Phosphorylation of vasodilator-stimulated phosphoprotein contributes to myocardial ischemic preconditioning.

Ischemic preconditioning (IP) is a well-known strategy to protect organs against cell death following ischemia. The previous work has shown that vasodilator-stimulated phosphoprotein (VASP) is involved in cytoskeletal reorganization and that it holds significant importance for the extent of myocardial ischemia reperfusion injury. Yet, the role of VASP during myocardial IP is, to date, not known. We report here that VASP phosphorylation at serine157 and serine239 is induced during hypoxia in vitro and during IP in vivo. The preconditioning-induced VASP phosphorylation inactivates the GP IIb/IIIa integrin receptor on platelets, which results in the reduced formation of organ compromising platelet neutrophil complexes. Experiments in chimeric mice confirmed the importance of VASP phosphorylation during myocardial IP. When studying this in VASP-/- animals and in an isolated heart model, we were able to confirm the important role of VASP on myocardial IP. In conclusion, we were able to show that IP-induced VASP phosphorylation in platelets is a protective mechanism against the deleterious effects of ischemia.

Inhibition of Adenosine Kinase Attenuates Acute Lung Injury.

OBJECTIVES: Extracellular adenosine has tissue-protective potential in several conditions. Adenosine levels are regulated by a close interplay between nucleoside transporters and adenosine kinase. On the basis of the evidence of the role of adenosine kinase in regulating adenosine levels during hypoxia, we evaluated the effect of adenosine kinase on lung injury. Furthermore, we tested the influence of a pharmacologic approach to blocking adenosine kinase on the extent of lung injury. DESIGN: Prospective experimental animal study. SETTING: University-based research laboratory. SUBJECTS: In vitro cell lines, wild-type and adenosine kinase+/- mice. INTERVENTIONS: We tested the expression of adenosine kinase during inflammatory stimulation in vitro and in a model of lipopolysaccharide inhalation in vivo. Studies using the adenosine kinase promoter were performed in vitro. Wild-type and adenosine kinase+/- mice were subjected to lipopolysaccharide inhalation. Pharmacologic inhibition of adenosine kinase was performed in vitro, and its effect on adenosine uptake was evaluated. The pharmacologic inhibition was also performed in vivo, and the effect on lung injury was assessed. MEASUREMENTS AND MAIN RESULTS: We observed the repression of adenosine kinase by proinflammatory cytokines and found a significant influence of nuclear factor kappa-light-chain-enhancer of activated B-cells on regulation of the adenosine kinase promoter. Mice with endogenous adenosine kinase repression (adenosine kinase+/-) showed reduced infiltration of leukocytes into the alveolar space, decreased total protein and myeloperoxidase levels, and lower cytokine levels in the alveolar lavage fluid. The inhibition of adenosine kinase by 5-iodotubercidin increased the extracellular adenosine levels in vitro, diminished the transmigration of neutrophils, and improved the epithelial barrier function. The inhibition of adenosine kinase in vivo showed protective properties, reducing the extent of pulmonary inflammation during lung injury. CONCLUSIONS: Taken together, these data show that adenosine kinase is a valuable target for reducing the inflammatory changes associated with lung injury and should be pursued as a therapeutic option.

Adenosine Receptor Adora2b Plays a Mechanistic Role in the Protective Effect of the Volatile Anesthetic Sevoflurane during Liver Ischemia/Reperfusion.

BACKGROUND: Liver ischemia/reperfusion (IR) injury is characterized by hepatic tissue damage and an inflammatory response. This is accompanied by the formation and vascular sequestration of platelet-neutrophil conjugates (PNCs). Signaling through Adora2b adenosine receptors can provide liver protection. Volatile anesthetics may interact with adenosine receptors. This study investigates potential antiinflammatory effects of the volatile anesthetic sevoflurane during liver IR. METHODS: Experiments were performed ex vivo with human blood and in a liver IR model with wild-type, Adora2a, and Adora2b mice. The effect of sevoflurane on platelet activation, PNC formation and sequestration, cytokine release, and liver damage (alanine aminotransferase release) was analyzed using flow cytometry, luminometry, and immunofluorescence. Adenosine receptor expression in liver tissue was analyzed using immunohistochemistry and real-time polymerase chain reaction. RESULTS: Ex vivo experiments indicate that sevoflurane inhibits platelet and leukocyte activation (n = 5). During liver IR, sevoflurane (2 Vol%) decreased PNC formation 2.4-fold in wild-type (P < 0.05) but not in Adora2b mice (n ≥ 5). Sevoflurane reduced PNC sequestration 1.9-fold (P < 0.05) and alanine aminotransferase release 3.5-fold (P < 0.05) in wild-type but not in Adora2b mice (n = 5). In Adora2a mice, sevoflurane also inhibited PNC formation and cytokine release. Sevoflurane diminished cytokine release (n ≥ 3) and increased Adora2b transcription and expression in liver tissue of wild-types (n = 4). CONCLUSIONS: Our experiments highlight antiinflammatory and tissue-protective properties of sevoflurane during liver IR and reveal a mechanistic role of Adora2b in sevoflurane-associated effects. The targeted use of sevoflurane not only as an anesthetic but also to prevent IR damage is a promising approach in the treatment of critically ill patients.

