Pulmonary endothelium is a major metabolic organ affecting pulmonary and systemic vascular homeostasis. Brain death (BD)-induced physiologic and metabolic derangements in donors’ lungs, in the absence of overt lung pathology, may cause pulmonary dysfunction and compromise post-transplant graft function. To explore the impact of BD on pulmonary endothelium, we estimated pulmonary capillary endothelium-bound (PCEB)-angiotensin converting enzyme (ACE) activity, a direct and quantifiable index of pulmonary endothelial function, in eight brain-dead patients and ten brain-injured mechanically ventilated controls. No subject suffered from acute lung injury or any other overt lung pathology. Applying indicator-dilution type techniques, we measured single-pass transpulmonary percent metabolism (%M) and hydrolysis (v) of the synthetic, biologically inactive, and highly specific for ACE substrate 3H-benzoyl-Phe-Ala-Pro, under first order reaction conditions, and calculated lung functional capillary surface area (FCSA). Substrate %M (35 ± 6.8%) and v (0.49 ± 0.13) in BD patients were decreased as compared to controls (55.9 ± 4.9, P = 0.033 and 0.9 ± 0.15, P = 0.033, respectively), denoting decreased pulmonary endothelial enzyme activity at the capillary level; FCSA, a reflection of endothelial enzyme activity per vascular bed, was also decreased (BD patients: 1,563 ± 562 mL/min vs 4,235 ± 559 in controls; P = 0.003). We conclude that BD is associated with subtle pulmonary endothelial injury, expressed by decreased PCEB-ACE activity. The applied indicator-dilution type technique provides direct and quantifiable indices of pulmonary endothelial function at the bedside that may reveal the existence of preclinical lung pathology in potential lung donors.
angiotensin converting enzyme; brain death; pulmonary endothelium
Lung transplantation is the only definitive therapy for many forms of end-stage lung diseases. However, the success of lung transplantation is limited by many factors: (1) Too few lungs available for transplantation due to limited donors or injury to the donor lung; (2) current methods of preservation of excised lungs do not allow extended periods of time between procurement and implantation; (3) acute graft failure is more common with lungs than other solid organs, thus contributing to poorer short-term survival after lung transplant compared with that for recipients of other organs; (4) lung transplant recipients are particularly vulnerable to pulmonary infections; and (5) chronic allograft dysfunction, manifest by bronchiolitis obliterans syndrome, is frequent and limits long-term survival. Scientific advances may provide significant improvements in the outcome of lung transplantation. The National Heart, Lung, and Blood Institute convened a working group of investigators on June 14–15, 2004, in Bethesda, Maryland, to identify opportunities for scientific advancement in lung transplantation, including basic and clinical research. This workshop provides a framework to identify critical issues related to clinical lung transplantation, and to delineate important areas for productive scientific investigation.
allograft dysfunction; infection; ischemia-reperfusion injury; lung transplantation; obliterative bronchiolitis; rejection
Primary graft dysfunction, formerly termed reperfusion pulmonary edema, is the leading cause of short-term complications after lung transplantation. New evidence shows that alveolar type I epithelial cells play an active role in alveolar fluid transport and are therefore presumed to be critical in the absorption of pulmonary edema. We tested the potential relevance of a novel marker of alveolar type I cell injury, the receptor for advanced glycation end products (RAGE), to short-term outcomes of lung transplantation.
Prospective, observational cohort study of 20 patients undergoing single lung, bilateral lung, or combined heart-lung transplantation. Plasma biomarkers were measured 4 hours after allograft reperfusion.
Higher plasma RAGE levels were associated with both a longer duration of mechanical ventilation and longer intensive care unit length of stay, in contrast to markers of alveolar type II cell injury, endothelial injury, and acute inflammation. Specifically, for every doubling in plasma RAGE levels, the duration of mechanical ventilation increased on average by 26 hours, adjusting for ischemia time (95% CI 7.4-44.7 hours, p=0.01). Likewise, for every doubling of plasma RAGE levels, intensive care unit length of stay increased on average by 1.8 days, again adjusting for ischemia time (95% CI 0.13-3.45; p=0.04). In contrast, the clinical diagnosis of primary graft dysfunction was not predictive of these short-term outcomes.
Higher levels of plasma RAGE measured shortly after reperfusion predicted poor short-term outcomes from lung transplantation. Elevated plasma RAGE levels may have both pathogenetic and prognostic value in patients following lung transplantation.
