolar epithelial cells (AECs), cellular signal transduction occurs via a biochemical response through Toll-like receptors. Alarmins, a subgroup of molecules of a larger set called damage-associated molecular patterns, are released in response to damaged and dying AECs, and local macrophages activate and recruit immune cells through binding to Toll-like receptors, interleukin-1 receptors, and receptors of advanced glycation end products (RAGEs), amplifying proinflammatory signaling pathways. (3)

These signals converge to induce transcription of nuclear factor-[kappa]B and activator protein-1, which ultimately drive the formation and deposition of collagen fibers by lung fibroblasts.

Histologically, the diffuse alveolar damage (DAD) pattern represents most cases of ARDS, so DAD morphology is equal to ARDS clinically. (17)

The morphologic appearance of DAD varies significantly, depending on the phase of lung injury. In general, 2 main phases are recognized, although those phases occur on a biologic continuum and both can be encountered simultaneously in various combinations, particularly when repetitive episodes of injury and repair are taking place. (2,17,18)

The initial exudative phase is characterized by alveolar septal edema, reactive changes of type II AECs, edema with intraalveolar fibrin, and sometimes, hyaline membranes and infiltration of macrophages and neutrophils. The exudative phase is followed or associated with a fibroproliferative phase, characterized by fibroblastic proliferation and type II AECs hyperplasia, which culminates in restoration of alveolar integrity and architecture or fibrotic scars in which the alveolar wall has been destroyed. (2,17,18)

Herein, we present concepts and information on major and additional biomarkers that are being investigated experimentally, are currently used clinically, and are available for laboratory use. We focus on biomarkers that reflect their primary function for academic and practicing pathologists who are developing and validating new assays.

BIOMARKERS OF INFLAMMATION

Experimental Models

Experimental models provide insight into the molecular basis of ARDS and have suggested several biomarkers that may be prognostically useful in ARDS. Table 2 summarizes major biomarkers of inflammation in experimental models of ARDS.

Activated neutrophils have a major role in ARDS and mediate microvascular damage. Interleukins also have a critical role in this process, and interleukin (IL)-8 has been identified as an important cytokine in ARDS because it is described as the main chemotactic factor for neutrophils in the blood and has also been identified in BAL from patients with ARDS. (19)

Moreover, high levels of IL-8 in the BAL fluid from patients with ARDS predict poor outcome. (19)

In one study, (20) cytokine levels and neutrophilic infiltration were analyzed in BAL fluid, and neutrophilic apoptosis of the lung parenchyma was examined in a model of pulmonary (p) and extrapulmonary (exp) ARDS.

The pARDS group presented a 3-fold increase in IL-8 and a 2-fold increase in IL-6 in BAL, when compared with expARDS. Neutrophils in the BAL were more frequent in pARDS than they were in expARDS. Patients with pARDS showed more extensive injury in alveolar epithelium, in intact capillary endothelium, and in apoptotic neutrophils, whereas the expARDS group presented with interstitial edema and intact type I and II AECs and endothelial layers. The authors concluded that direct insult yielded more-pronounced local inflammatory responses, which induced more-important ultrastructural changes. In another article, (21) that same group studied effects of methylprednisolone on lung mechanics and inflammatory response in endotoxin in pARDS and expARDS groups.

They found that methylprednisolone decreased mechanical and morphometric changes, IL-6, IL-8, and transforming growth factor (TGF)-[beta] cytokine levels, and tumor necrosis factor-[alpha] (TNF-[alpha]), macrophage migration inhibitory factor, interferon-[gamma], and TGF-[beta]2 messenger RNA (mRNA) only in pARDS animals but had similar effects at reducing collagen-fiber content, regardless of ARDS etiology.

Recent work by Araujo et al (22) demonstrated that cell-based therapy may be a promising treatment for ARDS and might act differently on lung and distal organs. The authors demonstrated that bone marrow-derived mononuclear cell therapy led to reductions in collagen fiber content and cell apoptosis in lung, kidney, and liver. In addition, serum concentrations of IL-6, the [KC.sub.murine] functional homolog to IL-8 and IL-10, were decreased. Moreover, reductions were noted in (1) mRNA, insulin-like growth factor-1, platelet-derived growth factor, and TGF-[beta], as well as repair of basement membrane, epithelium, and endothelium, regardless of ARDS etiology; and (2) elevations in vascular endothelial growth factor (VEGF) levels in BAL and mRNA in lung tissue in both ARDS groups. (22)

Clinical Practice

Clinical studies have also provided insight into potential prognostic biomarkers in ARDS. The inflammatory response has been widely investigated in patients with ARDS. Table 2 summarizes potential biomarkers of inflammation in ARDS clinical practice.