Semaphorin 7A is protective during inflammatory peritonitis through integrin receptor signaling.

Introduction: The study explores the role of endothelial Semaphorin 7A (SEMA7A) in inflammatory processes. SEMA7A is known for enhancing inflammation during tissue hypoxia and exhibiting anti-inflammatory properties in the intestinal system during colitis. This research extends the understanding of SEMA7A's function by examining its role in inflammatory peritonitis and intestinal inflammation. Methods: The research involved inducing peritonitis in SEMA7A knockout (SEMA7A-/-) and wild-type (WT) animals through Zymosan A (ZyA) injection. The inflammatory response was assessed by measuring cell count and cytokine release. In parallel, the study investigated the expression of SEMA7A in intestinal epithelial cells under inflammatory stimuli and its impact on interleukin 10 (IL-10) production using an in vitro co-culture model of monocytes and epithelial cells. Additionally, the distribution of SEMA7A target receptors, particularly ITGAV/ITGB1 (CD51/CD29), was analyzed in WT animals. Results: The results revealed that SEMA7A-/- animals exhibited increased inflammatory peritonitis compared to the WT animals. Inflammatory conditions in intestinal epithelial cells led to the induction of SEMA7A. The co-culture experiments demonstrated that SEMA7A induced IL-10 production, which depended on integrin receptors and was independent of PLXNC1 expression. Furthermore, ITGAV/ITGB1 emerged as the predominant SEMA7A receptor in the intestinal area of WT animals. Discussion: These findings underscore the multifaceted role of SEMA7A in inflammatory processes. The differential responses in peritonitis and intestinal inflammation suggest that SEMA7A's function is significantly influenced by the expression and distribution of its target receptors within different organ systems. The study highlights the complex and context-dependent nature of SEMA7A in mediating inflammatory responses.

Optoacoustic Imaging Offers New Insights into In Vivo Human Skin Vascular Physiology.

Functional imaging with new photoacoustic tomography (PAT) offers improved spatial and temporal resolution quality in in vivo human skin vascular assessments. In the present study, we followed a suprasystolic reactive hyperemia (RH) maneuver with a multi-spectral optoacoustic tomography (MSOT) system. A convenience sample of ten participants, both sexes, mean age of 35.8 ± 13.3 years old, was selected. All procedures were in accordance with the principles of good clinical practice and approved by the institutional ethics committee. Images were obtained at baseline (resting), during occlusion, and immediately after pressure release. Observations of the RH by PAT identified superficial and deeper vascular structures parallel to the skin surface as part of the human skin vascular plexus. Furthermore, PAT revealed that the suprasystolic occlusion impacts both plexus differently, practically obliterating the superficial smaller vessels and evoking stasis at the deeper, larger structures in real-time (live) conditions. This dual effect of RH on the skin plexus has not been explored and is not considered in clinical settings. Thus, RH seems to represent much more than the local microvascular reperfusion as typically described, and PAT offers a vast potential for vascular clinical and preclinical research.

Platelets and the Cybernetic Regulation of Ischemic Inflammatory Responses through PNC Formation Regulated by Extracellular Nucleotide Metabolism and Signaling.