Primary graft dysfunction; reperfusion pulmonary edema; biomarkers; alveolar epithelium; acute lung injury
Substances released by platelets during blood clotting are essential participants in events that link hemostasis and angiogenesis and ensure adequate wound healing and tissue injury repair. We assessed the participation of sphingosine 1-phosphate (Sph-1-P), a biologically active phosphorylated lipid growth factor released from activated platelets, in the regulation of endothelial monolayer barrier integrity, which is key to both angiogenesis and vascular homeostasis. Sph-1-P produced rapid, sustained, and dose-dependent increases in transmonolayer electrical resistance (TER) across both human and bovine pulmonary artery and lung microvascular endothelial cells. This substance also reversed barrier dysfunction elicited by the edemagenic agent thrombin. Sph-1-P–mediated barrier enhancement was dependent upon Giα-receptor coupling to specific members of the endothelial differentiation gene (Edg) family of receptors (Edg-1 and Edg-3), Rho kinase and tyrosine kinase-dependent activation, and actin filament rearrangement. Sph-1-P–enhanced TER occurred in conjunction with Rac GTPase- and p21-associated kinase–dependent endothelial cortical actin assembly with recruitment of the actin filament regulatory protein, cofilin. Platelet-released Sph-1-P, linked to Rac- and Rho-dependent cytoskeletal rearrangement, may act late in angiogenesis to stabilize newly formed vessels, which often display abnormally increased vascular permeability.
Rationale: Obesity has been linked to acute lung injury and is a risk factor for early mortality after lung transplantation.
Objectives: To examine the associations of obesity and plasma adipokines with the risk of primary graft dysfunction after lung transplantation.
Methods: We performed a prospective cohort study of 512 adult lung transplant recipients with chronic obstructive pulmonary disease or interstitial lung disease enrolled in the Lung Transplant Outcomes Group Study. In a nested case-control study, we measured plasma leptin, adiponectin, and resistin before lung transplantation and 6 and 24 hours after lung transplantation in 40 cases of primary graft dysfunction and 80 control subjects. Generalized linear mixed models and logistic regression were used to estimate risk ratios and odds ratios.
Measurements and Main Results: Grade 3 primary graft dysfunction developed within 72 hours of transplantation in 29% participants. Obesity was associated with a twofold increased risk of primary graft dysfunction (adjusted risk ratio 2.1; 95% confidence interval, 1.7–2.6). The risk of primary graft dysfunction increased by 40% (confidence interval, 30–50%) for each 5 kg/m2 increase in body mass index after accounting for center, diagnosis, cardiopulmonary bypass, and transplant procedure. Higher plasma leptin levels were associated with a greater risk of primary graft dysfunction (sex-adjusted P = 0.02). The associations of both obesity and leptin with primary graft dysfunction tended to be stronger among those who did not undergo cardiopulmonary bypass.
Conclusions: Obesity is an independent risk factor for primary graft dysfunction after lung transplantation.
acute lung injury; leptin; lung transplantation; obesity; primary graft dysfunction
The therapeutic options for ameliorating the profound vascular permeability, alveolar flooding, and organ dysfunction that accompanies acute inflammatory lung injury (ALI) remain limited. Extending our previous finding that the intravenous administration of the sphingolipid angiogenic factor, sphingosine 1–phosphate (S1P), attenuates inflammatory lung injury and vascular permeability via ligation of S1PR1, we determine that a direct intratracheal or intravenous administration of S1P, or a selective S1P receptor (S1PR1) agonist (SEW-2871), produces highly concentration-dependent barrier-regulatory responses in the murine lung. The intratracheal or intravenous administration of S1P or SEW-2871 at < 0.3 mg/kg was protective against LPS-induced murine lung inflammation and permeability. However, intratracheal delivery of S1P at 0.5 mg/kg (for 2 h) resulted in significant alveolar–capillary barrier disruption (with a 42% increase in bronchoalveolar lavage protein), and produced rapid lethality when delivered at 2 mg/kg. Despite the greater selectivity for S1PR1, intratracheally delivered SEW-2871 at 0.5 mg/kg also resulted in significant alveolar–capillary barrier disruption, but was not lethal at 2 mg/kg. Consistent with the S1PR1 regulation of alveolar/vascular barrier function, wild-type mice pretreated with the S1PR1 inverse agonist, SB-649146, or S1PR1+/− mice exhibited reduced S1P/SEW-2871–mediated barrier protection after challenge with LPS. In contrast, S1PR2−/− knockout mice as well as mice with reduced S1PR3 expression (via silencing S1PR3-containing nanocarriers) were protected against LPS-induced barrier disruption compared with control mice. These studies underscore the potential therapeutic effects of highly selective S1PR1 receptor agonists in reducing inflammatory lung injury, and highlight the critical role of the S1P delivery route, S1PR1 agonist concentration, and S1PR1 expression in target tissues.
