Lucas G. Fernandez

Tissue-derived proinflammatory effect of adenosine A2B receptor in lung ischemia–reperfusion injury

OBJECTIVE Ischemia-reperfusion injury after lung transplantation remains a major source of morbidity and mortality. Adenosine receptors have been implicated in both pro- and anti-inflammatory roles in ischemia-reperfusion injury. This study tests the hypothesis that the adenosine A(2B) receptor exacerbates the proinflammatory response to lung ischemia-reperfusion injury. METHODS An in vivo left lung hilar clamp model of ischemia-reperfusion was used in wild-type C57BL6 and adenosine A(2B) receptor knockout mice, and in chimeras created by bone marrow transplantation between wild-type and adenosine A(2B) receptor knockout mice. Mice underwent sham surgery or lung ischemia-reperfusion (1 hour ischemia and 2 hours reperfusion). At the end of reperfusion, lung function was assessed using an isolated buffer-perfused lung system. Lung inflammation was assessed by measuring proinflammatory cytokine levels in bronchoalveolar lavage fluid, and neutrophil infiltration was assessed via myeloperoxidase levels in lung tissue. RESULTS Compared with wild-type mice, lungs of adenosine A(2B) receptor knockout mice were significantly protected after ischemia-reperfusion, as evidenced by significantly reduced pulmonary artery pressure, increased lung compliance, decreased myeloperoxidase, and reduced proinflammatory cytokine levels (tumor necrosis factor-α; interleukin-6; keratinocyte chemoattractant; regulated on activation, normal T-cell expressed and secreted; and monocyte chemotactic protein-1). Adenosine A(2B) receptor knockout → adenosine A(2B) receptor knockout (donor → recipient) and wild-type → adenosine A(2B) receptor knockout, but not adenosine A(2B) receptor knockout → wild-type, chimeras showed significantly improved lung function after ischemia-reperfusion. CONCLUSIONS These results suggest that the adenosine A(2B) receptor plays an important role in mediating lung inflammation after ischemia-reperfusion by stimulating cytokine production and neutrophil chemotaxis. The proinflammatory effects of adenosine A(2B) receptor seem to be derived by adenosine A(2B) receptor activation primarily on resident pulmonary cells and not bone marrow-derived cells. Adenosine A(2B) receptor may provide a therapeutic target for prevention of ischemia-reperfusion-related graft dysfunction in lung transplant recipients.

The Maturing Kidney: Development and Susceptibility

Kidney morphogenesis is accomplished by the coordinated interaction of molecular signals that culminate in the production of an organ that is architecturally and functionally ready for extrauterine, free life. In humans, nephrogenesis is completed before birth. However the kidney continues to mature both from a functional and anatomical point of view. Throughout its development, the kidney is susceptible to a variety of injurious agents. This brief review considers the basic mechanisms of kidney organogenesis and functional maturation. To illustrate some concepts, the renal alterations caused by interference with a normal regulatory system, the renin-angiotensin system is discussed.

Adenosine A3 Receptor Activation Attenuates Lung Ischemia-Reperfusion Injury

BACKGROUND Severe ischemia-reperfusion (IR) injury leads to primary graft dysfunction after lung transplantation. Adenosine receptors modulate inflammation after IR, and the adenosine A3 receptor (A3R) is expressed in lung tissue and inflammatory cells. This study tests the hypothesis that A3R agonism attenuates lung IR injury by a neutrophil-dependent mechanism. METHODS Wild-type and A3R knockout (A3R-/-) mice underwent 1-hour left lung ischemia followed by 2-hours reperfusion (IR). A selective A3R agonist, Cl-IB-MECA, was administered (100 μg/kg intravenously) 5 minutes prior to ischemia. Study groups included sham, IR, and IR+Cl-IB-MECA (n = 6/group). Lung injury was assessed by measuring lung function, pulmonary edema, histopathology, and proinflammatory cytokines, and myeloperoxidase levels in bronchoalveolar lavage fluid. Parallel in vitro experiments were performed to evaluate neutrophil chemotaxis, and neutrophil activation was measured after exposure to acute hypoxia and reoxygenation. RESULTS Treatment of wild-type mice with Cl-IB-MECA significantly improved lung function and decreased edema, cytokine expression, and neutrophil infiltration after IR. The Cl-IB-MECA had no effects in A3R-/- mice; Cl-IB-MECA significantly decreased activation of wild-type, but not A3R-/-, neutrophils after acute hypoxia and reoxygenation and inhibited chemotaxis of wild-type neutrophils. CONCLUSIONS Exogenous activation of A3R by Cl-IB-MECA attenuates lung dysfunction, inflammation, and neutrophil infiltration after IR in wild-type but not A3R-/- mice. Results with isolated neutrophils suggest that the protective effects of Cl-IB-MECA are due, in part, to the prevention of neutrophil activation and chemotaxis. The use of A3R agonists may be a novel therapeutic strategy to prevent lung IR injury and primary graft dysfunction after transplantation.