Bauer et al (23) studied clinical and physiologic data and serum concentrations of TNF-[alpha], IL-1[beta], and IL-6, obtained from 46 patients with ARDS and 20 patients with pneumonia, compared with 10 control subjects with no inflammatory lung disease. They found that serum TNF-[alpha] levels were higher in patients with ARDS than in those with pneumonia or control patients. IL-1[beta] and IL-6 were not statistically significant between patients with ARDS, patients with pneumonia, and control subjects. They suggested that systemic TNF-[alpha] and IL-1[beta] levels reflect the severity of the lung injury, rather than its diagnosis. (23)

Inflammatory cytokines have also been associated with development of ARDS, shock, and multiple organ dysfunction syndrome. Meduri et al (24) tested the hypothesis that an unfavorable outcome in patients with ARDS was related to the presence of a persistent inflammatory response. They evaluated the behavior of inflammatory cytokines during progression of ARDS and the relationship of plasma and BAL inflammatory cytokines with clinical parameters and patient outcome. Their findings indicate that unfavorable outcomes in ALI were related to the degree of inflammatory response early in, and throughout, the course of ARDS. Patients with higher plasma levels of TNF-[alpha], IL-1[beta], IL-6, and IL-8 on day 1 of ARDS seemed to have persistently elevated levels of those inflammatory cytokines during the disease course and ultimately passed away from their disease. In contrast, surviving patients tended to have lesser elevations in plasma inflammatory cytokines on day 1 of ARDS and a rapid reduction over time.

Complementing the Meduri study, (24) the Calfee et al (14) ARDS Network (National Heart, Lung, and Blood Institute, Bethesda, Maryland) data looked at slightly different markers (but with significant overlap) and looked at about 50 times as many patients. They identified a hyperinflammatory subphenotype (phenotype 2), which was characterized by higher plasma levels of inflammatory biomarkers, a greater prevalence of vasopressor use, lower serum bicarbonate, and a higher prevalence of sepsis, compared with patients identified with phenotype 1. Patients in the phenotype 2 group had greater mortality and fewer ventilator-free and organ failure-free days in both cohorts (cohorts from 2 clinical trials: the ARMA [lower tidal volume ventilation] and ALVEOLI [higher and lower PEEP] trials). In the second cohort (the ALVEOLI cohort), the effects of ventilation strategy on mortality and ventilator- and organ failure-free days differed significantly by phenotype.

Although severe acute exacerbations of idiopathic pulmonary fibrosis is not a standalone ARDS, lung histology in those acute cases is typified by the superimposed presence of DAD, which is also a characteristic finding in ARDS and in acute-exacerbation idiopathic pulmonary fibrosis. Auto antibody-mediated lung diseases can also manifest with ALI in the absence of extrinsic causes, as demonstrated by Donahoe et al. (25) That group conducted a pilot trial to test the hypothesis that autoantibody-targeted therapies may also benefit patients with acute-exacerbation idiopathic pulmonary fibrosis. They used anti-HEp2 antibodies and matrix metallopeptidase (MMP)-7 as potential biomarkers and successfully treated patients with plasmapheresis. The results of the trial indicated autoantibody-reductive therapies may benefit some patients with this otherwise refractory and highly lethal syndrome. (25)

Additional Biomarkers of Inflammation

Table 3 shows additional inflammatory biomarkers of inflammation in ARDS. Higher levels of IL-18 are correlated with disease severity and mortality in patients with ARDS. (26) Among 38 patients with acute respiratory failure, Makabe et al (27) demonstrated that those with ARDS had significantly higher serum levels of IL-18. Circulating IL-1 receptor antagonist (IL-1RA) levels were increased in patients with ARDS and were associated with morbidity and mortality. (28) Trauma-associated ARDS differed from ARDS caused by other clinical disorders, with lower levels of soluble TNF receptors (sTNF-RI) in patients with trauma as a primary cause of ARDS. (29) Interleukin-10 inhibited release of proinflammatory mediators by alveolar macrophages in patients with ARDS who presented with lower plasma and BAL levels of IL-10. (30) High mobility group box nuclear protein 1 (HMGB1) is a DNA nuclear-binding protein produced by monocytes and macrophages. Plasma levels of HMGB1 were increased in patients with ARDS and were associated with outcome. (31) Villar et al (32) demonstrated that lipopolysaccharide binding protein (LBP) is a protein strongly associated with increased mortality and the development of ARDS in serum from patients with severe sepsis. Nitric oxide (NO), a marker of oxidative stress, was evaluated in patients with ARDS who presented higher levels of NO, and it was associated with mortality rates. (33)

C-reactive protein (CRP) is a marker of systemic inflammation; however, higher levels of CRP were associated with better survival among patients with ARDS. (34) One study (35) found that albumin levels, rather than CRP, may predict and monitor the severity and course of ARDS in critically ill patients with fever. In this same study, (35) lactate dehydrogenase levels predicted mortality but without association with severity.