SEW-2871; LPS; SB-649146; S1P; lung edema
Acute lung injury represents the result of multiple pathways initiated by local or systemic insults and is characterized by profound vascular permeability, pulmonary edema, and life-threatening respiratory failure. Permeability-reducing therapies are of potential clinical utility but are currently unavailable. We hypothesized that polyethylene glycol (PEG) compounds, inert and non-toxic polymers that serve as a surrogate mucin lining in intestinal epithelium, may attenuate agonist-mediated lung endothelial cell (EC) barrier dysfunction. High molecular weight PEG (PEG15-20) produced rapid, dose-dependent increases in transendothelial electrical resistance (TER) in human lung endothelium cultured on gold microelectrodes, reflecting increased paracellular integrity. The maximal effective concentration of 8% PEG induced a sustained 125% increase in TER (40 hrs), results similar to barrier-enhancing agonists such as sphingosine 1-phosphate (40% increase in TER). Maximal PEG barrier enhancement was achieved at 45–60 min and PEG effectively reversed both thrombin- and LPS-induced EC barrier dysfunction. Consistent with the increase in TER, immunofluorescent studies demonstrated that PEG produced significant cytoskeletal rearrangement with formation of well-defined cortical actin rings and lamellipodia containing the actin-binding proteins, cortactin and MLCK, known participants in cell-matrix and cell-cell junctional adhesion. Finally, PEG challenge induced rapid alterations in levels of MAP kinase and MLC phosphorylation. In summary, PEG joins a number of EC barrier-regulatory agents which rapidly activate barrier-enhancing signal transduction pathways which target the cytoskeleton and provides a potential therapeutic strategy in inflammatory lung injury.
PEG; LPS; thrombin; endothelium; barrier function
Donor brain death is the first injurious event which can produce inflammatory dysfunction after pulmonary transplantation. This study was designed to determine whether stimulation of the toll-like receptor system contributes to the changes produced by brain death.
Materials and Methods
Rats were repeatedly treated with specific agonists for toll-like receptor 4 or toll-like receptor 2/6 to desensitize these receptors. Brain death was then induced by inflation of a balloon catheter within the extradural space. Mean arterial pressure changes and inflammatory markers were measured serially by protein and mRNA analysis.
Both desensitizing pre-treatments prevented the neurogenic hypotension (p<0.001) and metabolic acidosis (p<0.001) observed in control animals after brain death. These treatments also reduced the levels of TNFα and CXCL1 in serum and bronchoalveolar lavage fluid, although desensitization of toll-like receptor 4 produced a greater inhibition than desensitization of toll-like receptor 2. Desensitization of toll-like receptor 4 also reduced (p<0.05) expression of the adhesive integrin CD11b on blood neutrophils after brain death. Examination of mRNA levels in lung tissue 5 hours after brain death showed that desensitization of TLR4 limited the expression of IFNγ, IFNβ and CXCL10, whilst desensitization of TLR2/6 only reduced the expression of IFNγ.
These results indicate that activation of toll-like receptor signalling pathways can contribute to the lung damage produced by brain death; this may increase subsequent graft injury following transplantation.
brain death; primary graft dysfunction; inflammation; toll-like receptors
Primary graft dysfunction (PGD) after lung transplantation may result from ischemia-reperfusion injury (IRI). The innate immune response to IRI may be mediated by Toll-like receptor and IL-1-induced long pentraxin-3 (PTX3) release. We hypothesized that elevated PTX3 levels were associated with PGD. We performed a nested case control study of lung transplant recipients with Idiopathic Pulmonary Fibrosis (IPF) or Chronic Obstructive Pulmonary Disease (COPD) from the Lung Transplant Outcomes Group cohort. PTX3 levels were measured pre-transplant, and 6 and 24 hours post reperfusion. Cases were subjects with grade 3 PGD within 72 of transplantation and controls were those without grade 3 PGD. Generalized estimating equations and multivariable logistic regression was used for analysis. We selected 40 PGD cases and 79 non-PGD controls. Plasma PTX3 level was associated with PGD in IPF but not COPD recipients (p for interaction<0.03). Among patients with IPF, PTX3 levels at 6 and 24 hours were associated with PGD (OR=1.6, p=0.02 at 6hrs; OR=1.4, p=0.008 at 24hrs). Elevated PTX3 levels were associated with the development of PGD after lung transplantation in IPF patients. Future studies evaluating the role of innate immune activation in IPF and PGD are warranted.