Ischemic events are associated with severe inflammation and are here referred to as ischemic inflammatory response (IIR). Recent studies identified the formation of platelet-neutrophil complexes (PNC) as key players in IIR. We investigated the role of extracellular platelet nucleotide signaling in the context of IIR and defined a cybernetic circle, including description of feedback loops. Cybernetic circles seek to integrate different levels of information to understand how biological systems function. Our study specifies the components of the cybernetic system of platelets in IIR and describes the theoretical progression of IIR passing the cybernetic cycle with positive and negative feedback loops based on nucleotide-dependent signaling and functional regulation. The cybernetic components and feedback loops were explored by cytometry, immunohistological staining, functional blocking antibodies, and ADP/ATP measurements. Using several ex vivo and in vivo approaches we confirmed cybernetic parameters, such as controller, sensor, and effector (VASP phosphorylation, P2Y12, ADORAs and GPIIb/IIIa activity), as well as set points (ADP, adenosine) and interfering control and disturbance variables (ischemia). We demonstrate the impact of the regulated platelet-neutrophil complex (PNC) formation in blood and the resulting damage to the affected inflamed tissue. Taken together, extracellular nucleotide signaling, PNC formation, and tissue damage in IIR can be integrated in a controlled cybernetic circle of platelet function, as introduced through this study.

Toll-like receptor 2 signaling triggers fatal arrhythmias upon myocardial ischemia-reperfusion.

OBJECTIVE: Restoration of myocardial blood flow after ischemia triggers an inflammatory response involving toll-like receptors. Toll-like receptor 2 deficiency is associated with a reduced infarct size after myocardial ischemia and reperfusion. Because a marked mortality was observed in C3HeN wild-type mice, which was absent in TLR2 mice, we tested whether cardiac arrhythmias are the underlying pathology and aimed to elucidate how toll-like receptor 2 ligation might prevent lethal arrhythmias. DESIGN: Experimental animal model. SETTING: University hospital research laboratory. SUBJECTS: Male C3HeN mice. INTERVENTIONS: Myocardial ischemia and reperfusion was surgically induced by ligation of the left anterior descending coronary artery for 20 mins followed by 24 hrs of reperfusion. Electrocardiography was continuously recorded during the observation period through an implantable telemetry transmitter to detect cardiac arrhythmias during reperfusion. MEASUREMENTS AND MAIN RESULTS: Toll-like receptor 2 expression was associated with a 51% mortality rate (23 of 45 mice died) after myocardial ischemia and reperfusion. Absence of toll-like receptor 2 improved survival toward 100% (17 of 17 mice survived). Electrocardiography diagnostics in conscious animals and histologic analysis revealed that absence of toll-like receptor 2 signaling prevented the formation of pathologic heart rate turbulence after myocardial ischemia and reperfusion and modulated the density of connexin 43-positive gap junctions in the ischemic area compared with wild-type hearts, indicating arrhythmia as the cause underlying the observed mortality. CONCLUSIONS: The results presented here indicate toll-like receptor 2 as a novel target for the prevention of lethal arrhythmic complications after myocardial ischemia and reperfusion.

Preconditioning by toll-like receptor 2 agonist Pam3CSK4 reduces CXCL1-dependent leukocyte recruitment in murine myocardial ischemia/reperfusion injury.

OBJECTIVE: To test whether preconditioning with a toll-like receptor (TLR) 2 agonist protects against myocardial ischemia and reperfusion by interfering with chemokine CXCL1 release from cardiomyocytes. DESIGN: C3H mice were challenged with vehicle or synthetic TLR2 agonist Pam3Cys-Ser-Lys4 (Pam3CSK4; 1 mg/kg) 24 hrs before myocardial ischemia (20 mins) and reperfusion (2 hrs or 24 hrs). Infarct size, troponin T release, and leukocyte recruitment were quantified. In murine cardiomyocytes (HL-1), we studied the expression/activation profile of TLR2 in response to stimulation with Pam3CSK4 (0.01-1 mg/mL). Furthermore, we studied the chemokine ligand 1 (CXCL1) response to Pam3CSK4 and ischemia/reperfusion in vivo and in vitro. SETTING: University hospital research laboratory. SUBJECTS: Anesthetized male mice and murine cardiomyocytes. MEASUREMENTS AND MAIN RESULTS: Preconditioning by Pam3CSK4 reduced infarct size and troponin T release. This was accompanied by a decreased recruitment of leukocytes into the ischemic area and an improved cardiac function. In HL-1 cells, TLR2 activation amplified the expression of the receptor in a time-dependent manner and led to CXCL1 release in a concentration-dependent manner. Preconditioning by Pam3CSK4 impaired CXCL1 release in response to a second inflammatory stimulus in vivo and in vitro. CONCLUSIONS: Preconditioning by TLR2 agonist Pam3CSK4 reduces myocardial infarct size after myocardial ischemia/reperfusion. One of the mechanisms involved is a diminished chemokine release from cardiomyocytes, which subsequently limits leukocyte infiltration.