BIOMARKERS OF ENDOTHELIAL CELL DAMAGE

Endothelial cell damage has an important role in the pathogenesis of ARDS, and several biomarkers of endothelial damage have shown promise in determining prognosis. Table 2 summarizes biomarkers of endothelial cell damage in experimental models and clinical practice in ARDS.

Systemic endothelial activation and injury have a fundamental role in multiorgan system failure. von Willebrand factor (VWF), adhesion molecules (such as E-selectin, L-selectin, intercellular adhesion molecule [ICAM], and vascular cell adhesion protein-1), thrombomodulin (TM), protein C, and plasminogen activator inhibitor-1 (PAI-1) are important markers of endothelial activation and injury.

Experimental Models

In the model of sepsis-induced ARDS, Silva et al (36) demonstrated the use of recruitment maneuvers, a ventilatory strategy to reopen collapsed alveoli during hypervolemia. They found that recruitment maneuvers reduced alveolar collapse and improved oxygenation and lung mechanics at the expense of alveolar capillary-membrane damage, increased edema, and greater gene expression of caspase-3, IL-6, IL-1[beta], type III procollagen, ICAM-1, and vascular cell adhesion protein-1. Their data suggest that hypervolemia may induce and potentiate lung damage after recruitment maneuvers. (36)

Clinical Practice

Ware et al (37) postulated that plasma levels of VWF, a marker of endothelial activation and injury, would be associated with clinical outcomes in ARDS. They found that baseline VWF levels were similar in patients with and without sepsis and were significantly higher in nonsurviving patients, compared with surviving patients, when controlling for the severity of the ARDS, sepsis, and ventilator strategy. Higher VWF levels were also associated with a reduction in organ failure-free days. Ventilator strategy had no effect on VWF levels. They concluded that the degree of endothelial activation and injury was strongly associated with outcomes in ARDS, regardless of the presence or absence of sepsis, and was not modulated by a protective ventilator strategy.

Peres e Serra et al (38) determined the nature of hyaline membranes in secondary (pulmonary and extrapulmonary ARDS) and idiopathic DAD (acute interstitial pneumonia). Regarding factor VIII, they found that idiopathic DAD presented larger amounts of immunostained hyaline membranes than did extrapulmonary, diffuse alveolar damage. They suggested that a local and specific lesion had different pathways (direct, indirect, or idiopathic), depending on the type of DAD.

Cowley et al (39) and Kayal et al (40) investigated soluble adhesion molecules (soluble E-selectin and soluble ICAM1) and VWF in the development of the systemic inflammatory response syndrome and organ dysfunction in patients with sepsis. The plasma level of adhesion molecules in the healthy volunteers were within reference range, whereas their concentrations were increased in the sepsis group. Higher levels of E-selectin, ICAM-1, and VWF were found in nonsurvivor patients with septic shock and sepsis. Initial plasma levels of E-selectin, ICAM-1, and VWF predicted survival with high sensitivity and specificity.

McClintock et al (41) measured plasma markers of inflammation, coagulation, and fibrinolysis simultaneously to assess whether those markers remained predictive in the era of lung-protective ventilation in ARDS. They found that all markers, except IL-6, were significantly different between survivors and nonsurvivors and were further from reference range in nonsurvivors. They concluded that higher levels of IL-8 and ICAM-1 were independently predictive of worse outcomes.

Thrombomodulin is a glycoprotein localized on the endothelial cell surface with the function of neutralizing the procoagulant effects of thrombin and accelerating activation of protein C. Ware et al (42) showed that levels of soluble TM (sTM) were higher in plasma from patients with ARDS. They found no association between plasma sTM and the development of ARDS, although higher levels of sTM were observed in patients at high risk for ARDS. The same authors demonstrated that, in patients with ARDS, higher plasma and alveolar levels of sTM correlated with the severity of illness and multiple organ failure. Sapru and colleagues (43) used the ARDS Network and found 449 patients who demonstrated that increased levels of plasma sTM were associated with increased mortality, probably reflecting an increased degree of inflammation in lungs and systemic endothelial damage.

Activated protein C controls coagulation and fibrinolysis and also decreases the levels of proinflammatory cytokines. Ware et al (42) showed that protein C levels were lower in the plasma from patients with ARDS compared with that from control subjects and those levels predicted worse survival with increased risk of multiple organ failure. Ware et al (44) also demonstrated, in a larger cohort of patients with early ARDS, that low plasma levels of protein C were again associated with mortality and adverse clinical outcomes.