Primary Graft Dysfunction; Lung transplantation; Long Pentraxin-3; Idiopathic Pulmonary Fibrosis
Neurogenic pulmonary edema (NPE) leading to cardiopulmonary dysfunction is a potentially life-threatening complication in patients with central nervous system lesions. This case report describes a 28-yr woman with life-threatening fulminant NPE, which was refractory to conventional respiratory treatment, following the rupture of an aneurysm. She was treated successfully with extracorporeal membrane oxygenation (ECMO), although ECMO therapy is generally contraindicated in neurological injuries such as brain trauma and diseases that are likely to require surgical intervention. The success of this treatment suggests that ECMO therapy should not be withheld from patients with life-threatening fulminant NPE after subarachnoid hemorrhage.
Extracorporeal Membrane Oxygenation; Pulmonary Edema; Subarachnoid Hemorrhage
Acute lung injury, sepsis, lung inflammation, and ventilator-induced lung injury are life-threatening conditions associated with lung vascular barrier dysfunction, which may lead to pulmonary edema. Increased levels of atrial natriuretic peptide (ANP) in lung circulation reported in these pathologies suggest its potential role in the modulation of lung injury. Besides well recognized physiological effects on vascular tone, plasma volume, and renal function, ANP may exhibit protective effects in models of lung vascular endothelial cell (EC) barrier dysfunction. However, the molecular mechanisms of ANP protective effects are not well understood. The recently described cAMP-dependent guanine nucleotide exchange factor (GEF) Epac activates small GTPase Rap1, which results in activation of small GTPase Rac-specific GEFs Tiam1 and Vav2 and Rac-mediated EC barrier protective responses. Our results show that ANP stimulated protein kinase A and the Epac/Rap1/Tiam/Vav/Rac cascade dramatically attenuated thrombin-induced pulmonary EC permeability and the disruption of EC monolayer integrity. Using pharmacological and molecular activation and inhibition of cAMP- and cGMP-dependent protein kinases (PKA and PKG), Epac, Rap1, Tiam1, Vav2, and Rac we linked ANP-mediated protective effects to the activation of Epac/Rap and PKA signaling cascades, which dramatically inhibited the Rho pathway of thrombin-induced EC hyper-permeability. These results suggest a novel mechanism of ANP protective effects against agonist-induced pulmonary EC barrier dysfunction via inhibition of Rho signaling by Epac/Rap1-Rac and PKA signaling cascades.
small GTPases; PKA; guanine nucleotide exchange factors; pulmonary endothelium; permeability
Aneurysmal subarachnoid hemorrhage (SAH) affects relatively young people and carries a poor prognosis with a case fatality rate of 35%. One of the major systemic complications associated with SAH is acute lung injury (ALI) which occurs in up to one-third of the patients and is associated with poor outcome. ALI in SAH may be predisposed by neurogenic pulmonary edema (NPE) and inflammatory mediators. The objective of this study was to assess the immunomodulatory effects of interferon-β (IFN-β) on inflammatory mediators in the lung after experimental SAH.
Male Wistar rats were subjected to the induction of SAH by means of the endovascular filament method. Sham-animals underwent sham-surgery. Rats received IFN-β for four consecutive days starting at two hours after SAH induction. After seven days, lungs were analyzed for the expression of inflammatory markers.
SAH induced the influx of neutrophils into the lung, and enhanced expression of the pulmonary adhesion molecules E-selectin, inter-cellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 compared to sham-animals. In addition, SAH increased the expression of the chemokines macrophage inflammatory protein (MIP)-1α, MIP-2, and cytokine-induced neutrophil chemoattractant (CINC)-1 in the lung. Finally, tumor necrosis factor-α (TNF-α) was significantly increased in lungs from SAH-animals compared to sham-animals. IFN-β effectively abolished the SAH-induced expression of all pro-inflammatory mediators in the lung.