Rapamycin Blocks Fibrocyte Migration and Attenuates Bronchiolitis Obliterans in a Murine Model

BACKGROUND Fibrocytes are integral in the development of fibroproliferative disease. The CXCL12/CXCR4 chemokine axis has been shown to play a central role in fibrocyte migration and the development of bronchiolitis obliterans (BO) after lung transplantation. Inhibition of the mammalian target of rapamycin (mTOR) pathway with rapamycin has been shown to decrease expression of both CXCR4 and its receptor agonist CXCL12. Thus, we hypothesized that rapamycin treatment would decrease fibrocyte trafficking into tracheal allografts and prevent BO. METHODS A total alloantigenic mismatch murine heterotopic tracheal transplant (HTT) model of BO was used. Animals were either treated with rapamycin or dimethyl sulfoxide (DMSO) for 14 days after tracheal transplantation. Fibrocyte levels were assessed by flow cytometry, and allograft neutrophil, CD3(+) T-cell, macrophage, and smooth muscle actin (SMA) levels were assessed by immunohistochemistry. Tracheal luminal obliteration was assessed on hematoxylin and eosin (H&E) stains. RESULTS Compared with DMSO-treated controls, rapamycin-treated mice showed a significant decrease in fibrocyte levels in tracheal allografts. Fibrocyte levels in recipient blood showed a similar pattern, although it was not statistically significant. Furthermore, animals treated with rapamycin showed a significant decrease in tracheal allograft luminal obliteration compared with controls. Based on immunohistochemical analyses, populations of α-SMA-positive (α-SMA(+)) cells, neutrophils, CD3(+) T cells, and macrophages were all decreased in rapamycin-treated allografts versus DMSO controls. CONCLUSIONS Rapamycin effectively reduces recruitment of fibrocytes into tracheal allografts and mitigates development of tracheal luminal fibrosis. Further studies are needed to determine the cellular and molecular mechanisms that mediate the protective effect of rapamycin against BO.

Inflammatory lung injury after cardiopulmonary bypass is attenuated by adenosine A2A receptor activation

OBJECTIVE Cardiopulmonary bypass has been shown to exert an inflammatory response within the lung, often resulting in postoperative pulmonary dysfunction. Several studies have shown that adenosine A(2A) receptor activation attenuates lung ischemia-reperfusion injury; however, the effect of adenosine A(2A) receptor activation on cardiopulmonary bypass-induced lung injury has not been studied. We hypothesized that specific adenosine A(2A) receptor activation by ATL313 would attenuate inflammatory lung injury after cardiopulmonary bypass. METHODS Adult male Sprague-Dawley rats were randomly divided into 3 groups: 1) SHAM group (underwent cannulation + heparinization only); 2) CONTROL group (underwent 90 minutes of normothermic cardiopulmonary bypass with normal whole-blood priming solution; and 3) ATL group (underwent 90 minutes of normothermic cardiopulmonary bypass with ATL313 added to the normal priming solution). RESULTS There was significantly less pulmonary edema and lung injury in the ATL group compared with the CONTROL group. The ATL group had significant reductions in bronchoalveolar lavage interleukin-1, interleukin-6, interferon-gamma, and myeloperoxidase levels compared with the CONTROL group. Similarly, lung tissue interleukin-6, tumor necrosis factor-alpha, and interferon-gamma were significantly decreased in the ATL group compared with the CONTROL group. There was no significant difference between the SHAM and ATL groups in the amount of pulmonary edema, lung injury, or levels of proinflammatory cytokines. CONCLUSION The addition of a potent adenosine A(2A) receptor agonist to the normal priming solution before the initiation of cardiopulmonary bypass significantly protects the lung from the inflammatory effects of cardiopulmonary bypass and reduces the amount of lung injury. Adenosine A(2A) receptor agonists could represent a new therapeutic strategy for reducing the potentially devastating consequences of the inflammatory response associated with cardiopulmonary bypass.

Comparison of Seer Sonorheometry With Rotational Thromboelastometry and Laboratory Parameters in Cardiac Surgery.