Plasminogen activator inhibitor-1 activates fibrinolysis through the conversion of plasminogen to plasmin, a fibrinolytic enzyme. During ARDS, AECs and activated macrophages overexpressed PAI-1, decreasing fibrinolytic activity. Prabhakaran et al (45) showed that patients with ARDS presented higher PAI-1 levels than did patients with cardiogenic lung edema, with greater rates of mortality and longer durations on mechanical ventilation. In a large, randomized controlled trial, Agrawal et al (46) showed that PAI-1 was associated with a decreased oxygenation index but not with mortality. In an ARDS Network study, Ware et al (44) demonstrated that higher levels of PAI-1 were independently associated with greater mortality, poor survival, and more organ failure. However, that association between PAI-1 levels and the development of multiorgan failure was not confirmed in a recent study of 100 patients with ARDS. (47)

Additional Biomarkers of Endothelial Cell Damage

Additional biomarkers of endothelial cell damage are shown in Table 3. Angiopoietin (Ang)-1 and Ang-2 improves angiogenesis, promoting vascular maturation and diminution of vascular permeability. In a large study from the ARDS Network trial, Calfee et al (14) demonstrated that plasma levels of Ang-2 were associated with high mortality in patients with noninfectious ARDS, whereas that association was absent in patients with infectious ARDS. Those authors further demonstrated that extrinsic lung injury was characterized by a molecular phenotype consistent with more-severe lung endothelial damage, as evaluated by plasma Ang-2, and less-severe AEC damage. (14) In a large study of 390 critically ill patients, Ware et al (48) demonstrated that the pulmonary edema fluid to plasma protein ratio, a noninvasive measure of alveolar-capillary barrier permeability, presented good discriminative value in differentiating ARDS from hydrostatic edema and was strongly associated with clinical outcomes. Bastarache et al (49) reported that tissue factor, a potent 47-kDa transmembrane glycoprotein stimulator of the extrinsic coagulation cascade to produce thrombin formation and fibrin deposition tissue factor levels, in lung edema fluid was higher than it was in plasma in patients with ARDS, suggesting a lung source for tissue factor in this situation. In another study, Bastarache et al (50) found that plasma and alveolar levels of tissue factor were higher in patients with ARDS than they were in patients with hydrostatic edema. Patients with ARDS present cell-free hemoglobin levels higher in the alveolar space than do critically ill patients with hydrostatic lung edema. (51)

BIOMARKERS OF EPITHELIAL CELL DAMAGE

In ARDS, disruption of the integrity of AECs and reconstitution of the basement membrane (BM) are important determinants of clinical outcome. (51) Injury to the lung parenchyma and loss of AECs lead to a deposition of collagen fibers. If the BM is intact and loss of AECs integrity is limited, the collagen fibers are reabsorbed, and reepithelialization by AECs occurs simultaneously. (51) Persistent injury to AECs causes loss of BM integrity and leads to epithelial mesenchymal transition, which may contribute to the progression of fibrosis. (52)

Experimental Models

Experimental models of pulmonary epithelial damage in ARDS have provided insights into this process and have suggested potential biomarkers. Phosphatase and tensin homolog (PTEN) is a multifunctional phosphatase that negatively regulates the PI3K/Akt pathway and exerts tumor suppression. Previous studies have reported the regulatory role of PTEN on fibroblasts in lung fibrosis and have shown that deletion of PTEN confers resistance to airway injury. (53)

Mice with postnatally deleted PTEN gene, specifically in AECs, were used by Miyoshi et al, (54) who demonstrated that an Akt inhibitor had a protective effect; and the epithelial PTEN gene is essential for prevention of ALI and lung fibrosis through regulation of AEC integrity. Under healthy conditions, AECs establish close contact with their neighbors through laterally located, intercellular junctional complexes (ie, tight junctions and adherent junctions) and reside on the BM. (54) The tight junctions of AECs provide intercellular sealing and are integral to the maintenance of the integrity of the alveolar-capillary barrier. (54) Seven days after administration of bleomycin, types I and II AECs were intimately associated with each other through tight junctions and adherent junctions. In bleomycin-treated, PTEN-mutant lungs, diminution of electron-dense materials, indicating disruptions of intercellular junctional complexes, and opening of cell-cell contacts of AECs were observed. (54) Using immunofluorescence staining of lung sections for claudin 4, E-cadherin, and laminin after bleomycin instillation, the authors showed that PTEN-mutant lungs presented a marked decrease in the staining intensity for those markers when compared with the controls. They also found that the BM was severely degraded in PTEN-mutant lungs. The response to damage of the AEC barrier is mainly represented by increased barrier permeability to protein-rich edema, recruitment of neutrophil exudate from the alveolar capillary in response to chemokines, and the release of several cytokines, chemokines, proteases, eicosanoids, and growth factors into the extracellular space. (3) During that stage, AEC death from apoptosis (in response to released mediators) and/or necrosis (caused by toxins and proteases) results in exposure of the BM of the alveolar epithelium. (3) Immunofluorescent staining of cultured cells at 48 hours show that TGF-[beta]1-treated, PTEN-deficient-mutant cells exhibited decreased expressions of zonula occludens-1 and claudin 4 compared with green fluorescent protein and PTEN wild types. (54) In addition, epithelial PTEN gene deficiency, isolated from mutant mice, increased the number of epithelial-derived myofibroblasts after injury. These epithelial-derived fibroblasts exhibited increased proliferative capacity, migration capacity, and resistance to apoptosis compared with non-epithelial-derived fibroblasts isolated from the PTEN wild mice after injury. Miyoshi et al (54) highlighted the epithelial PTEN gene as a crucial gatekeeper controlling ALI and lung fibrosis by modulating AEC integrity and the PTEN/PI3K/Akt pathway as a potential therapeutic target in these intractable diseases.