IFN-β strongly reduces lung inflammation after experimental SAH and may therefore be an effective drug to prevent SAH-mediated lung injury.
A defining feature of acute lung injury (ALI) is the increased lung vascular permeability and alveolar flooding, which leads to associated morbidity and mortality. Specific therapies to alleviate the unremitting vascular leak in ALI are not currently clinically available; however, our prior studies indicate a protective role for sphingosine-1-phosphate (S1P) in animal models of ALI with reductions in lung edema. As S1P levels are tightly regulated by synthesis and degradation, we tested the hypothesis that inhibition of S1P lyase (S1PL), the enzyme that irreversibly degrades S1P via cleavage, could ameliorate ALI. Intratracheal instillation of LPS to mice enhanced S1PL expression, decreased S1P levels in lung tissue, and induced lung inflammation and injury. LPS challenge of wild-type mice receiving 2-acetyl-4(5)-[1(R),2(S),3(R),4-tetrahydroxybutyl]-imidazole to inhibit S1PL or S1PL+/− mice resulted in increased S1P levels in lung tissue and bronchoalveolar lavage fluids and reduced lung injury and inflammation. Moreover, down-regulation of S1PL expression by short interfering RNA (siRNA) in primary human lung microvascular endothelial cells increased S1P levels, and attenuated LPS-mediated phosphorylation of p38 mitogen-activated protein kinase and I-κB, IL-6 secretion, and endothelial barrier disruption via Rac1 activation. These results identify a novel role for intracellularly generated S1P in protection against ALI and suggest S1PL as a potential therapeutic target.
intracellular sphingosine-1-phosphate; sphingosine-1-phosphate lyase; IL-6; transendothelial resistance; acute lung injury
Ovarian transplantation is one of the key approaches to restoring fertility in women who became menopausal as a result of cancer treatments. A major limitation of human ovarian transplants is massive follicular loss during revascularization. Here we investigated whether sphingosine-1-phosphate or its receptor agonists could enhance neoangiogenesis and follicle survival in ovarian transplants in a xenograft model. Human ovarian tissue xenografts in severe-combined-immunodeficient mice were treated with sphingosine-1-phosphate, its analogs, or vehicle for 1–10 days. We found that sphingosine-1-phosphate treatment increased vascular density in ovarian transplants significantly whereas FTY720 and SEW2871 had the opposite effect. In addition, sphingosine-1-phosphate accelerated the angiogenic process compared to vehicle-treated controls. Furthermore, sphingosine-1-phosphate treatment was associated with a significant proliferation of ovarian stromal cell as well as reduced necrosis and tissue hypoxia compared to the vehicle-treated controls. This resulted in a significantly lower percentage of apoptotic follicles in sphingosine-1-phosphate-treated transplants. We conclude that while sphingosine-1-phosphate promotes neoangiogenesis in ovarian transplants and reduces ischemic reperfusion injury, sphingosine-1-phosphate receptor agonists appear to functionally antagonize this process. Sphingosine-1-phosphate holds great promise to clinically enhance the survival and longevity of human autologous ovarian transplants.
Neurogenic pulmonary edema (NPE) is a well-known complication of acute central neurologic injury, particularly aneurysmal subarachnoid hemorrhage. Both increased intracranial pressure and severe over-activation of the sympathetic nervous system seem to be pathogenetic for the onset of NPE. Although intracranial endovascular therapy is minimally invasive, it may affect brain stem regions and result in sympathetic activation. We now report the case of a 70-year-old woman who suddenly developed pulmonary edema during coil embolization of a ruptured aneurysm. During the intervention, oxygen saturation declined suddenly and a chest radiograph revealed pulmonary edema. The delayed appearance of NPE in this patient implies a risk for sympathetically mediated NPE during endovascular therapy.
Endovascular procedure; Pulmonary edema; Subarachnoid hemorrhage
Lung transplantation is commonly used for patients with end-stage lung disease. However, there is continuing debate on the optimal operation for patients with chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis. Single-lung transplantation (SLT) provides equivalent short- and medium-term results compared with bilateral lung transplantation (BLT), but long-term survival appears slightly better in BLT recipients (especially in patients with COPD). The number of available organs for lung transplantation also influences the choice of operation. Recent developments suggest that the organ donor shortage is not as severe as previously thought, making BLT a possible alternative for more patients. Among the different complications, re-implantation edema, infection, rejection, and bronchial complications predominate. Chronic rejection, also called obliterative bronchiolitis syndrome, is a later complication which can be observed in about half of the patients. Improvement in graft survival depends greatly in improvement in prevention and management of complications. Despite such complications, graft survival in fibrosis patients is greater than spontaneous survival on the waiting list; idiopathic fibrosis is associated with the highest mortality on the waiting list. Patients should be referred early for the pre-transplantation work-up because individual prognosis is very difficult to predict.