BACKGROUND:The Quantra Hemostasis Analyzer is a novel diagnostic device that uses an ultrasound-based technology, sonic estimation of elasticity via resonance (SEER) sonorheometry, to characterize the dynamic changes in viscoelastic properties of a blood sample during coagulation. Cardiac surgery utilizing cardiopulmonary bypass (CPB) is associated with a significant impact on the coagulation system and can result in perioperative coagulopathy. The aim of this study was to correlate SEER sonorheometry results to corresponding rotational thromboelastometry (ROTEM) and laboratory parameters obtained before, during, and after CPB. METHODS:The Quantra uses a multiwell cartridge that performs 4 independent measurements with different combination of reagents. The output test results include Clot Time, Clot Stiffness, Fibrinogen and Platelet Contribution, Clot Time Ratio, and Heparinase Clot Time. Clot Time was compared with ROTEM INTEM clotting time and the adjusted partial thromboplastin time. Clot Stiffness was compared with ROTEM EXTEM. The Fibrinogen Contribution to the Clot Stiffness was correlated to ROTEM FIBTEM as well as fibrinogen concentration by the Clauss method. The Platelet Contribution to Clot Stiffness was compared with absolute platelet count and ROTEM-determined clot elasticity attributable to platelets. RESULTS:Fifty-five patients undergoing elective cardiac surgery were enrolled in this prospective observational study. Clot Time exhibited good correlation with ROTEM INTEM clotting time (pre-CPB r = 0.84, post-CPB r = 0.65) and adjusted partial thromboplastin time (pre-CPB r = 0.72, post-CPB r = 0.89); however, the majority of values were within a narrow normal range. Clot Stiffness exhibited significant correlation with ROTEM EXTEM A10 throughout the course of the study in all samples (r = 0.84). Fibrinogen Contribution correlated strongly with FIBTEM A10 (r = 0.85), and moderately with the fibrinogen concentration (r = 0.73) determined with the Clauss assay. The Platelet Contribution to Clot Stiffness showed moderate correlation to absolute platelet counts (r = 0.48). However, the correlation between Platelet Contribution and ROTEM-determined clot elasticity attributable to platelets was stronger (r = 0.78) than platelet number. All of the correlation coefficients were statistically significant with P < .001. CONCLUSIONS:SEER sonorheometry demonstrates significant correlation with ROTEM for determining Clot Stiffness and assessing Fibrinogen Contribution. SEER sonorheometry results can provide valuable information about the coagulation status in patients undergoing cardiac surgery using CPB.

Comparison of Coagulation Parameters in Arterial and Venous Blood in Cardiac Surgery Measured Using the Quantra System

Objective: Perioperative coagulation testing often is performed with arterial samples even though device reference ranges typically are established in venous samples. Although limited studies exist comparing coagulation parameters across sampling sites, viscoelastic testing devices have demonstrated some differences. The objective of this study was to compare coagulation parameters determined using the Quantra System for venous and arterial samples. Design: Prospective, observational study. Setting: Tertiary care university hospital. Participants: The study comprised 30 adult patients undergoing cardiac surgery. Interventions: Paired arterial and venous samples were obtained at 2 of the following time points: baseline, during bypass, or after protamine reversal of heparin. Quantra measurements included Clot Time (CT), Heparinase Clot Time (CTH), Clot Time Ratio (CTR), Clot Stiffness (CS), and Fibrinogen (FCS) and Platelet (PCS) Contributions to clot stiffness. Measurements and Main Results: The relationship and agreement between matched data pairs were established and statistical analysis was performed via paired t tests. CTR, CS, FCS, and PCS were unaffected by the sampling site, whereas CT and CTH demonstrated statistically significant differences between arterial and venous samples (p < 0.001). Arterial clot times were prolonged relative to the venous ones with a mean percent error of 14.2 % and 11.9 %, respectively. These results are in general agreement with those reported for other viscoelastic testing devices. Conclusions: This study demonstrates that Quantra clot stiffness-based parameters (CS, FCS, PCS) are unaffected by sampling site, whereas the clot time parameters (CT and CTH) show good correlation in the presence of a bias. CTR, a ratio of CT and CTH, also is unaffected.

Compensatory Lung Growth After Pneumonectomy

Pneumonectomy, the surgical removal of a lung, elicits a number of anatomical changes within the thoracic cavity that augments the diffusion capacity of the remaining lung. Pneumonectomy directs the entire cardiac output into the remaining lung and creates an empty hemithorax that results in a shift of the mediastinum toward the vacated thoracic compartment. In a number of experimental animal models, pneumonectomy initiates compensatory, regenerative growth of the remaining lung tissue that restores normal mass, structure and function. This growth process, called compensatory lung growth (CLG), is qualitatively similar across species, but differs with gender, age and hormonal status. CLG involves unique structure-function interactions not seen in solid organs. Little is known about the regenerative potential of human lungs. Although CLG has been reported in children after major lung resection, CLG in adults rarely occurs and remains a significant challenge. Mechanical feedback between the lung and thorax constitutes a major signal that sustains both post-natal lung development as well as post-pneumonectomy CLG. After pneumonectomy, increased mechanical stress and strain on the remaining lung induce adaptive responses to augment oxygen transport, including 1) recruitment of alveolarcapillary reserves, 2) remodeling of existing tissue, and 3) regenerative growth of acinar tissue when strain exceeds a critical threshold. This chapter will discuss the clinical aspects of pneumonectomy and will primarily review cellular and molecular mechanisms of CLG via experimental pneumonectomy models, which offers powerful insights into regenerative organ growth.