Clinical Practice

Clinical studies in patients with ARDS have also suggested biomarkers of epithelial cell damage that may be useful for determining prognosis. Table 2 summarizes potential biomarkers of epithelial cell damage in ARDS. Biomarkers of epithelial cell damage include RAGE, a member of the immunoglobulin superfamily involved in propagating inflammatory responses for type I AECs, surfactant protein D (SP-D) and Krebs von den Lungen (KL)-6, a mucinlike glycoprotein expressed on the surface of type II AECs and epithelial cells of the respiratory bronchioles for type II AECs and Clara cell secretory protein (CC16).

A study performed by Calfee et al (55) showed that higher baseline plasma RAGE was associated with increased lung injury severity. In addition, higher baseline RAGE was associated with increased mortality and fewer ventilator-free and organ failure-free days in patients randomized to higher tidal volumes. Matsuse et al (56) investigated the presence and distribution of RAGE in DAD in lung tissue from 7 necropsy cases by immunohistochemistry with a monoclonal antibody that was specific for RAGE. They found that all the specimens from cases of DAD showed strong RAGE expression on macrophages, whereas lung specimens from healthy parenchyma showed positive RAGE immunoreactivity on macrophages from only 2 of 7 cases (29%). They suggested that RAGE-modified proteins accumulated in alveolar macrophages in patients with DAD. (56)

KL-6 levels in plasma and serum reflect the severity of lung injury and neutrophilic inflammation. Nathani et al (57) investigated the KL-6 antigen as a marker of alveolar inflammation in patients with ARDS and demonstrated that increased KL-6 levels in BAL and plasma reflected the severity of the lung injury, which was associated with a poor prognosis. They also suggested KL-6 was a sensitive indicator of type II pneumocyte damage, increased permeability of the air-blood barrier, and destruction of the healthy lung parenchyma. Ohtsuki et al (58) reported linear and continuous immunostaining and immunogold for KL-6 on the cell surface of regenerating type II pneumocytes in patients with ARDS, but only discontinuous staining in healthy lung tissues.

Surfactant-associated protein D (SP-D) has a role in the lung immune system, improving phagocytosis of bacteria and virus. It also regulates AEC integrity. In a cohort of 38 patients, reduced SP-D in pulmonary edema fluid at the onset of ARDS was associated with a poor prognosis. (59) Nevertheless, in a study of 259 patients from the ARDS Network trial of low versus high PEEP in ARDS (ALVEOLI) as well as in 75 patients enrolled in a randomized trial for ARDS, plasma SP-D was not associated with 28-day mortality or ventilator-free days. (44)

CC16, an anti-inflammatory protein secreted by the Clara cells of the distal respiratory epithelium, has been proposed as a biomarker of lung epithelial injury. Kropski et al (60) tested the diagnostic and prognostic utility of CC16 in patients with non-trauma-related ARDS, compared with a control group of patients with acute cardiogenic pulmonary edema. They found that patients with ARDS had lower median CC16 levels in plasma and pulmonary edema fluid. Relative to the total protein concentration in pulmonary edema fluid, median CC16 levels were lower in patients with ARDS. Although neither plasma nor edema fluid CC16 levels predicted mortality, the number of days of unassisted ventilation, or intensive care unit length of stay, they concluded that CC16 was a promising diagnostic biomarker for helping to discriminate ARDS from cardiogenic pulmonary edema. (60)