Lung transplantation; chronic obstructive pulmonary disease
Sphingosine 1–phosphate (S1P) is a key endogenous regulator of the response to lung injury, maintaining endothelial barrier integrity through interaction with one of its receptors, S1P1. The short-term administration of S1P or S1P1 receptor agonists enhances endothelial monolayer barrier function in vitro, and attenuates injury-induced vascular leak in the lung and other organ systems in vivo. Although S1P1 agonists bind to and activate S1P1, several of these agents also induce receptor internalization and degradation, and may therefore act as functional antagonists of S1P1 after extended exposure. Here we report on the effects of prolonged exposure to these agents in bleomycin-induced lung injury. We demonstrate that repeated administration of S1P1 agonists dramatically worsened lung injury after bleomycin challenge, as manifested by increased vascular leak and mortality. Consistent with these results, prolonged exposure to S1P1 agonists in vitro eliminated the ability of endothelial cell monolayers to respond appropriately to the barrier-protective effects of S1P, indicating a loss of normal S1P–S1P1 signaling. As bleomycin-induced lung injury progressed, continued exposure to S1P1 agonists also resulted in increased pulmonary fibrosis. These data indicate that S1P1 agonists can act as functional antagonists of S1P1 on endothelial cells in vivo, which should be considered in developing these agents as therapies for vascular leak syndromes. Our findings also support the hypothesis that vascular leak is an important component of the fibrogenic response to lung injury, and suggest that targeting the S1P–S1P1 pathway may also be an effective therapeutic strategy for fibrotic lung diseases.
lung injury; vascular leak; fibrosis; sphingosine 1–phosphate; S1P1
Brain death is associated with dramatic and serious pathophysiologic changes that adversely affect both the quantity and quality of organs available for transplant. To fully optimise the donor pool necessitates a more complete understanding of the underlying pathophysiology of organ dysfunction associated with transplantation. These injurious processes are initially triggered by catastrophic brain injury and are further enhanced during both brain death and graft transplantation. The activated inflammatory systems then contribute to graft dysfunction in the recipient. Inflammatory mediators drive this process in concert with the innate and adaptive immune systems. Activation of deleterious immunological pathways in organ grafts occurs, priming them for further inflammation after engraftment. Finally, posttransplantation ischaemia reperfusion injury leads to further generation of inflammatory mediators and consequent activation of the recipient's immune system. Ongoing research has identified key mediators that contribute to the inflammatory milieu inherent in brain dead organ donation. This has seen the development of novel therapies that directly target the inflammatory cascade.
Although alveolar epithelial injury is a major determinant of outcome in patients with acute lung injury, there is no reliable biological marker of alveolar epithelial injury. The primary objective was to determine whether elevated levels of the receptor for advanced glycation end products (RAGE), a marker of alveolar epithelial injury, reflect impaired alveolar fluid clearance (AFC) in an ex vivo perfused human lung preparation. A second objective was to determine whether levels of a marker of endothelial injury, von Willebrand factor antigen (vWF:Ag), are associated with impaired AFC.
Human lungs (N = 30) declined for transplantation by the California Transplant Donor Network were perfused at a constant pulmonary artery pressure of 12 mm Hg. Following rewarming to 36°C, the lungs were inflated with a continuous positive airway pressure of 10 cm H2O. RAGE and vWF:Ag levels and AFC rates were then measured.
The rate of AFC was inversely correlated with RAGE levels in the alveolar fluid (p < 0.005). Similarly, the concentration of RAGE in the alveolar fluid was significantly higher in lungs with submaximal AFC, defined in a prespecified analysis as ≤ 14%/h, when compared with lungs with preserved AFC (median 0.82 vs 0.43 μg/mL; p < 0.05). In contrast, vWF:Ag levels did not correlate with the rate of AFC.
RAGE may be a useful biological marker of alveolar epithelial injury and impaired AFC in donor lungs prior to transplant and perhaps in patients with acute lung injury.
acute lung injury; alveolar epithelial fluid transport; alveolar epithelial permeability; ARDS; lung transplantation; primary graft failure; receptor for advanced glycation end products; von Willebrand factor
Novel therapeutic strategies are needed to reverse the loss of endothelial cell (EC) barrier integrity that occurs during inflammatory disease states such as acute lung injury. We previously demonstrated potent EC barrier augmentation in vivo and in vitro by the platelet-derived phospholipid, sphingosine 1-phosphate (S1P) via ligation of the S1P1 receptor. The S1P analogue, FTY720, similarly exerts barrier-protective vascular effects via presumed S1P1 receptor ligation. We examined the role of the S1P1 receptor in sphingolipid-mediated human lung EC barrier enhancement. Both S1P and FTY induced sustained, dose-dependent barrier enhancement, reflected by increases in transendothelial electrical resistance (TER), which was abolished by pertussis toxin indicating Gi-coupled receptor activation. FTY-mediated increases in TER exhibited significantly delayed onset and intensity relative to the S1P response. Reduction of S1P1R expression (via siRNA) attenuated S1P-induced TER elevations whereas the TER response to FTY was unaffected. Both S1P and FTY rapidly (within 5 minutes) induced S1P1R accumulation in membrane lipid rafts, but only S1P stimulated S1P1R phosphorylation on threonine residues. Inhibition of PI3 kinase activity attenuated S1P-mediated TER increases but failed to alter FTY-induced TER elevation. Finally, S1P, but not FTY, induced significant myosin light chain phosphorylation and dramatic actin cytoskeletal rearrangement whereas reduced expression of the cytoskeletal effectors, Rac1 and cortactin (via siRNA), attenuated S1P-, but not FTY-induced TER elevations. These results mechanistically characterize pulmonary vascular barrier regulation by FTY720, suggesting a novel barrier-enhancing pathway for modulating vascular permeability.
FTY720; sphingosine 1-phosphate; vascular permeability; Rac; cytoskeleton; G-coupled receptors
Pulmonary edema and associated impaired oxygenation are a major reason for rejection of donor lung allografts offered for transplantation. Clearance of pulmonary edema can be up-regulated by stimulation of ϐ adrenergic receptors (ϐAR). Single nucleotide polymorphisms (SNPs) in ϐAR genes have functional effects in vitro and in vivo. We hypothesized that SNPs in ϐAR genes would be associated with rates of utilization of donor lung allografts offered for transplantation.
951 organ donors were genotyped for 4 amino-acid coding SNPs in the ϐAR genes. Lung allograft utilization was compared among donors stratified by genotypes.
Utilization of donor lung allografts was 55% vs. 35% (p = 0.02) among donors with GG vs. AA/AG genotypes of the Ser49Gly SNP, 39% vs. 32% (p = 0.04) with GG vs. AA/AG genotype of Gly16Arg SNP and 37% vs. 32% (p = 0.1) with CC vs. GC/GG genotype of the Arg389Gly SNP. In combined analysis, donors carrying 0–1 associated genotypes had a utilization rate of 33%, whereas donors carrying two or three associated genotypes had utilization rate of 44% and 58% respectively (p=0.008). There was a stepwise decrease in chest radiograph infiltrates and increase in the PaO2/FiO2 with increasing number of these associated genotypes.
Genetic variants in the ϐAR genes among organ donors are associated with higher rates of lung allograft utilization. The increased utilization may be related to increased clearance of pulmonary edema and improved oxygenation in donors with favorable genotypes and suggests ϐAR agonists may have a role in donor management.
Primary graft dysfunction (PGD) is the leading cause of early post-transplant morbidity and mortality after lung transplantation. Clara cell secretory protein (CC16) is produced by the non-ciliated lung epithelium and may serve as a plasma marker of epithelial cell injury. We hypothesized that elevated levels of CC16 would be associated with increased odds of PGD. We performed a prospective cohort study of 104 lung transplant recipients. Median plasma CC16 levels were determined at three time points: pre-transplant and 6 and 24 hours post transplant. The primary outcome was the development of grade 3 PGD within the first 72 hours after transplantation. Multivariable logistic regression was performed to evaluate for confounding by donor and recipient demographics and surgical characteristics. Twenty-nine patients (28%) developed grade 3 PGD within the first 72 hours. The median CC16 level 6 hours after transplant was significantly higher in patients with PGD (13.8 ng/ml (IQR 7.9, 30.4 ng/ml)) than in patients without PGD (8.2 ng/ml (IQR 4.5, 19.1 ng/ml)), p = 0.02. Elevated CC16 levels were associated with increased odds of PGD after lung transplantation. Damage to airway epithelium or altered alveolar permeability as a result of lung ischemia and reperfusion may explain this association.