Additional Biomarkers of Epithelial Cell Damage

Additional biomarkers for epithelial cell damage are shown in Table 3. Human type I cell-specific membrane protein (HTI56) is a glycosylated lung protein, which is specific to the apical membrane of type I AECs. Patients with ARDS presented higher levels of HTI56 in both lung edema fluid and plasma compared with patients with hydrostatic lung edema. (61) However, no study evaluating the association between HTI56 levels and prognosis in patients with ARDS has, to our knowledge, been published. Growth factors promote repair of AECs, including keratinocyte growth factor, hepatocyte growth factor, fibroblast growth factor, and TGF-[alpha]. Divergent effects involve growth factors during ARDS: tyrosine kinase receptor mediation (eg, keratinocyte growth factor, hepatocyte growth factor, fibroblast growth factor, and VEGF) and serine-threonine kinase receptors, such as TGF-1[beta], which tend to have an opposite effect on the upregulation that occurs when the tyrosine kinase receptor pathway is involved. (15) The Fas/FasL system has an important role in the regulation of apoptosis. Both soluble Fas and soluble FasL were associated with outcome and were higher in the lung edema fluid from patients with ARDS, compared with patients with hydrostatic pulmonary edema. (62)

MAJOR BIOMARKERS OF FIBROGENESIS

Injury to the lung parenchyma and loss of AECs lead to a deposition of temporary extracellular matrix (ECM), which is favorable to the ingrowth of fibroblasts. (54) Repeated injury to AECs causes the loss of BM integrity and activation of myofibroblasts, which results in abnormal remodeling processes leading to scarring. Fibrogenic mediators in ALI may also represent useful biomarkers and include type III procollagen, myofibroblasts, fibrocytes, and proteoglycans (PGs; Table 3).

Experimental Models

Tissue stiffness is determined by collagen and elastin fibers, the main mechanical load-bearing components of the ECM. (63) Those fibers are embedded in a matrix of PGs, which are composed of glycosaminoglycan (GAG) chains covalently linked to a protein core. The negatively charged GAGs generate repulsive electrostatic forces, which contribute to the compressive bearing and shear resistance of the ECM. (63) During tissue deformation, collagen and elastin fibers unfold and reorient; that process is opposed by the PGs surrounding those fibers. (63) Therefore, PGs contribute to the stress-strain properties of the ECM, by preventing the alveolar structure from collapse in the healthy lung. (64) There are 2 main types of GAGs: nonsulfated GAG (hyaluronic acid) and sulfated GAG (heparan sulfate and heparin, chondroitin sulfate, dermatan sulfate, keratin sulfate, versican, and decorin). Except for hyaluronic acid, GAGs are usually covalently attached to a protein core, forming an overall structure referred to as PGs. (63) Computational models of alveolar stability and rupture incorporating the mechanical properties of fibers and PGs were developed. (63) Although absolute tissue stiffness was reduced from reference range, changes in relative stiffness and alveolar shape distortion from changes in tonicity were increased in a model of porcine pancreatic elastase injury. (64) The researchers found that the amount of GAG per unit of alveolar wall length, which is responsible for PG stiffness, was greater in mice treated with porcine pancreatic elastase than in control mice. Specifically, matrix-expression versican PG increased in the tissue, but matrix-expression decorin PG decreased. They concluded that the rate of tissue deterioration was reduced when PG stiffness was increased. They suggested that the loss of PGs observed in human diseases contributed to disease progression, whereas treatments that promoted PG deposition in the ECM would slow the progression. (64)

Clinical Practice

Meduri et al (65) investigated the relationship between procollagen types I and III, pulmonary and extrapulmonary organ dysfunction, and outcome during the natural course of ARDS and in response to prolonged methylprednisolone treatment. They found that methylprednisolone promoted reduction in procollagen levels by day 8 to 15, providing evidence of biologic efficacy in unresolving ARDS.

Synenki et al (66) determined whether BAL from patients with ARDS could induce myofibroblast differentiation and whether that induction was associated with outcome. Bronchoalveolar lavage fluid was incubated with healthy human lung fibroblasts in vitro, and a-smooth muscle actin expression was assessed by reverse transcription-polymerase chain reaction. They found that BAL-induced myofibroblast differentiation was partially attributable to TGF-P1 and that an optimal level of myofibroblast induction was associated with a better outcome.

Fibrocytes were quantified in BAL fluid from 122 patients with ARDS by Quesnel et al. (67) After 28 days of inclusion, 37 patients (30%) died with BAL fibrocyte percentages greater than 6%, identifying patients at highest risk of an adverse outcome.

Additional Biomarkers of Fibrogenesis

Laminins are ECM proteins with high molecular weights that are deposited in basal membranes involved in cell processes, such as cellular adhesion, growth, differentiation, and remodeling of epithelial tissue. Higher levels of laminins were found in the plasma and lung edema fluid from 17 patients with ARDS onset than those levels were in survivors, and survivors had decreasing levels of the marker over time, suggesting that its secretion is suppressed during ARDS recovery. (68) Elastin is a protein of the ECM responsible for lung elastic recoil. During lung epithelial and endothelial injury, elastin can be broken down by neutrophil elastase and excreted in the urine. A large cohort that included 579 patients with ARDS showed that patients who were ventilated with lower tidal volumes had lower urine elastin levels, reflecting reduced ECM breakdown; however, no correlation was found with mortality in patients with ARDS. (69) The MMPs are involved in degradation, turnover, and remodeling of the ECM, digesting type I collagen. (15) Elevated MMP-2, MMP-8, and MMP-9 levels in the BAL fluid from patients with ARDS were associated with patterns of acute inflammation but with poor outcome. (70)

CLINICAL PERSPECTIVES

Regardless of improvements in the identification of biomarkers involved in ARDS pathogenesis, no single clinical or biologic marker reliably predicts clinical outcomes in ARDS. The combination of clinical and biologic markers may improve the sensitivity and/or the specificity of the test, as showed by Ware et al. (71) The ARDS Network trial that enrolled 549 patients was designed to measure 8 biomarkers involved in endothelial and epithelial cell damage, inflammation, and coagulation: VWF, SP-D, TNF-R1, IL-6, IL-8, ICAM-1, protein C, and PAI-1. (71) They found that clinical biomarkers predicted mortality with an area under the receiver operating characteristic curve of 0.82, whereas a combination of those 8 biomarkers and the clinical predictors presented an area under the curve of 0.85. The best biomarkers were IL-8 and SP-D, emphasizing that inflammation and damage to alveolar epithelial cells have fundamental roles in the pathogenetic pathways in ARDS. Recently, a panel of biomarkers of lung epithelial cell damage combined with inflammation (SP-D, sRAGE, IL-8, CC16, and IL-6) provided value for diagnosis of ARDS in patients with sepsis. (72) Therefore, the use of such biomarker panels may be useful for selecting patients for clinical trials designed to reduce lung epithelial cell damage. (31)

CONCLUSIONS

Biomarkers provide an important translational association for comprehension of lung pathophysiology. Biomarker testing has expanded into the pathogenic role of epithelial cell dysfunction, inflammation, and fibrosis in ARDS. In addition, biomarker studies may help us to explore the immune and molecular mechanisms of various therapeutic strategies for ARDS. Because of the complexity of the underlying pathobiology of ARDS, it is questionable whether a single biomarker will be found for ARDS, but the development of biomarker panels reflecting each important lung-injury pathway would provide valuable predictive and prognostic responses for both clinicians and pathologists. Although biomarkers are not now recommended for use in clinical practice with ARDS, biomarkers may be promising in developing and applying targeted therapies and in identifying candidates for clinical trials of novel therapies for ARDS.

We thank Patricia Rieken Macedo Rocco, MD, PhD, head of the Laboratory of Pulmonary Investigation and professor of physiology at Federal University in Rio de Janeiro, Brazil, for her useful comments. Research was supported by the Sao Paulo Research Foundation (FAPESP) and the CNPq National Council for Scientific and Technological Development (grants 471939/2010-2 and 483005/2012-6).

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Vera Luiza Capelozzi, MD, PhD; Timothy Craig Allen, MD, JD; Mary Beth Beasley, MD; Philip T. Cagle, MD; Don Guinee, MD; Lida P. Hariri, MD, PhD; Aliya N. Husain, MD; Deepali Jain, MD, DNB, FIAC; Sylvie Lantuejoul, MD, PhD; Brandon T. Larsen, MD, PhD; Ross Miller, MD; Mari Mino-Kenudson, MD; Mitra Mehrad, MD; Kirtee Raparia, MD; Anja Roden, MD; Frank Schneider, MD; Lynette M. Sholl, MD; Maxwell Lawrence Smith, MD

Accepted for publication May 3, 2017.

Published as an Early Online Release June 14, 2017.

From the Department of Pathology, University of Sao Paulo, Sao Paulo, Brazil (Dr Capelozzi); the Department of Pathology, the University of Texas Medical Branch, Galveston (Dr Allen); the Department of Pathology, Mount Sinai Medical Center, New York, New York (Dr Beasley); the Department of Pathology and Genomic Medicine, Houston Methodist Hospital, Houston, Texas (Drs Cagle and Miller); the Department of Pathology, Virginia Mason Medical Center, Seattle, Washington (Dr Guinee); the Department of Pathology, Massachusetts General Hospital, Boston (Drs Hariri and Mino-Kenudson); the Department of Pathology, University of Chicago, Chicago, Illinois (Dr Husain); the Department of Pathology, All India Institute of Medical Sciences, New Delhi, India (Dr Jain); the Department of Pathology, INSERM Unit, Centre Leon Berard, Lyon, France (Dr Lantuejoul) and the Universite Joseph Fourier INSERM Unit, Grenoble, France (Dr Lantuejoul); the Department of Laboratory Medicine and Pathology, Mayo Clinic, Scottsdale, Arizona (Drs Larsen and Smith); the Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee (Dr Mehrad);Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois (Dr Raparia); the Department of Laboratory Medicine and Pathology, Mayo Clinic Rochester, Rochester, Minnesota (Dr Roden); the Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania (Dr Schneider); and the Department of Pathology, Brigham and Women's Hospital, Boston (Dr Sholl).