Primary Graft Dysfunction; Lung transplantation; Clara Cell Secretory Protein
Neurogenic pulmonary edema (NPE) can result from various central nervous system disorders such as brain malignancies, traumatic brain injuries, infections, and seizures. Although the pathogenesis is not completely understood, NPE creates an increase in pulmonary interstitial and alveolar fluid. It has been reported with prolonged seizure activity. Treatment for NPE is largely supportive. If unrecognized, it can lead to hypoxia and respiratory arrest. We report a case of NPE in a middle-aged female patient following a breakthrough seizure in whom an immunological cause for respiratory findings was high on the differential list, based on her past medical history and chronicity of symptoms. Rapid symptomatic and radiological improvement following hospitalization led to the correct diagnosis.
Development of primary graft dysfunction (PGD) is associated with poor outcomes after transplantation. We hypothesized that Receptor for Advanced Glycation End-products (RAGE) levels in donor lungs is associated with the development of PGD. Furthermore, we hypothesized that RAGE levels would be increased with PGD in recipients after transplantation.
We measured RAGE in bronchoalveolar lavage fluid (BALf) from 25 donors and 34 recipients. RAGE was also detected in biopsies (TBBX) from recipients with and without PGD.
RAGE levels were significantly higher in donor lungs that subsequently developed sustained PGD vs transplanted lungs that did not display PGD. Donor RAGE level was a predictor of recipient PGD (odds ratio = 1.768 per 0.25 ng/ml increase in donor RAGE level). In addition, RAGE levels remained high at 14 days in those recipients that developed severe graft dysfunction.
Recipients may be at higher risk for developing PGD if they receive transplanted organs that have higher levels of soluble RAGE prior to explantation. Moreover, the clinical and pathologic abnormalities associated with PGD post-transplantation are associated with increased RAGE expression. These findings also raise the possibility that targeting the RAGE signaling pathway could be a novel strategy for treatment and/or prevention of PGD.
Primary Graft Dysfunction; Lung transplantation; Reperfusion injury; Receptor for advanced glycation end products; RAGE
The main goal of adequate organ preservation is to avoid further cellular metabolism during the phase of ischemia. However, modern preservation solutions do rarely achieve this target. In donor organs hypoxia and ischemia induce a broad spectrum of pathologic molecular mechanisms favoring primary graft dysfunction (PGD) after transplantation. Increased hypoxia-induced transcriptional activity leads to increased vascular permeability which in turn is the soil of a reperfusion edema and the enhancement of a pro-inflammatory response in the graft after reperfusion. We hypothesize that inhibition of the respiration chain in mitochondria and thus inhibition of the hypoxia induced mechanisms might reduce reperfusion edema and consecutively improve survival in vivo. In this study we demonstrate that the rotenoid Deguelin reduces the expression of hypoxia induced target genes, and especially VEGF-A, dose-dependently in hypoxic human lung derived cells. Furthermore, Deguelin significantly suppresses the mRNA expression of the HIF target genes VEGF-A, the pro-inflammatory CXCR4 and ICAM-1 in ischemic lungs vs. control lungs. After lung transplantation, the VEGF-A induced reperfusion-edema is significantly lower in Deguelin-treated animals than in controls. Deguelin-treated rats exhibit a significantly increased survival-rate after transplantation. Additionally, a downregulation of the pro-inflammatory molecules ICAM-1 and CXCR4 and an increase in the recruitment of immunomodulatory monocytes (CD163+ and CD68+) to the transplanted organ involving the IL4 pathway was observed. Therefore, we conclude that ischemic periods preceding reperfusion are mainly responsible for the increased vascular permeability via upregulation of VEGF. Together with this, the resulting endothelial dysfunction also enhances inflammation and consequently lung dysfunction. Deguelin significantly decreases a VEGF-A induced reperfusion edema, induces the recruitment of immunomodulatory monocytes and thus improves organ function and survival after lung transplantation by interfering with hypoxia induced signaling.