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Timothy Craig Allen, MD, JD, University of Texas Medical Branch, Department of Pathology, 301 University Blvd, Galveston, TX 77555 (email: tcallen@utmb.edu).
Table 1. Clinical Causes Associated With Acute
Respiratory Distress Syndrome

                                           Indirect
Direct (Pulmonary) Injury           (Extrapulmonary) Injury

* Pneumonia                         * Sepsis
* Aspiration of gastric contents    * Severe trauma with
* Pulmonary contusion               shock and multiple
* Fat emboli                        transfusions
* Near drowning                     * Cardiopulmonary
* Inhalatory injury                 bypass
* Reperfusion pulmonary             * Drug overdose
edema after transplantation         * Acute pancreatitis
or pulmonary embolectomy            * Transfusion of blood
                                    products

Table 2. Summary of Published Findings for Biomarker Routine
Laboratory Testing in Clinical and Experimental Acute
Respiratory Distress Syndromea, (b)

Biomarkers     Experimental      Clinical       BAL   Plasma    Lung
                                                               Tissue

Inflammation   IL-6, IL-8,    TNF-[alpha],       X      X        X
               TGF-[beta]     IL-1[beta],
               (20-22-41}     IL-6 (23-24,36)

Endothelial    VEGF (22)      Von-Willebrand     X      X        X
cell damage                   Factor (37-41)

                              E-selectin and            X
                              ICAM-1 (36,41)

                              Thrombomodulin            X
                              (42-43)

                              Protein C                 X
                              (41-42)

                              Plasminogen               X
                              Activator
                              Inhibitor 1
                              (44-45)

Epithelial     PTEN           RAGE (55-56)              X        X
cell damage    deficiency     KL-6 (57,58)              X        X
               (54)           SP-D (59)                 X
                              CC16 (60)                 X

Fibrogenesis   Versican       Procollagen               X        X
               (63,64)        III (65)

               Decorin63      Myofibroblasts            X        X
               (64)           (66)
                              Fibrocytes (67)           X        X

Abbreviations: BAL, bronchoalveolar lavage; CC16, Clara cell
secretory protein; IL, interleukin; KL-6, Krebs von den
Lungen-6; RAGE, receptor of advanced glycation end
products; SP-D, surfactant protein; TGF, transforming growth
factor; VEGF, vascular endothelial growth factor.

(a) X indicates the increase in the biomarker according to the
investigation in routine laboratory testing.

(b) Empty cells indicate that the biomarker was not
investigated as routine laboratory testing.

Table 3. Summary of Published Findings for Additional
Biomarkers in Routine Laboratory Testing in Clinical Acute
Respiratory Distress Syndrome (a,b)

Biomarkers                 Clinical            BAL   Plasma

Inflammation       IL-18 (27)                          X
                   IL-1RA (28)                         X
                   sTNF-RI/sTNF-RII (29)               X
                   IL-10 (30)                          X
                   HMGB1 (31)                   X      X
                   LBP (32)                            X
                   NO (33)                             X
                   CRP (34)                            X
                   Albumin (35)                        X
                   LDH (35)                            X

Endothelial cell   Ang-1, Ang-2 (14)                   X
damage             EF/PL ratio (48)                    X
                   Tissue factor (49,50)        X      X
                   Cell-free hemoglobin (51)           X

Epithelial cell    HTI (61)                            X
damage             KGF (15)                            X
                   HGF (15)                            X
                   Fas/FasL (62)                       X

Fibrogenesis       Laminin (68)                        X
                   Elastin/desmosine (69)              X
                   MMPs (70)                           X

Abbreviations: Ang, angiopoietin; BAL, bronchoalveolar lavage; CRP,
C-reactive protein; EF/PL, edema fluid-to-plasma protein; HGF,
hepatocyte growth factor; HMGB1, high mobility group box nuclear
protein 1; HT156, human type I cell-specific membrane protein; IL,
interleukin; KGF, keratinocyte growth factor; LBP, lipopolysaccharide
binding protein; LDH, lactate dehydrogenase; MMP, metalloproteinases;
NO, nitric oxide; sTNF, soluble tumor necrosis factor receptors.

(a) X indicates the increase in the biomarker according to the
investigation in routine laboratory testing.

(b) Empty cells indicate that the biomarker was not investigated as
routine laboratory testing.
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Title Annotation:Special Article
Author:Capelozzi, Vera Luiza; Allen, Timothy Craig; Beasley, Mary Beth; Cagle, Philip T.; Guinee, Don; Hari
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Report
Date:Dec 1, 2017
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